In Vitro Fossilization for High Spatial Resolution Quantification of Elements in Plant-Tissue Using LA-ICP-TOFMS.

Laser ablation in combination with an inductively coupled plasma time-of-flight mass spectrometer (LA-ICP-TOFMS) is an upcoming method for rapid quantitative element mapping of various samples. While widespread in geological applications, quantification of elements in biotissues remains challenging. In this study, a proof-of-concept sample preparation method is presented in which plant-tissues are fossilized in order to solidify the complex biotissue matrix into a mineral-like matrix. This process enables quantification of elements by using silicone as an internal standard for normalization while also providing consistent ablation processes similar to minerals to reduce image blurring. Furthermore, it allows us to generate a quantitative image of the element composition at high spatial resolution. The feasibility of the approach is demonstrated on leaves of sunflowers (Helianthus annuus), soy beans (Glycine max), and corn (Zea mays) as representatives for common crops, which were grown on both nonspiked and cadmium-spiked agricultural soil. The quantitative results achieved during imaging were validated with digestion of whole leaves followed by ICP-OES analysis. LA-ICP-TOFMS element mapping of conventionally dried samples can provide misleading trends due to the irregular ablation behavior of biotissue because high signals caused by high ablation rates are falsely interpreted as enrichment of elements. Fossilization provides the opportunity to correct such phenomena by standardization with Si as an internal standard. The method demonstrated here allows for quantitative image acquisition without time-consuming sample preparation steps by using comparatively safe chemicals. The diversity of tested samples suggests that this sample preparation method is well-suited to achieve reproducible and quantitative element maps of various plant samples.

Plant-soil interactions alter nitrogen and phosphorus dynamics in an advancing subarctic treeline.

Treelines advance due to climate warming. The impacts of this vegetation shift on plant-soil nutrient cycling are still uncertain, yet highly relevant as nutrient availability stimulates tree growth. Here, we investigated nitrogen (N) and phosphorus (P) in plant and soil pools along two tundra-forest transects on Kola Peninsula, Russia, with a documented elevation shift of birch-dominated treeline by 70 m during the last 50 years. Results show that although total N and P stocks in the soil-plant system did not change with elevation, their distribution was significantly altered. With the transition from high-elevation tundra to low-elevation forest, P stocks in stones decreased, possibly reflecting enhanced weathering. In contrast, N and P stocks in plant biomass approximately tripled and available P and N in the soil increased fivefold toward the forest. This was paralleled by decreasing carbon (C)-to-nutrient ratios in foliage and litter, smaller C:N:P ratios in microbial biomass, and lower enzymatic activities related to N and P acquisition in forest soils. An incubation experiment further demonstrated manifold higher N and P net mineralization rates in litter and soil in forest compared to tundra, likely due to smaller C:N:P ratios in decomposing organic matter. Overall, our results show that forest expansion increases the mobilization of available nutrients through enhanced weathering and positive plant-soil feedback, with nutrient-rich forest litter releasing greater amounts of N and P upon decomposition. While the low N and P availability in tundra may retard treeline advances, its improvement toward the forest likely promotes tree growth and forest development.

Hydroponic fodder as alternative feeds for ruminants to reduce ruminal methane emissions: an in vitro study.

Malate, a precursor in the ruminal propionate production pathway, competes with methanogenesis for metabolic hydrogen, offering a way to reduce ruminal methane (CH4) production in ruminants. However, cost considerations hinder widespread use of malate in ruminant diets. An alternative approach involves utilizing transient malate levels generated during seed germination via the glyoxylate cycle. This study investigated the methane-mitigating potential of malate-containing hydroponic fodder. Fodder samples with peak malate concentrations from alfalfa, forage pea, Italian ryegrass, rye, soybean, triticale, and wheat during germination were subjected to in vitro rumen fermentation using the Hohenheim gas test. The basal diet of in vitro fermentation comprised 40% grass silage, 40% maize silage, 15% hay, and 5% concentrate on a dry matter basis, with nutritional characteristics including 42.1% neutral detergent fiber (NDF), 25.0% acid detergent fiber, 14.0% starch, 12.7% crude protein, and 3.5% ether extract (EE), on a dry matter basis. Experimental treatments were fodder inclusion involved replacing 20% of the basal diet (20R), and additionally, 100% replacement of the silages with alfalfa d 10 and rye d 9 (SR), the 2 high-malate fodders. Reductions in CH4 production were observed with soybean (20R, 6.7% reduction), alfalfa (20R, 6.6% reduction), and increased with rye (20R, 6.3% increase). In the setup replacing silages with high-malate fodders (SR), alfalfa decreased CH4 production (17.7%) but increased ammonia (174%), while rye increased CH4 production (35.8%). Organic matter digestibility increased with SR rye (12.6%). Marginal effects of dietary variables were analyzed in a Generalized Additive Model. A negative relationship between dietary malate content and CH4 production was observed, whereas dietary NDF and starch content were positive correlated with CH4 production. In conclusion, malate within the hydroponic fodder could potentially reduce CH4 emissions in ruminants. However, achieving sufficient efficacy requires high malate content. Additionally, use of hydroponic fodder may increase the risk of nitrogen emissions. Animal studies are required for further investigation.

