Horticulture

Differences in total root length, surface and volume density, average root diameter, and root tips and fork densities were analyzed using a split-plot design with genotypes as main plots and depth as subplot. This is a conservative statistical analysis because it reduces the df for genotype from 3 to 1. Therefore, we also compared the two same pairs of genotypes using statistical contrasts in an ANOVA including all four genotypes. To account for the inability to randomize depths, we used a conservative estimate of the df for subplots and for the interaction between subplot and main plot. Conservative df were calculated by dividing their df by the number of subplots. This strategy is similar to that used for repeated measures in time and does not affect comparisons among main plots , which are the main objective of this study. Homogeneity of variance and normality of the residuals was confirmed for all the individual ANOVAs performed at each depth for all parameters. When necessary, data was transformed using power transformations to satisfy ANOVA assumptions.The experiments were performed in 13 L hydroponic tanks containing the nutrient solution. Twenty-four seedlings were placed in each tank in a six by four pattern. All genotypes were included in one tank and, if necessary, multiple tanks were used as replications. In the experiments to study the effect of different nitrogen sources and concentrations ,vertical growing systems seedlings were grown in normal growth solution for seven days and then transferred to four separate tanks with each of the four nitrogen sources for 10 days .

Roots were measured at 22 DAG. Each tank included 6 replications of each of the four genotypes organized in a completely randomized design. The results were analyzed in a 2 x 2 factorial ANOVA with distal and proximal 1RS regions as factors and wheat or rye chromosome segments as levels. For the analysis of distances between the first lateral roots and the RAM, the four genotypes were grown in a tank with normal nitrogen conditions in a completely randomized design . Lines carrying distal rye or wheat segments were compared using a t-test. Chascomús, Buenos Aires Argentina: The CaCl2 from the germination tank was replaced by nutrient solution on the 4th day. On the 5 th day, plants were transferred to 350 mL pots containing nutrient solution, with each pot being a replicate. Pots were rotated every two days to ensure that they occupied different positions within the growth chamber. For the root elongation time course, the length of the second longest seminal root was measured daily four hours after the start of the light period, starting 6 DAG. Within each experiment, data was analyzed as repeated measures . A combined ANOVA was performed using experiments as blocks.The NBT staining experiments were performed using 5 cm root sections excised from the second longest root of 1RS and 1RSRW plants 17 DAG. This root segment was placed for 90 min in a 0.1 mg mL-1 NBT solution dissolved in 200 mM potassium phosphate buffer, pH 7.6, in darkness. For the DCF-DA staining similar root segments were placed in the same buffer supplemented with 10 µM DCF-DA for 60 min, in darkness. Roots segments stained with NBT or DCF-DA were washed in the same buffer for 30 min and placed on a slide. Roots were observed using a Zeiss Discovery.V20 stereo microscope equipped with a coaxial fluorescence mechanism. Pictures were obtained with an Axiocam 512 color . The images were processed with the ImageJ software obtaining a longitudinal profile of color and fluorescence intensities.

These experiments were performed in Chascomús. Previous studies have shown an association between the introgression of the rye 1RS arm in wheat and improved resistance to water stress . In three of these studies, the 1RS.1BL lines showed increased root biomass compared to the non-1RS control lines in large pot or sand-tube experiments. However, these differences were not validated in the field. In this study we showed that differences in grain yield and biomass between plants carrying a complete 1RS translocation and NILs with an introgressed distal wheat chromosome segment are associated with differences in total root length density and average root diameter in the field. Field excavations of the four different 1RS NILs provided an opportunity to visualize the differences in their root systems and to quantify these differences using horizontal soil cores at consistent depths. This experiment confirmed the hypothesis that the 1RSxR lines have a higher root density throughout the soil profile, with roots that reach deeper in the soil than the 1RSxW lines . The more extensive root system of the 1RSxR lines relative to the 1RSxW lines may have contributed to their better tolerance to drought and water logging conditions in the experiments presented in this study , and to the higher carbon isotope discrimination and increased stomatal conductance values detected in a previous study . Through their deeper root system, the 1RSxR plants can access more stored soil moisture and nutrients, keep their stomata open longer, and generate additional photosynthetic products and biomass than the 1RSxW plants. However, we cannot rule out the possibility that the genes in the distal 1BS introgression may have a more direct effect on aerial biomass or on other anatomical and/or physiological root differences known to impact tolerance to water logging and drought . The differences in root depth observed between the Hahn 1RSxR and 1RSxW NILs in the field were paralleled by drastic changes in seminal root length in hydroponic cultures . These differences were robust across experiments and were detected with different nitrogen sources and concentrations .

We hypothesize that these early differences in seminal root length may have contributed to the observed differences in total root length density observed in the deepest soil core samples in the field . The early and consistent differences in root growth under controlled conditions provided the opportunity to study the process in detail. During the first week of development, root growth occurred at the same rate for both genotypes, suggesting that the differences were not primarily associated with embryonically determined differences in root elongation. Instead, differences in root growth consistently manifested during the second week across multiple experiments. The growth rate of the seminal roots of the 1RSxW plants gradually decreased during the second and third week, to come close to zero by the end of the third week, whereas growth continued in the 1RSxR plants . The consistent timing of these events suggests that these changes are developmentally regulated. The growth arrest of the seminal roots in the 1RSxW plants was accompanied by the proliferation of lateral roots in close proximity to the RAM, suggesting important changes in the RAM. The RAM consists of a quiescent center surrounded by stem cells that generate new daughter cells, which undergo additional divisions in the proximal region of the meristem and differentiatein the transition zone . At a cellular level, a balance between cell proliferation and cell elongation/differentiation determines root growth rate . The arrest of the growth of seminal roots in 1RSxW plants suggests a modification in cell proliferation and/or cell elongation/differentiation. Additional studies will be required to determine if this arrest involves changes in the QC and/or modifications in the root regions adjacent to the meristem. In any case,outdoor vertical plant stands the dramatic reduction in seminal root growth and increased lateral root proliferation close to the RAM argues for an early developmental program switch in the regulation of the RAM in the 1RSxW plants. The transition from cell proliferation to cell elongation and differentiation and the subsequent development of lateral roots depends on the distribution of ROS along the root axis, specifically on the opposing gradients of superoxide and hydrogen peroxide.