Environmental parameters and microbial community profiles as indication towards microbial activities and diversity in aquaponic system compartments.

BACKGROUND: An aquaponic system couples cultivation of plants and fish in the same aqueous medium. The system consists of interconnected compartments for fish rearing and plant production, as well as for water filtration, with all compartments hosting diverse microbial communities, which interact within the system. Due to the design, function and operation mode of the individual compartments, each of them exhibits unique biotic and abiotic conditions. Elucidating how these conditions shape microbial communities is useful in understanding how these compartments may affect the quality of the water, in which plants and fish are cultured. RESULTS: We investigated the possible relationships between microbial communities from biofilms and water quality parameters in different compartments of the aquaponic system. Biofilm samples were analyzed by total community profiling for bacterial and archaeal communities. The results implied that the oxygen levels could largely explain the main differences in abiotic parameters and microbial communities in each compartment of the system. Aerobic system compartments are highly biodiverse and work mostly as a nitrifying biofilter, whereas biofilms in the anaerobic compartments contain a less diverse community. Finally, the part of the system connecting the aerobic and anaerobic processes showed common conditions where both aerobic and anaerobic processes were observed. CONCLUSION: Different predicted microbial activities for each compartment were found to be supported by the abiotic parameters, of which the oxygen saturation, total organic carbon and total nitrogen differentiated clearly between samples from the main aerobic loop and the anaerobic compartments. The latter was also confirmed using microbial community profile analysis.

Zinc Absorption From Agronomically Biofortified Wheat Is Similar to Post-Harvest Fortified Wheat and Is a Substantial Source of Bioavailable Zinc in Humans.

BACKGROUND: Limited data exist on human zinc absorption from wheat biofortified via foliar (FBW) or root (hydroponically fortified wheat, HBW) zinc application. Stable isotope labels added at point of consumption (extrinsic labeling) might not reflect absorption from native zinc obtained by intrinsic labeling. OBJECTIVES: We measured fractional and total zinc absorption (FAZ, TAZ) in FBW and HBW wheat, compared with control wheat (CW) and fortified wheat (FW). The effect of labeling method was assessed in HBW (study 1), and the effect of milling extraction rate (EXR, 80% and 100%) in FBW (studies 2 and 3). METHODS: Generally healthy adults (n = 71, age: 18-45 y, body mass index: 18.5-25 kg/m2) were allocated to 1 of the studies, in which they served as their own controls. In study 1, men and women consumed wheat porridges colabeled intrinsically and extrinsically with 67Zn and 70Zn. In studies 2 and 3, women consumed wheat flatbreads (chapatis) labeled extrinsically. Zinc absorption was measured with the oral to intravenous tracer ratio method with a 4-wk wash-out period between meals. Data were analyzed with linear mixed models. RESULTS: In study 1 there were no differences in zinc absorption from extrinsic versus intrinsic labels in either FW or HBW. Similarly, FAZ and TAZ from FW and HBW did not differ. TAZ was 70-76% higher in FW and HBW compared with CW (P < 0.01). In studies 2 and 3, TAZ from FW and FBW did not differ but was 20-48% higher compared with CW (P < 0.001). Extraction rate had no effect on TAZ. CONCLUSIONS: Colabeling demonstrates that extrinsic zinc isotopic labels can be used to accurately quantify zinc absorption from wheat in humans. Biofortification through foliar zinc application, root zinc application, or fortification provides higher TAZ compared with unfortified wheat. In biofortified wheat, extraction rate (100-80%) has a limited impact on total zinc absorption. These studies were registered on clinicaltrials.gov (NCT01775319).