Superoxide is predominant in dividing cells in the meristematic zone, while hydrogen peroxide is predominant in elongated cells in the differentiation zone . The balance between these ROS modulates the transition between root proliferation and differentiation zones. Seventeen days after germination, the apical region of 1RS seminal roots showed opposing gradients of superoxide and hydrogen peroxide characteristic of elongating roots . A different ROS distribution was detected in the arrested 1RSxW roots, where superoxide was restricted to the distal ~700 m and increased levels of DCF-DA fluorescence were detected between 250-950 m in the cell proliferation zone . The contrasting patterns of ROS distribution reflect the major developmental changes that differentiate the seminal roots of the 1RS and 1RSxW genotypes. Studies in Arabidopsis have shown that changes in ROS distribution can be triggered by the altered expression of major genes that control the size of the meristematic zone. These genes include UPBEAT1 , a basic helix-loop-helix transcription factor that regulates the meristematic zone size by restricting H2O2 distribution in the elongation zone . In addition, ROOT MERISTEM GROWTH FACTOR 1and the transcription factor RGF1 INDUCIBLE TRANSCRIPTION FACTOR 1that mediates RGF1 signaling can modulate the distribution of ROS along the root developmental zones leading to enhanced stability of PLETHORA2 . Reduced expression of PLETHORA in the root apical region or changes in its distribution have been associated with impaired root growth. To test if these Arabidopsis results are applicable to wheat, we are initiating expression studies of these genes in the 1RSxR and 1RSxW lines. It remains unknown if the differential pattern of ROS distribution in the roots of the 1RSxW plants is the result of changes in the wheat homologs of these central developmental genes or a more direct effect on genes affecting the redox balance in different developmental root zones. The differences in superoxide and hydrogen peroxide distribution between the seminal roots of the 1RSRW and 1RS plants were measured after the arrest in root growth . Therefore, we currently do not know if the changes in ROS distribution are a cause or consequence of the changes observed in root growth and lateral root proliferation close to the RAM. Strawberry frigoplants cv. Elsanta were cultivated in hydroponics. The variation in N-form started when the plants switched over from the vegetative to the generative growth phase. During the vegetative phase, all plants were fed with NO3-N. After changing to the generative stage, plants were divided into three different nutrition treatments, containing two ammonium treatments and one nitrate treatment. The solution pH was stabilized to pH 3.5 and 5.5 by pH-stat. The nutrient solution was changed every 3 d.The concentrations of the main organic acids in the fruits were measured using high performance liquid chromatography . Seeds of E. camaldulensis were germinated on acid-washed and sterilized sand in a growth chamber . Seedlings were watered daily with a nutrient solution and grown for 3 to 4 months. After seedlings were cultured hydroponically for 5 days, roots of seedlings were treated with 0.35 mM CaCl2 solution containing 0 or 1 mM AlCl3 for 24 h. The 5 mm apical portions of roots were excised, freeze-thawed, and centrifuged on an ultrafilter to obtain soluble compounds from root-tip cells. Al-binding compounds which were soluble in the form of complex with Al were separated by gel-filtration chromatography. The cellular extract added with Al was subjected to a gel-filtration column to separate the complexes of Al-binding compounds with Al from free Al. The Al concentration of each fraction was determined with an inductively coupled plasma atomic emission spectrometer . In addition, Al-binding compounds which became insoluble in the form of complex with Al were separated by HPLC equipped with a reversed-phase column. The cellular extract added with or without Al was subjected to HPLC analysis. Chromatographic peaks whose area was decreased by the addition of Al were judged to be those of Al-binding compound. When Al was added to the cellular extract from roots cultivated without Al and the extract was subjected to the gel-filtration column, one peak was observed by monitoring the Al concentration in each fraction. Two peaks were found when the roots were treated with 1 mM Al. These results indicate that roots of E. camaldulensis contain Al-binding compounds which are soluble in the form of complex with Al, and the compounds increase with Al treatment. We also measured organic acids in each fraction, and found that the peak of citrate corresponded to that of the Al-complex. The increase of Al-binding compound can be explained partly by the increase of citrate.

Interestingly, plants can be divided into four categories based on the quantity of B required: 1) Lactifers, contain the highest amount of B ; 2) Cole crops have the second highest B concentrations; 3) Legumes and the lily family of monocots are in the third group and 4) Graminaceous plants contain the least amount of B . When graminaceous plants flower, their B requirements increase. Now we know that, except for the lactifers, the B content of plants is closely aligned with the amount of pectin in their cell walls . Cell wall scientists have discovered that the RGII fraction of cell wall pectin contains B, and that cell wall structures in plants differ among species.Cell walls of grasses are much lower in pectin, and therefore these plants contain less B . Since each carbon fixed in photosynthesis is released to the Calvin cycle in bundle sheath cells by Mn-activated NAD-malic enzyme in this sub-type of C4 plant, perhaps the Mn requirement of these plants would be higher than that of C3 or NADP-malic enzyme plants . We tested this hypothesis by using hydroponic solutions where Mn concentrations could be carefully controlled. A survey of plant nutrient solution recipes indicated that most nutrient solutions contain around 2µM Mn. In this experiment, we compared the growth and photosynthetic rates of two NAD-malic enzyme C4 plants, Pearl millet and amaranthus, with two NADP-malic enzyme C4 plants,vertical hydroponic corn and sorghum, and two C3 plants, squash and wheat. Corn, sorghum, squash and wheat produced maximum biomass with the normal 2 µM Mn concentration in the hydroponic medium.

On the other hand, NAD-malic enzyme C4 plants, Pearl millet and amaranthus, produced maximum biomass with ~50 µM Mn in the nutrient solution . Photosynthetic rate responses of each species to nutrient solution Mn concentration were similar to their biomass responses. These results clearly show that when all of the carbon going into photosynthesis goes through a single Mn-activated enzyme, plant growth response is dependent on high levels of available Mn. Legumes are some of the highest protein crops grown, and they utilize N from the atmosphere rather that relying on N fertilizer to produce this protein. There are two major types of leguminous plants when it comes to root nodules and forms of N transported from these nodules to leaves and developing pods . There are determinate nodules, which tend to be round, with life spans of about 35 days. These are nodules formed on roots of warm season legumes and contain bacteroids that fix atmospheric N and use the fixed N to synthesize the ureide molecule, allantoate, for transport in xylem to leaves and developing pods. Allantoate contains 4N’s and 4C’s, and is a very efficient molecule for transporting N. Cool season legumes have indeterminate nodules that are elongated and often form a Y-shape. Bacteroids in these nodules fix N and, in general, synthesize the amide, asparagine, for transport in xylem to leaves and pods. Asparagine contains 2N’s and 4N’s. The fixed-N is released in leaves and developing pods of ureide-transporting legumes by an enzyme called allantoate amidohydrolase, and one interesting feature of this enzyme is that it is activated by Mn .

Therefore, according to the soil N status, a large proportion of the total N, mostly protein-N in the harvested legume, comes through this Mn-activated enzyme. As with the Mn-activated NADmalic enzyme plants, perhaps ureide-utilizing leguminous plants, like soybean, cowpea and lespedeza, will require higher Mn nutritional levels than asparagine-transporting legumes, like alfalfa and clover or all legumes grown on nitrate-N. To my knowledge, the Mn-requirements of ureide-transporting, amide-transporting and nitrate-fed leguminous plants have not be directly compared. In addition to a Mn-ureide metabolism connection, there is a Mn-bacteroid connection inside the root nodule. Bacteroids depend on their host legume for a source of energy to support the nitrogen fixation process. Although plants usually transport sucrose via the phloem from leaves to root nodules, root nodule cells metabolize the sucrose and provide bacteroids organic acids, like malate, as an energy source. Bacteroids in nodules of some species, like soybean, use the Mn-activated NAD-malic enzyme in the initial step of malate utilization . Therefore, a Mn-enzyme plays a central role in root nodule/legume N metabolism! The knowledge gained on structure and metabolism of a wide range of plant species over the past few years allows us to predict special nutrient needs. High protein plants require large quantities of K because components involved in protein synthesis must be bathed in high K concentrations in order to maintain the proper configurations. Plus, K+ is used to balance the negative charges of asparagine and glutamine in proteins produced. Plants with pectin-rich cell walls have high B contents, and thus plants with low pectin cell walls have low B contents. NAD-malic enzyme sub-type C4 plants have high Mn requirements for maximum growth and photosynthesis rates since every C fixed is released in bundle sheath cells by this Mn-activated enzyme. Based on their ureide metabolism with the Mn-activated enzyme, allantoate amidohydrolase, and with malate as the primary C source for bacteroids via NAD-malic enzyme, N-fixing soybeans may have a higher Mn requirement than nitrate-N grown plants.