The Fate of Zn in Agricultural Soils: A Stable Isotope Approach to Anthropogenic Impact, Soil Formation, and Soil-Plant Cycling.

The supplementation of Zn to farm animal feed and the excretion via manure leads to an unintended Zn input to agricultural systems, which might compromise the long-term soil fertility. The Zn fluxes at three grassland sites in Switzerland were determined by a detailed analysis of relevant inputs (atmospheric deposition, manure, weathering) and outputs (seepage water, biomass harvest) during one hydrological year. The most important Zn input occurred through animal manure (1076-1857 g ha-1 yr-1) and Zn mass balances revealed net Zn accumulations (456-1478 g ha-1 yr-1). We used Zn stable isotopes to assess the importance of anthropogenic impacts and natural long-term processes on the Zn distribution in soils. Soil-plant cycling and parent material weathering were identified as the most important processes, over the entire period of soil formation (13 700 years), whereas the soil pH strongly affected the direction of Zn isotopic fractionation. Recent anthropogenic inputs of Zn only had a smaller influence compared to the natural processes of the past 13 700 years. However, this will probably change in the future, as Zn stocks in the 0-20 cm layer will increase by 22-68% in the next 100 years, if Zn inputs remain on the same level as today.

Understanding fertilizer adoption and effectiveness on maize in Zambia.

Increased fertilizer use will likely be crucial for raising and sustaining farm productivity in Africa, but adoption may be limited by ineffectiveness under certain conditions. This article quantifies the impacts of soil characteristics on maize response to fertilizer in Zambia using a nationally representative sample of 1453 fields, combining economic, farm management and soil analysis data. Depending on soil regimes, average maize yield response estimates range from insignificant (0) to 7 maize kg per fertilizer kg. For the majority of farmers, the estimated average value cost ratio is between 1 and 2, meaning fertilizer use would be fiscally rational, barring uncertainty and transfer costs. Since transfer costs exist and outcomes are uncertain, however, many farmers may sensibly pause before deciding whether to adopt fertilizer. This suggests shifting the emphasis of chronically low fertilizer use in Africa away from explanations of "market failure" toward greater emphasis on improving fertilizer efficacy.

Identification of Heterotrophic Zinc Mobilization Processes among Bacterial Strains Isolated from Wheat Rhizosphere (Triticum aestivum L.).

Soil and plant inoculation with heterotrophic zinc-solubilizing bacteria (ZSB) is considered a promising approach for increasing zinc (Zn) phytoavailability and enhancing crop growth and nutritional quality. Nevertheless, it is necessary to understand the underlying bacterial solubilization processes to predict their repeatability in inoculation strategies. Acidification via gluconic acid production remains the most reported process. In this study, wheat rhizosphere soil serial dilutions were plated on several solid microbiological media supplemented with scarcely soluble Zn oxide (ZnO), and 115 putative Zn-solubilizing isolates were directly detected based on the formation of solubilization halos around the colonies. Eight strains were selected based on their Zn solubilization efficiency and siderophore production capacity. These included one strain of Curtobacterium, two of Plantibacter, three strains of Pseudomonas, one of Stenotrophomonas, and one strain of Streptomyces In ZnO liquid solubilization assays, the presence of glucose clearly stimulated organic acid production, leading to medium acidification and ZnO solubilization. While solubilization by Streptomyces and Curtobacterium was attributed to the accumulated production of six and seven different organic acids, respectively, the other strains solubilized Zn via gluconic, malonic, and oxalic acids exclusively. In contrast, in the absence of glucose, ZnO dissolution resulted from proton extrusion (e.g., via ammonia consumption by Plantibacter strains) and complexation processes (i.e., complexation with glutamic acid in cultures of Curtobacterium). Therefore, while gluconic acid production was described as a major Zn solubilization mechanism in the literature, this study goes beyond and shows that solubilization mechanisms vary among ZSB and are strongly affected by growth conditions.IMPORTANCE Barriers toward a better understanding of the mechanisms underlying zinc (Zn) solubilization by bacteria include the lack of methodological tools for isolation, discrimination, and identification of such organisms. Our study proposes a direct bacterial isolation procedure, which prevents the need to screen numerous bacterial candidates (for which the ability to solubilize Zn is unknown) for recovering Zn-solubilizing bacteria (ZSB). Moreover, we confirm the potential of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a quick and accurate tool for the identification and discrimination of environmental bacterial isolates. This work also describes various Zn solubilization processes used by wheat rhizosphere bacteria, including proton extrusion and the production of different organic acids among bacterial strains. These processes were also clearly affected by growth conditions (i.e., solid versus liquid cultures and the presence and absence of glucose). Although highlighted mechanisms may have significant effects at the soil-plant interface, these should only be transposed cautiously to real ecological situations.