In addition to these examples, there are other specific nutrient requirements that can be predicted based on our knowledge of plant structure and metabolism. Both Casparian strips and suberin lamellae, two extracellular hydrophobic barriers located in the wall of endodermal cells of the root, are thought to play important roles in restricting the free difusion of solutes and water . Casparian strips act as apoplastic barriers not only to block solutes moving into the xylem through the free space between cells, but also to prevent their back fow from the stele to the apoplast of the cortex. Suberin lamellae, due to their deposition between the endodermal plasma membrane and secondary cell wall, do not block aploplastic transport but rather limit transcellular transport of nutrients and possibly water at the endodermis. Cross talk between the Casparian strip and suberin lamellae exists, with suberin being deposited in response to disruption of Casparian strips.These extracellular barriers are therefore at a cross-road between control of mineral nutrient and water uptake. However, the mechanisms that allow plants to integrate both these barrier functions to enable the simultaneous uptake of sufcient water and mineral nutrients remain under explored. Te dirigent-like protein Enhanced Suberin1 functions in the correct formation of Casparian strips by allowing the lignin, deposited at the Casparian Strip Domain through the action of Peroxidase 64 and the Respiratory Burst Oxidase Homolog F, to form into a continuous ring. In the absence of this dirigent-like protein defective Casparian strips are formed along with enhanced and early deposition of suberin in the endodermis. A similar pattern of Casparian strip disruption and response is also observed when the Casparian Strip Domain is disrupted through the loss of Casparian Strip Domain Proteins. Tese changes lead to systematic alterations in the profle of mineral nutrients and trace elements accumulating in leaves, and this phenotype provided the frst tool for identification of genes involved in Casparian strip development. Detection of the difusible vasculature-derived peptides CASPARIAN STRIP INTEGRITY FACTORS 1 & 2 through interaction with the SCHENGEN3 receptor-like kinase is what drives this endodermal response to loss of Casparian strip integrity. Here, we report that detection of a loss of Casparian strip integrity at the root endodermis by the CIFs/SGN3 pathway leads to an integrated local and long-distance response. This response rebalances water and mineral nutrient uptake,what is vertical farming compensating for breakage of the Casparian strip apoplastic seal between the stele and the cortex. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquaporins, and limitation of ion leakage through deposition of suberin in endodermal cell walls. This local root-based response is also coupled to a reduction in water demand in the shoot driven by ABA-mediated stomatal closure.The dirigent-like protein Enhanced Suberin1 functions in the formation of Casparian strips by allowing the correct deposition of lignin at the Casparian strip domain. The enhanced deposition of suberin in the esb1-1 mutant with disrupted Casparian strips can clearly be observed using the lipophilic stain Fluorol Yellow 088 close to the root tip , and this can be quantifed by counting the number of endodermal cells afer the onset of cell expansion to the frst appearance of yellow fuorescence . This early deposition of suberin is also verifed by the clear correspondence of FY 088 staining with enhanced promoter activity of known suberin biosynthetic genes, including GPAT5 monitored through both GUS staining and GFP fuorescence , and also others through GUS staining . This is further reinforced by the enhanced expression of known suberin biosynthetic genes in esb1-1 relative to wild-type . To better understand the causal link between Casparian strip integrity and enhanced deposition of suberin, we performed a reciprocal grafing experiment that revealed that the esb1-1 mutation is only required in the root to drive enhanced deposition of suberin at the endodermis, placing the function of ESB1 and the driver for increased suberin in the same tissue . To determine the cause and effect relationship between damaged Casparian strips and enhanced suberin we carefully monitored the first appearance of both Casparian strips and enhanced suberin in esb1-1. Using lignin staining in the Casparian strip marker line pCASP1::CASP1::GFP, we are able to observe that damaged Casparian strips are visible 2.5 days after sowing .

This is at least 12hr before the first indication of enhanced suberin biosynthesis, which we monitor using promoter activity of suberin biosynthetic genes GPAT5, FAR4, FAR1 and FAR5 . This was also verified by the direct observation of suberin deposition with FY 088 . The observation that treatment with the CIF2 peptide, normally leaked from the stele through loss of Casparian strip integrity, can enhance suberin deposition in wild-type plants supports our interpretation that enhanced suberin deposition is a response to loss of integrity of the Casparian strip-based apoplastic diffusion barrier. Furthermore, loss-of-function of the receptor-like kinase SGN3, required for sensing of CIFs, blocks the enhanced deposition of suberin in esb1-1 and casp1-1casp3-1 based on a chemical analysis of suberin in esb1-1 , and also on FY 088 staining. We conclude that Casparian strip defects sensed by the CIFs/SGN3 surveillance system lead to enhanced deposition of suberin in the endodermis.Te observation that enhanced suberin is deposited as a response to loss of integrity of the endodermal-based diffusion barrier between stele and cortex, raises the question, what is the function of this increased suberin deposition? Previously, the extent of endodermal suberin has been shown to be part of the response to nutrient status. We therefore tested the selectivity to solutes σNaCl, in roots varying in the extent of suberin deposition and the functionality of Casparian strips. For this, we measured solute leakage into xylem sap of pressurized roots at increasing sodium chloride concentrations in the solution bathing the roots. Taken individually, σNaCl of roots of esb1-1, sgn3-3 and wild-type were not significantly diferent from one another , which is surprising given the disruption of the Casparian strip-based apoplastic diffusion barrier in both mutants. However, removal of suberin in esb1-1, by endodermal-specific ectopic expression of a cutinase, caused a signifcant decrease in σNaCl compared to wild-type plants , and a similar tendency when compared to esb1-1 . This supports the notion that enhanced suberin deposition at the endodermis helps prevent passive solute leakage caused by defects in the Casparian strips of the esb1-1 mutant. We also observed a significant decrease in σNaCl in the double mutant esb1-1sgn3-3 compared to both wild-type and sgn3-3 . It is known that SGN3 is required for the enhanced deposition of suberin that occurs at the endodermis in esb1-1. Our observation that removal of this enhanced suberin in esb1-1sgn3-3 decreases σNaCl further supports our conclusion that the role of this increased suberin deposition is to limit solute leakage where Casparian strip barriers are disrupted.It has also been suggested that endodermal suberin may impact water permeability, though how is still unclear. To further address the role of enhanced endodermal suberin, we investigated root hydraulic conductivity of esb1-1 and observed a significant reduction by 62% with respect to wild-type.Importantly, this difference in esb1-1 Lpr originates mainly from a reduction in an aquaporin-mediated water transport pathway.

The BEST program also sponsored a phytoremediation workshop for BEST investigators and students that was attended by more than 60 participants. Additional workshops are planned for the coming year. In this report, the research is organized by subject area, and two-page briefs are presented for each of 28 BEST projects. The projects presented provide a good representation of the state-of-the-science research being done with students in the BEST program – the best of BEST.Over the next 75 years, the U.S. government will undertake what has been called the largest civil works project in world history to restore the environment damaged by previous activities at federal sites, e.g., Department of Defense military bases and Department of Energy nuclear facilities. Legislative action, resulting from concern over the accumulating hazards, has mandated pollution control measures and environmental restoration of hazardous waste at all sites. Estimates of total cleanup costs range from $230 billion to more than half a trillion dollars. Given the trend of diminishing budgets throughout the federal government,vertical home farming future generations could inherit both an environmental and budgetary disaster. The imprecision of the cost estimates results from the lack of knowledge of how “clean” the contaminated sites will need to be. Some of the environmental damage is permanent—cleanup technologies either do not exist or are incapable of remediating the contamination.

For DoD bases being closed by the Base Realignment and Closure Program, all toxic sites must be remediated before the site is returned to public use. The projected costs of site restoration using existing technologies are staggering: the estimated cleanup cost is at least $24.5 billion for the 7,313 identified U.S. sites . The pollutants at these sites include chlorinated hydrocarbons, metals, petroleum products, explosives, mixed waste and other organics. DOE also has substantial remediation costs—estimated to be from $90 billion to $200 billion . The domestic private sector presents yet another huge set of remediation problems, dwarfed only by the international problems in Eastern Europe and Russia . There is clearly a need for new cost-effective treatment technologies. Bio-remediation, the use of microorganisms to detoxify hazardous waste, promises to provide economical and ecologically sound clean-up strategies. An Office of Technology Assessment analysis concluded that the U.S. does not possess a sufficient pool of qualified environmental professionals, i.e., the trained scientific personnel required to support this rapidly developing multidisciplinary field. In response to these national environmental needs, the Bio-remediation Education, Science and Technology Program, funded by DoD, was established in 1996. In a few short years, BEST has pioneered a new and successful model for environmental science and education. This partnership has a highly integrated programmatic focus on the scientific and workforce needs of DoD. Since the inception of the BEST program, a significant number of major milestones and deliverables have been achieved. They are described below. The BEST program has made these dramatic accomplishments by using an approach that combines a training-education element with an integrated research project, described later in this introduction.

Successful restoration of DoD hazardous waste sites and the growth of the bio-remediation industry is dependent on a cadre of trained scientists, engineers and technicians. By the year 2005, thousands of trained professionals will be required to meet DoD and national environmental needs. The BEST partnership continues to build upon accomplishments and successful programs developed with DoD support. The training-educational element continues to provide career development opportunities for underrepresented groups with bio-remediation curricula, courses and fellowships. The training-educational experience is personalized to provide students with a meaningful bio-remediation curriculum, financial support, an extensive mentorship network, and research and field training. The shared resources of the BEST partnership institutions aid faculty in the development of curricula, courses and environmental research projects. The training-education programs span the continuum from community college outreach to faculty development, but focuses on undergraduate and graduate students. Innovative features of the BEST program are the use of distributed learning technologies and a Rotating Scholar Program, integrated with a coordinated academic and video seminar curriculum.The primary objective of the BEST program is to provide hands-on training in bio-remediation, phytoremediation and ecotox/risk assessment for underrepresented minorities. Students will obtain the necessary skills and knowledge to enable them to either enter the environmental science workforce with a bachelor’s degree or enter graduate school at the master’s or doctoral level. Another objective is to ensure a continued supply of skilled workers to address the diverse environmental restoration needs at DoD sites.

DoD sites throughout the United States contain highly contaminated soils, groundwater and sediments. These properties pose direct and indirect exposure hazards to humans and wildlife. Conventional remedial solutions for contaminated soils and sediments or groundwater are slow and expensive, increase inputs to hazardous waste disposal sites, and can increase human exposure to contaminants. Bio-remediation — the use of microo ganisms to destroy hazardous contaminants or to convert them to harmless forms — is an emerging treatment technology that can in many instances restore contaminated environments more quickly, at lower cost and at lower human risk than alternative remediation technologies. Bio-remediation can operate in either an in situ mode where contaminants are treated in place, or in an ex situ mode where contaminants are removed from a contaminated zone for treatment . In situ bio-remediation can be used when excavation is impractical — under buildings, highways, runways, etc. In situ bio-remediation can simultaneously treat soil and groundwater in one step, without the generation of hazardous waste products. In situ contaminant degradation can be achieved by either intrinsic or enhanced bio-remediation. Intrinsic bio-remediation exploits the innate capabilities of indigenous microbial communities to degrade pollutants. Enhanced bio-remediation seeks to accelerate in situ microbial activity by isolating and controlling the contaminated site so that the microbial environment can be purposely manipulated to correct nutritional or gas phase limitations. Ex situ treatment seeks to further control the remedial environment by placing the contaminants in an engineered treatment system. Phytoremediation, a process in which plants and associated microbial communities are used for contaminant biodegradation or bioimmobilization, is an important and rapidly developing mode of bio-remediation. To realize the full potential benefits of plant and microbial treatment systems at DoD sites, these bio-technologies must be developed and optimized for remediation of DoD priority contaminants by an expanded pool of qualified professionals. It was in response to these DoD environmental needs that the BEST partnership of institutions was established.

In order to determine whether plants can stimulate the degradation of PAHs in soil, plant species found in literature on phytoremediation of metal-contaminated sites were selected to measure the removal of PAHs in artificially contaminated soil over a period of 62 days. The plant species used for this experiment were alfalfa , barley , tall fescue and orchard grass . The PAHs were phenanthrene and anthracene, in a mixture of 600 ppm each. As shown in Figures 1 and 2,vertical growers phenanthrene and anthracene were removed from the soils with plants after 62 days. More than 98% of the phenanthrene was removed during that period while the anthracene removal was found to be between 70 and 90%. The results suggest that the rate of disappearance of phenanthrene in soil was greater than anthracene under the same conditions. From the results, it is also indicated that the disappearance of PAHs in soil depends on the bio-availability of the compounds. Because phenanthrene is approximately 10 times more soluble in water than anthracene, it was expected to be more readily available to microbial degradation than anthracene. Plant-assisted degradation of PAHs is thought to be more effective on PAHs with a higher number of rings and higher molecular weights, such as benzopyrene. Anthracene removal in the soil planted with alfalfa was greater than in the soil without plants, while all the other plants have minimal to no effect on anthracene removal compared to the control soil. Phenanthrene was removed to a greater extent in the soil with alfalfa and tall fescue compared to the control without plants . However, both barley and orchard grass showed no effects of the removal of phenanthrene during that period when compared to the soil without plants. Overall, plants had minimum effect on phenanthrene degradation while anthracene degradation was more dependent on plant species. In order to determine the effect of PAH degradation by plants on bacterial numbers in soil, bacteria were counted in soil during the course of the experiment.The result suggests that the degradation of PAHs by plants is not affected by differences in bacterial biomass in the soil. Sterile controls will be used in the future in order to assess the role of bacteria in the degradation process.Wheat is a crop of major importance and together with other staple cereals supply the bulk of calories and nutrients in the diets of a large proportion of the world population . Cereals are inherently low in protein and mineral micro-nutrients such as Fe and Zn . A major focus of wheat breeders has been grain protein concentration as it affects bread- and pasta-making quality, but micro-nutrient improvement has received less attention. Approximately half of the world’s population suffers from Fe and/or Zn deficiencies and millions of children suffer from protein-energy malnutrition . As such, the improvement of nutritional quality of wheat could benefit the nutritional status of millions of people. A common agronomic practice to increase grain protein concentration is the use of N fertilization. However, this practice is expensive and excess fertilizer run-off is a potential environmental contaminant . A substantial percentage of the N in wheat grain is supplied by amino acids remobilized from vegetative tissue .

Much of this N content is derived from proteins that are disassembled and recycled during the leaf senescence stage of development . Likewise, Fe and Zn have been shown to be remobilized from vegetative tissues in several plants , although the specific sources are unknown. Zinc fertilization has been a successful strategy to improve wheat grain Zn concentration , and improvement in the partitioning or remobilization of Zn to grain could make fertilization efforts more efficient. Wheat grain with higher Zn concentration has been demonstrated to produce more vigorous crops . Thus, breeding or transgenic approaches that result in plants with increased partitioning of minerals to grain could be useful for both nutritional bio-fortification and reduced fertilizer application. Chromosome 6B from wild emmer wheat was identified as a potential source of genetic variation for grain protein , Zn, and Fe concentration . A quantitative trait locus for grain protein concentration was mapped on chromosome arm 6BS and later mapped as a single Mendelian locus, Gpc-B1 . In near-isogenic lines of this locus, increased grain protein was associated with the increased remobilization of amino acids from the flag leaf , higher grain Fe and Zn concentrations , and accelerated leaf yellowing, indicating accelerated senescence . A NAC transcription factor, NAM-B1, was identified as the causal gene for Gpc-B1 by positional cloning . Other members of the NAC family are known to regulate developmental processes , including leaf senescence . In transgenic wheat NAM RNA interference lines in which NAM-B1 and its homeologous genes had decreased expression, leaf yellowing was delayed, and grain protein, Fe, and Zn concentrations were greatly decreased . These results, together with higher N, Fe, and Zn concentrations in RNAi line flag leaves at maturity, suggested a role for NAM-B1 homeologues in the remobilization of N compounds, Fe, and Zn. However, without taking organ mass, nutrient concentrations at prior time points, and total nutrient accumulation of other organs into account, this model could not be confirmed. In addition, the body of literature does not contain sufficient data regarding sources of grain minerals to support the idea that remobilization alone could account for the differences observed. Because a whole-plant partitioning profile has not been undertaken in plants differing in NAM-B1 expression, it is currently unclear whether this gene directly affects remobilization , alters partitioning of nutrients within the plant, alters total plant uptake of these nutrients, or influences a combination of these processes. The current study uses multiple time point sampling of an expanded profile of mineral concentrations and contents of all shoot organs in NAM knockdown and control lines. This sampling allows the quantification of N and mineral remobilization as contributors to final grain protein and mineral content, and provides a better understanding of the physiological effects of the NAM genes.

Arabidopsis is known to synthesize four GDP-linked sugars: GDP-L-fucose, GDP-L-galactose, GDP-D-glucose and GDP-D-mannose. GDP-mannose for the glycosylation of glycosylinositolphosphorylceramides is transported into the Golgi by GONST1 , whereas GDP-fucose is transported by GONST4, which has been renamed GDPfucose transporter1 . No Golgi-localized GDP-L-galactose transporters have been identified to date. GDP-L-galactose is synthesized from GDP-mannose in the cytosol by GDP-mannose epimerase . Most GDP-L-galactose is then converted, via L-galactose, into L-ascorbate , which is important for maintaining redox balance in the cell, particularly under abiotic or biotic stress . However, some GDP-Lgalactose is required for cell wall polysaccharide synthesis since L-galactose is present in side chain A of RG-II, in the side-chains of xyloglucan from a limited number of plant species  and in corn bran glucuronoarabinoxylan . Here we provide evidence that GONST3 probably encodes a Golgi-localized GDP-L-galactose transporter, which we rename Golgi GDP-L-galactose transporter1 . We used RNA interference to suppress GGLT1 expression in Arabidopsis, since complete loss of GGLT1 is lethal. Plants with decreased GGLT1 expression have growth defects, which are rescued by increasing the amount of borate in their growth medium. Chemical analysis of the cell walls of GGLT1 knock-down plants revealed a substantial reduction in the L-galactose decoration of RG-II,vertical gardening in greenhouse which is correlated with a decrease in the proportion of RG-II dimer in the wall and a decrease in the stability of the cross link. Our results underscore the importance of RG-II to plant survival, and highlight an unexpectedly critical role for L-galactose in borate cross linking of this unusual pectic polysaccharide.

Publicly available gene expression data reveal that GGLT1 is a ubiquitously expressed gene, with a level of expression that is slightly lower than GONST1 and GFT1 . In an earlier study, the subcellular localization of GGLT1 was not determined because tagged GGLT1 could not be expressed in vivo . To overcome this issue, the full-length GGLT1 coding sequence tagged with a fluorescent protein was introduced into onion epidermal cells by biolistic transformation. Confocal imaging revealed that the fluorescently tagged GGLT1 gave a punctate signal that co-localized with a Golgi marker .No Arabidopsis lines carrying a T-DNA insertion in the GGLT1 open reading frame have been reported. A singleT-DNA line , with an insertion 841 bp upstream of the start of transcription was obtained, but we were unable to identify any plants homozygous for the TDNA insertion despite screening at least 30 different seedlings. Therefore, we took a targeted gene-knockdown approach and generated RNAi transgenic lines with a hairpin RNA construct, which specifically targeted GGLT1. Forty independent hpGGLT1 transformants were screened, and four were selected for characterization . These lines all had rosettes which were smaller than the empty vector control . Quantitative real-time PCR showed that in the rosette leaves of the hpRNAi lines 1–3 the levels of GGLT1 silencing were similar . These data, together with the lack of T-DNA lines, suggest that stronger suppression of GGLT1 or null mutants will produce plants that are not viable. The expression of GFT1, the closest homolog of GGLT1, was not affected in lines 3 and 4 but was decreased by up to 50% in lines 1 and 2 . The monosaccharide compositions of the walls, including fucose, were not significantly altered in any of the hpRNAi lines , indicating that their phenotypes do not result from altered fucosylation of cell wall glycans and are thus a consequence of GGLT1 silencing.

Moreover, the shortened petiole phenotype that is characteristic of silenced GFT1 plants as well as mur1 plants, which also have cell walls with reduced fucose , was not observed in our hpGGLT1 knock-down lines .L-Galactose replaces L-fucose in the xyloglucan formed by mur1 and GFT1-silenced plants where GDP-fucose synthesis or transport is perturbed . Since GGLT1 and GFT1 are closely related NSTs, we first determined if xyloglucan fucosylation is altered in hpGGLT1 . No differences were discernible in the matrix-assisted laser desorption–ionization time-of-flight mass spectra of the oligosaccharides generated by enzymatic fragmentation of the xyloglucan from hpGGLT1 and EV control lines . The presence of fucosylated side chains, together with no substantial increase in the abundance of galactosylated side-chains in the hpGGLT1 lines, supports our assertion that GDP-fucose transport is unaffected in the silenced plants. GGLT1 is in the same NST subclade as GONST1, which provides GDP-mannose specifically for GIPC glycosylation, as opposed to polysaccharide biosynthesis . Although glycosylation of GIPCs is still poorly understood, it is possible that other GDP-sugars, in addition to GDP-mannose are required. Therefore, we used thin layer chromatography and LC-MS to determine the GIPC glycan composition of hpGGLT1. No major differences were discernible between hpGGLT1 and EV GIPCs. The overall sphingolipidomic composition was also unchanged . Together, the combined results of these studies show that GGLT1 does not encode a Golgi-localized protein involved in the transport of GDP-L-fucose or GDP-D-mannose. Thus, we next investigated if the L-galactose content of the wall was altered in the GGLT1 suppressed lines.No significant differences were detected in the wall monosaccharide compositions of leaves from soil-grown EV and hpGGLT1 lines . This is not surprising since in primary cell walls D-galactose is far more abundant than L-galactose . Moreover, D-galactose and L-galactose are not separated when the monosaccharide composition of the cell wall is determined by high-performance anion exchange chromatography with pulsed amperometric detection . Rhamnogalacturonan-II is the only known L-galactose containing polysaccharide present in wild-type Arabidopsis cell walls, so we next determined whether the structure of RG-II differed in hpGGLT1 and EV plants. Material enriched in pectic polysaccharides, including RG-II, was obtained by extracting hpGGLT1 and EV leaf alcohol-insoluble residue with ammonium oxalate, a calcium chelator. This material was then treated with endopolygalacturonase and the products separated by size-exclusion chromatography . This separates RG-II from RG-I and oligogalacturonides, and also separates the RG-II monomer and dimer. In EV control plants the dimer accounts for 77% of the total RG-II isolated from the wall. Somewhat unexpectedly, the dimer accounts for only 49% of the hpGGLT1 RG-II, and makes up only 6% of the RG-II in mur1-1 .

This led us to suspect that the ability of hpGGLT1 RG-II to form dimers or the stability of those dimers had been altered.Under these conditions, in the absence of a chelating agent, the dimer accounted for 97% of the RG-II in the EV control plants , 87% of the RG-II in the hpGGLT1 lines and 70% of the mur1-1 . These results, together with data showing that calcium chelators partially convert the RG-II dimer to the monomer , strongly suggest that both the extent of formation and the stability of the borate cross-link in RG-II are affected in the hpGGLT1 lines. The differences in dimer abundance in the EPG and oxalate fractions were most pronounced with mur1-1 plants. This mutant produces RG-II that lacks L-galactose because its A side-chain is truncated , which led us to suspect that the L-galactose content of side-chain A of the RG-II from the hpGGLT1 lines may also be reduced. To determine if RG-II structure is indeed altered in the hpGGLT1 lines we isolated the total RG-II from the silenced and EV plants. Glycosyl residue composition analyses showed that D/L-galactose was reduced by about 35% in the most strongly affected hpGGLT1 lines . We then treated the RG-II with warm trifluoroacetic acid  to release side chains A and B. The MALDI-TOF MS analysis showed that a substantial portion of side-chain A from hpGGLT1 RG-II existed as a heptasaccharide whereas virtually all the A chain from the EV control was present as an octasaccharide . The A side-chains produced by hpGGLT1 and EV plants differ in mass by 162 Da, corresponding to a hexose residue, which we consider likely to be L-galactose. The side-chain B of RG-II contains a D-galactose residue . However, no differences were discernible in the structures of this side-chain from RG-II of hpGGLT1 and EV plants . Our structural data provide compelling evidence that the abundance of terminal L-galactose present on the A side-chain of RG-II is specifically affected in hpGGLT1 plants. To confirm the identity of the missing hexose in sidechain A,greenhouse vertical farming the RG-II monomers generated from the hpGGLT1 and EV plants were treated with a recently identified a-Lgalactosidase 95 from Bacteroides thetaiotaomicron that specifically removes the terminal L-galactose from side-chain A of RGII . Galactose was the only monosaccharide detected by HPAEC-PAD following hydrolysis of EV control RG-II with the a-L-galactosidase . Less galactose was released from the RG-II of the hpGGLT1- silenced lines relative to the control . The MALDI-TOF MS analysis of side-chain A, released by mild TFA hydrolysis following a-L-galactosidase treatment of RG-II monomer, revealed that the predominant oligosaccharides in the EV control plants correspond to side-chain A lacking L-galactose . The L-galactose was almost completely removed as only low-intensity signals corresponding to L-galactosylated A side-chains were discernible . The mass spectra of side-chain A from both hpGGLT1 silenced lines are similar to that of the EV control , demonstrating that the mass difference of 162 Da between the EV control and hpGGLT1 lines in Figure 3 is due to the specific loss of L-galactose. It has been proposed that pectin domains may be linked covalently to each other or to other cell wall components . To investigate whether the altered RG-II structure in the hpGGLT1 silenced lines had affected other pectic domains, the oxalate cell wall fraction was used to perform immune dotblots with a panel of antibodies raided against different pectin epitopes .

However, no difference was observed between the EV control and the silenced lines. In combination with the monosaccharide composition data and the xyloglucan data we conclude that the reduction in GGLT1 expression does not affect non-RG-II polymers. These data provide strong evidence that silencing of GGLT1 leads to a reduction in the abundance of L-galactose on side-chain A of RG-II, and provides additional evidence that the absence of this sugar leads to a decrease in the ability of the RG-II monomer to self-assemble into a borate cross-linked dimer. Moreover, this L-galactosedepleted dimer is less stable in the presence of calcium chelators than its wild-type counterpart, a result consistent with the notion that interactions of borate and calcium with RG-II are important for plant growth .Several growth phenotypes, including the dwarf phenotype of mur1, that have been attributed to defects in RG-II structure and cross-linking have been reported to be rescued by supplementing the growth medium with additional borate . To further explore the observed growth phenotypes of the hpGGLT1 lines , plants were grown hydroponically to control the availability of all macro- and micro-nutrients, including borate. In low-borate media the hpGGLT1 lines are severely stressed, and their rosette diameter is about 70% smaller than that of EV control plants . However, this phenotype is not observed when the silenced plants are grown in high-borate media . The amount of borate in the growth medium did not affect GGLT1 expression, thereby excluding a potential effect of borate deficiency or supplementation on transgene expression and silencing strength . Therefore, we conclude that partial loss of the RG-II L-galactose decoration in hpGGLT1 reduces the rate of RG-II borate-dependent dimerization, directly affecting plant development.Since hpGGLT1 plants grown in the presence of 1 mM boric acid or no added boric acid had different phenotypes,we were curious to know if altering the structure and dimerization of RG-II in hpGGLT1 led to changes in other cell wall components. Therefore we determined the monosaccharide composition of destarched leaf AIR from plants grown under different borate concentrations . No significant visible differences were discernible in hpGGLT1 and EV plants grown with 1 mM borate . However, we saw increases in the abundance of several neutral monosaccharides, in particular glucose , in the walls of plants grown with no added borate. No differences in aniline blue staining of the walls of EV and hpGGLT1 lines were observed, suggesting that the increase in non-cellulosic glucose in plants which appear severely stressed is not due to callose deposition . Finally, we performed Saeman hydrolysis of the TFA-resistant AIR to determine the amount of glucose derived from crystalline cellulose. A substantial increase in cellulose-derived glucose was detected in the hpGGLT1 lines grown with no added borate but not in plants grown under high-borate conditions .

Ideally, the internal levee should maintain a factor of safety of above 1.4, and as such, the maximum back flooding of the system will be will be MSL + 6 ft. Table 3 shows that for different river water levels increasing the back flood water levels results in a increase in the the factor of safety of the levees. While there are minor variations to this trend, the levee becomes more stable with increased back flooding. This result supports the theory that the hydraulic exit gradient resulting in piping and slope instability can be reduced by the Water Management System. The optimum back flooding levels from simulations of various elevated river conditions are highlighted in Table 3. Significantly higher factors of safety for the existing sections of the Sherman Island levees can be achieved with these levels of back flooding resulting in greater levee stability and lower risk of failure from river flooding. The driving forces for levee and piping resulting in slope failure stems from high exit gradients.As pressure builds up on one side of the levee, stacking flower pot tower water is forced towards the backside of the levee and thus increases the hydraulic gradient. This increases the already high hydraulic gradient of the peat layer even further.

The addition of the temporary flood storage zone allow for siphoning water to balance water levees on each side on each side of the level. Therefore the exit gradient and pore pressures are decreased leading to a significant increase of the stability of the levees. Figure 7 compares the exit gradient for an 8+MSL flood level and back flooding of 2+MSL which indicates a reduction of the exit gradient by approximately half.Site work estimates consider the construction costs of internal cutoff levee and necessary road improvements for site access. The cutoff levee requires approximately 440,000 cubic yards of fill at $15 per cubic yard, therefore the total cost of the levee is approximately $6.6 million . For project access, revitalization of a portion of the former Victory Highway is the most likely option. The approximate length of road to be improved is 0.54 miles with an estimated cost of $1.8 million per mile constructed . This value reflects construction equivalent to a one-foot asphalt concrete layer and gives a total of approximately $972,000. An alternative route involving revitalization of a larger portion of Victor Highway stretching roughly 2.17 miles would cost $3.9 million, which is four times the cost of the first option. The total materials cost for the aquaponics system is approximately $23 million. This includes 600 aquaponics subsystems at $30,000 to $50,000 each, with an average of approximately $38,500. The prices of hydroponics and aquaculture components, including: tanks, pumps, anchors, and rafts per subsystem.

The prices are based on data from UVI Aquaponics, CropKing Hydroponics, Dock Float Supply, Dock Builders Supply and Seaflex Anchors. Labor and construction costs are not included as further design and bidding by licensed aquaponics contractors are needed. The cost of wetland development is extremely variable depending on the scale of the project and quality of labor and materials used. According to Dr. Alexander Horne, estimates for wetland construction range from $2,000 to $75,000 dollars per acre . For the purposes of this project, Dr. Horne recommends a value of $25,000 per acre of wetland constructed. Therefore for 700 acres of wetland restoration there is an initial cost of approximately $17-18 million. Operations & Maintenance The economic analysis of sturgeon production by Logan estimates costs of operations and maintenance per aquaculture system at approximately $3,500 to $4,000. This includes cost of labor, feed, electricity, and medicine. Assuming a 25 percent increase in these costs to account for agricultural labor, this results in operating and maintenance costs between $4,000 and $5,000 per subsystem. Consequently for the 600 systems proposed there is an annual cost of approximately $2.8 million. For the proposed system on Sherman Island, there are three chief sources of revenue. The first is the sale of agricultural yields from the hydroponics component. The second is the sale of fish yields from the aquaculture component. The third source is the economic value placed on various wetland services.

Income generated by agriculture is dependent upon the type of crop produced as well as the frequency of harvesting cycles. For the purposes of this model, it is assumed that average yield is approximately 11,000 pounds of crop per year per system. Price indices for crops typically used in aquaponic operations indicate that cilantro has the highest economic benefit of $88,000 per year, with onion having the lowest value of $1,100 per year. On average, each year of production will generate roughly $25,000 to $30,000 in revenue per subsystem, resulting in nearly $17 million of income over a two-year time period. The aquaculture component of this business model uses price indices developed for similar systems rearing sturgeon. Numbers generated from previous examples include considerations for 5, 10, and 15 brood stocks. There is a harvest time of 18 months for each system, and production will likely be staggered to produce maximum output and more continuous income for the project. Considering a time period of two years, fish production gives estimated revenue of roughly $17 million assuming a sale value of $4.5 per pound of sturgeon. Wetland value is based on a sum of averages of the following services: gas regulation, disturbance regulation, water regulation, water supply, waste treatment, habitat, food production, raw materials, recreation, and cultural ecosystem services . Revenue for these services is presented in Table 4.The wetland component of the system will provide carbon sequestration. Carbon sequestration is the capture of atmospheric CO2 by plants such as bulrushes and cattails through photosynthesis. The Bulrush wetland can grow approximately 500 g C/ m2 per year, half of which is preserved. The CO2 is absorbed by the plants and then incorporated into the soil biomass as the plants decay over time. A wetland can sequester as much as 25 metric tons of CO2 per acre per year. This helps to reduce carbon emissions while reversing subsidence . The system actively stops and reverses subsidence. By flooding the area with water the peat layer preserved because it is wet. As the plant biomass from the wetland decomposes it increase the peat layer and therefore reverses subsidence. Wetland vegetation and non-sellable crop yields from the hydroponics system can be used to restore peat. Similar subsidence reversal projects have been proposed and approve on and around Sherman Island. One such project includes Twitchell Island Ponds where an increase of 1.6 inches/year in addition to preventing subsidence of approximately 1 inch/year has been demonstrated. During flooding even there will be sediment will be transported into the system and then settle out before the water in release back into river. Therefore sediment can slowly build up with flooding events and also reverse subsidence .

In addition the wetland restores natural habitats for birds and aquatic species. The fish a reptile species that would take advantage of this wetland ecosystem include carp, crappie, striped bass, white catfish, and pond turtles. Along with hundreds of different bird species including the mallard duck, red-tailed hawk, eagle, Canadian goose, cinnamon teal, ring-necked pheasant, ruddy duck, wood duck and many more. The wetlands will serve to restore the delta wetland habitats as well as to sustain future bio-diversity of wetland species . Water contamination and waste is reduced by the aquaponics system because nutrients and water are recycled back into the system through water recirculation. This separation of the system from the surrounding environment reduces water waste due to runoff and agricultural pollution when compared to terrestrial agriculture. Terrestrial agriculture requires a large amount of water,ebb and flow the majority of which is not absorbed by plants but leaves the land via groundwater runoff. Agricultural runoff promotes algal blooms and eutrophication, which lower the oxygen concentration dissolved in the water and leads to death of aquatic species. The potential environmental concerns of the wetland include the production of methane from biomass decomposition. While wetlands sequester carbon and hence reduce carbon dioxide, they release other GHGs, primarily methane and nitrous oxide . The concern is that Methane has a Global warming potential 21 times that of CO2, and N2O has a GWP 310 times that of CO2. Most of the GWP of CH4 occurs in the first twelve to fifteen years after its release, whereas the GWP of CO2 lasts decadnto the water within the system and the transport of such toxins into the surrounding environment. The soil at Sherman Island has been exposed to pesticides, fertilizers, and dredged soils for the bay which were contaminated with heavy metals. Therefore because the proposed design is to partially flood the soil the toxins could leach from the soil. In order to mitigate this possible issue a test flood plot an monitor the toxin release levels. Leach test or Barrel tests can be used to mimic the conditions and will give results that can be used to assess further needs of mitigation. Additionally, the heavy metals such as the metals off of ships including copper, zinc, and nickel can be immobilized in wet peat soils.

Organic compounds will bind to peat and once the wetlands have been established the plants will also remove toxins from the water. Finally because the flood storage zone will contain a large volume of water the toxins will be diluted. There may be short term effects from pesticides so to prevent birds from being exposed to these toxins the use of bird scaring devices can be implemented until toxins diffuse. It is important to monitor the toxins before, during and after the installation of the wetland in order to understand the mitigation strategies that must be considered and implemented. Many of the environmental concerns can be monitored at demonstration sites including Mayberry Farms Subsidence Reversal and Carbon Sequestration Project and the Twitchell Island Ponds which both contain many of the same environmental considerations that will be faced in the flood storage zone. Fatty acid composition was determined in leaves of purslane plants exposed to saline treatments for 30 days. A sample of 50 g of fresh tissue leaves was dehydrated in a incubator at 60 ºC for 48 h. The dehydrated tissue was macerated and stored in 15 ml vials at room temperature, protected from light. An aliquot of 1 g of each sample was used to determine fatty acid composition, according with the procedures described by Liu, et al. . After hexane extraction, the samples were vortex-mixed, centrifuged, and the upper phase was collected prior to GC analysis. Samples were injected via an auto sampler onto a fused-silica capillary column in a HP 6890 gas chromatograph system fitted with a flame ionization detector and eluted with helium at 44.0 ± 1 ml/min, with a split ratio of 1:17. The injector and detector were heated to 250 ºC. The column was temperature programmed from 130 ºC to 180 ºC at 25 ºC/min, and then to 230 ºC at 2.5 ºC/min. Fatty acid methyl esters were identified by comparing GC retention times with those of a mixture of standard fatty acids methyl esters Mix C14-C22, . Fatty acids were quantified using peak areas integration against internal standard. In GL and GR cultivars, submitted to 30 days of saline treatment, the main fatty acids detected were C16:0, C18:3 and C18:2. Such high amounts of palmitic acid, has it was found in the P. oleracea leaves of control plants is unusual, previous studies reported an proportion of 17% of this fatty acid in the total FAMEs detected . Palmitic acid, the most abundant fatty acid in the human diet, causes oxidative DNA damage, DNA strand breakage, necrosis and apoptosis in human cells in vitro, but when consumed with others fatty acids, like PUFAs, is unlikely to have any significant impact on human health. The amount of unsaturated fatty acids was also a little bit lower than expected, although the proportions between UFAs and SFAs were maintained ; it has been reported in other studies that the linolenic acid content in P. oleracea leaves was circa 50% of total fatty acids and linoleic acid content 3- 4 times lower.

However, only photosynthesis rates during leaf senescence of plant pre-cultured at low N supply reflected leaf senescence score during reproductive growth and N efficiency in the field experiments. Therefore, cultivar differences in leaf senescence during reproductive growth can only partly be reproduced in a short-term nutrient-solution experiment. Several differences between vegetative and reproductive growth might influence the induction and development of leaf senescence: first, although leaf senescence might be induced by N shortage both in hydroponics and under field conditions, the timing of N shortage is dependent upon different factors. In the field, the exploration of N sources in deeper soil layers might play the most important role for N uptake during reproductive growth . Thus in the field, root growth and morphology are the most important plant traits, which play only a minor role for N uptake in hydroponics. Secondly, source-sink relationships differ distinctly between vegetative and reproductive growth, both for carbohydrates and as a consequence also for N. The changes in assimilate flows might influence the development of leaf senescence, or at least the parameters used to characterize leaf senescence. However,potted blueberries the fact that photosynthesis rate during late stages of leaf senescence was significantly correlated to leaf senescence in the field experiments and to grain yield at limiting N supply suggests that cultivar differences in specific steps of leaf senescence related to the breakdown of the photosynthetic apparatus contribute to N efficiency in the field.

Life support systems are what make human travel a possibility. In long range space travels, such as the travel to Mars, life support cannot depend upon storage alone, it requires a fully regenerative system as well, i.e. waste must be reclaimed for reuse. Steam reformation, supercritical water oxidation, electrochemical oxidation, and incineration are a few of the solid waste reclamation technologies that are being developed and tested for use in space travel. Currently though, it seems that incineration might be the best choice among the previously mentioned, in providing a fully regenerative system. Through rapid conversion, incineration of the inedible parts of wastes and crops produces carbon dioxide, water, and minerals. Incineration is already the most thoroughly developed technology for use in a terrestrial environment. However, with the use of incineration in a closed environment, there is the eventual buildup of pollutants that are emitted in the process. The resulting NOx and SO2 pollutants need to be removed and recovered for reuse by a flue gas cleanup system. Important things to consider when developing a flue gas clean up technology for use in long range space travels are safety, energy requirements, sustainability, and doable under a micro-gravity condition. Due to the sensitivity and restrictions of space missions, a flue gas clean up system lacking in any of these considerations can be hazardous and could potentially compromise the missions. Technologies requiring things such as expendables or the use of catalysts are unsuitable for space missions due to the loss of valuable resources and the possibility of catalyst poisoning thus limiting the life-span of a catalyst. Also, due to the micro-gravity, it is difficult to use wet processes that handle liquids, such as spray absorbers.

Consequently, even though there are numerous flue gas clean up technologies developed , taking into consideration the limitation each provides, the number of reliable and applicable systems seem to be dwindling. Commercial activated carbon, made mostly from materials such as coconut shells and coal, has been studied for the adsorption and/or reduction of NOx and SO2 . In this paper, we study the use of the activated carbon prepared from hydroponic grown wheat straw and sweet potato stem for the control of air pollutants that are a result of incineration during space travel. Both wheat straw and sweet potato stalk are inedible biomass that can be continuously produced in the space vehicle. It was found that there is actually a minuscule amount of SO2 in the flue gas from the incineration of hydroponic biomass, and that most of the sulfur from the biomass ends up as sulfate in flyash. Since SO2 should have already reacted with the alkali metal, the technique entails the carbonization of the wheat straw and sweet potato stalk, resulting in an activated carbon for the adsorption of NOx and then a reduction of the adsorbed NOx by carbon to form N2. Since most NOx in flue gas from combustion is in the form of NO, and NO2 is readily adsorbed on the activated carbon, this paper concentrates on the removal of NO. Parametric studies on the production of activated carbon and the adsorption of NO by the carbon have been conducted. The optimal conditions and effectiveness of this procedure to regulate NO emissions have been determined.After the biomass was shredded, it was packed tightly into a stainless cylinder for pyrolysis and activation. Nitrogen and Carbon dioxide were used as the pyrolysis and activation gases, respectively. The gas flow rate for pyrolysis was 0.5-1.0L nitrogen per minute and activation was 0.25L carbon dioxide per minute.

The amount of biomass used was approximately 50.0-60.0g for each batch. Pyrolysis and activation temperature and times were varied during carbonization in order to determine optimal activation conditions. The activation temperature was 50°C higher than the pyrolysis temperature. The notation of the activated carbon “WS-2-600-1-650” and “SP-2-600-1-650” indicate that the activated carbon was prepared from wheat straw and sweet potato stalk, respectively with a 2 hrs pyrolysis time at 600°C followed by 1 hr of activation at 650°C. Once activation was complete, CO2 was supplied to the sample until it could be sealed in a container. Biomass on a space mission would likely come from a hydroponic system. The total mineral content of hydroponic biomass can be much higher than field-grown biomass if nutrients are supplied luxuriously to the hydroponic solution. A typical hydroponic plant solution has a nutrient ratio of N:K:Ca:P:S:Mg =16:6:4:2:1:1 and contains micro-nutrients of B, Mn, Zn, Cu, Co, Mo, and Fe. The concentration of potassium in the solution is about 6 mM. Thus, hydroponic activated carbon adsorption efficiencies may differ significantly from field-grown biomass. Field grown biomass generally has less than 10% mineral content, while hydroponic biomass can have as much as 30% mineral content. Because of this, many of the experiments were performed using char, which had been soaked in deionized water to remove the soluble minerals. After soaking, the char was drained and dried at 200o C for one hour before use. Specific surface areas of samples were determined by gas adsorption. An automated adsorption apparatus was employed for these measurements. Nitrogen adsorption/desorption was measured isothermally at -196o C. Before any such analysis, the sample was degassed at 250o C in a vacuum at about 10-3 torr. The nitrogen isotherms were analyzed by the BET equation, to determine the surface areas of the chars. BJH adsorption cumulative surface areas of pores of the samples also were determined.Most of NOx in flue gas from combustion is in the form of NO. Also, NO2 is readily adsorbed on the activated carbon. Consequently, efforts were directed to determine conditions for maximal removal efficiency of NO. Adsorption efficiency of NO on the activated carbon was studied to evaluate the effects of temperature, oxygen composition,square plastic pot moisture and flow rate on the production of effective activated carbon. The adsorption experiments were performed by using a simulate flue gas with variable concentrations of N2, carbon dioxide, oxygen, NO and H2O. NO and NO2 concentrations were analyzed by a chemilum inescent NOx analyzer . The amount of NOx absorbed by the activated carbon was determined from the difference in NOx concentration of the inlet and outlet gases. It was assumed that missing NOx was adsorbed by the activated carbon.Preparation of activated carbon was conducted by heating hydroponic grown wheat straw and sweet potato stalk under anaerobic conditions. Nitrogen and carbon dioxide were used as the pyrolysis and activating gases, respectively. Time and temperature were varied during pyrolysis and activation to determine optimal carbonization conditions. In order to determine optimal carbonization temperatures, samples of wheat straw and sweet potato stalk were heated at 100o C intervals between 300 to 800o C for two hours during pyrolysis and between 350°C to 850o C for one hour during activation. Afterwards, percent burn off was measured. Higher carbonization temperatures caused larger portions of the samples to burn off, as shown in Fig.2.

The burn off was 68% at 600°C and 86% at 800°C for wheat straw, while 66% at 600°C and 92%at 800°C for sweet potato stalk. Both samples appear to follow the same burn off trend, the percent burn off increases only slightly with the increase of temperature between 400°C to 600o C. However, pyrolysis and activation temperatures above 700o C were observed to cause significant amounts of wheat straw and sweet potato stalk to burn to ash. It is thus not recommended that reactions be run at temperatures exceeding 600o C. The decrease in surface area beyond 600°C is caused by sample burning off. Using temperatures much lower than 600o C would compromise the maximum amount of effective adsorption surface area attainable . It is not only important to run reactions at temperatures low enough to prevent burn off and ash formation but also high enough to generate effective surface areas, which would be at 600o C for wheat straw. Fig.4 shows the BJH adsorption cumulative pore area of wheat straw activated carbon generated at different pyrolysis and activation times. It is evident from the plot that samples derived from longer pyrolysis and carbonization times exhibited a higher micropore count compared to shorter times. As with temperature generation optimum, however, too long pyrolysis and activation reaction times cause an adverse increase in burn off percentage. Though wheat straw carbonized with a pyrolysis time of six hours and activation time of 2 hours still demonstrated a higher cumulative pore area than the shorter times, it also produces larger amounts of burn off. Since reaction temperatures were kept below 600o C, ash formation that would have diluted effective surface area was prevented. Even though ash does not form, pyrolysis and activation times must still be chosen to create a balance between pore formation and burn off, one that would generate a high micro-pore count but at the same time, minimize material loss. The optimal pyrolysis and activation times for wheat straw are 2 hours and 1 hour respectively.Optimal pyrolysis and activation temperatures and times for carbon preparation were determined based on the amount of NOx that can be adsorbed by the activated carbon. The adsorption capacities of wheat straw activated carbons generated by different pyrolysis and activation temperatures are shown in Fig.5. A gas mixture containing 250ppm of NO, 5% O2, 10% CO2, with N2 as the balance was passed, at a flow rate of 250ml/min, through a turbular reactor containing 2g of activation carbon at 25o C. It is evident from the plots that the WS-2-600-1-650 activated carbon had the best adsorption efficiency. Samples carbonized above 600o C have higher ash concentrations than those carbonized below, while those carbonized below have lower percent micro-pore counts and surface area—both explaining why wheat straw generated at 600o C had the best adsorption efficiency. The NO adsorption efficiencies of wheat straw samples carbonized by differing pyrolysis and activation times are shown in Figure 6. It is evident from the plots that activated carbons carbonized by prolonged pyrolysis and activation times have better adsorption efficiencies than those carbonized by shorter times due to higher pore count and BET surface area. The micro-pore count and the surface area of activated carbon increases with an increase of the preparation time, which explains why the samples with the longest pyrolysis and activation times have the best adsorption efficiencies. However, prolong activation results in more burn off and the production of ash. A balance must be reached when setting reaction parameters, one that will generate the largest surface area without a significant burn off. We have found that the optimal pyrolysis and activation times for wheat straw are two and one hour, respectively. The hydroponic biomass possesses high mineral content. The effect of the minerals on the activated carbon on NO adsorption efficiency was studied. The activated carbon was first soaked in water to dissolve the soluble minerals and then dried to remove the moisture from the carbon particles. The adsorption experiments using the mineral-free activated carbon were performed and the results indicate that the NO adsorption efficiency was substantially improved .