Zinc isotope fractionation during grain filling of wheat and a comparison of zinc and cadmium isotope ratios in identical soil-plant systems.

Remobilization of zinc (Zn) from shoot to grain contributes significantly to Zn grain concentrations and thereby to food quality. On the other hand, strong accumulation of cadmium (Cd) in grain is detrimental for food quality. Zinc concentrations and isotope ratios were measured in wheat shoots (Triticum aestivum) at different growth stages to elucidate Zn pathways and processes in the shoot during grain filling. Zinc mass significantly decreased while heavy Zn isotopes accumulated in straw during grain filling (Δ66 Znfull maturity-flowering  = 0.21-0.31‰). Three quarters of the Zn mass in the shoot moved to the grains, which were enriched in light Zn isotopes relative to the straw (Δ66 Zngrain-straw -0.21 to -0.31‰). Light Zn isotopes accumulated in phloem sinks while heavy isotopes were retained in phloem sources likely because of apoplastic retention and compartmentalization. Unlike for Zn, an accumulation of heavy Cd isotopes in grains has previously been shown. The opposing isotope fractionation of Zn and Cd might be caused by distinct affinities of Zn and Cd to oxygen, nitrogen, and sulfur ligands. Thus, combined Zn and Cd isotope analysis provides a novel tool to study biochemical processes that separate these elements in plants.

Fate of Cd in Agricultural Soils: A Stable Isotope Approach to Anthropogenic Impact, Soil Formation, and Soil-Plant Cycling.

The application of mineral phosphate (P) fertilizers leads to an unintended Cd input into agricultural systems, which might affect soil fertility and quality of crops. The Cd fluxes at three arable sites in Switzerland were determined by a detailed analysis of all inputs (atmospheric deposition, mineral P fertilizers, manure, and weathering) and outputs (seepage water, wheat and barley harvest) during one hydrological year. The most important inputs were mineral P fertilizers (0.49 to 0.57 g Cd ha-1 yr-1) and manure (0.20 to 0.91 g Cd ha-1 yr-1). Mass balances revealed net Cd losses for cultivation of wheat (-0.01 to -0.49 g Cd ha-1 yr-1) but net accumulations for that of barley (+0.18 to +0.71 g Cd ha-1 yr-1). To trace Cd sources and redistribution processes in the soils, we used natural variations in the Cd stable isotope compositions. Cadmium in seepage water (δ114/110Cd = 0.39 to 0.79‰) and plant harvest (0.27 to 0.94‰) was isotopically heavier than in soil (-0.21 to 0.14‰). Consequently, parent material weathering shifted bulk soil isotope compositions to lighter signals following a Rayleigh fractionation process (ε ≈ 0.16). Furthermore, soil-plant cycling extracted isotopically heavy Cd from the subsoil and moved it to the topsoil. These long-term processes and not anthropogenic inputs determined the Cd distribution in our soils.

Evaluation of MALDI-TOF mass spectrometry for the competitiveness analysis of selected indigenous cowpea (Vigna unguiculata L. Walp.) Bradyrhizobium strains from Kenya.

Cowpea N2 fixation and yield can be enhanced by selecting competitive and efficient indigenous rhizobia. Strains from contrasting agro-ecologies of Kilifi and Mbeere (Kenya) were screened. Two pot experiments were established consisting of 13 Bradyrhizobium strains; experiment 1 (11 Mbeere + CBA + BK1 from Burkina Faso), experiment 2 (12 Kilifi + CBA). Symbiotic effectiveness was assessed (shoot biomass, SPAD index and N uptake). Nodule occupancy of 13 simultaneously co-inoculated strains in each experiment was analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to assess competitiveness. Strains varied in effectiveness and competitiveness. The four most efficient strains were further evaluated in a field trial in Mbeere during the 2014 short rains. Strains from bacteroids of cowpea nodules from pot and field experiments were accurately identified as Bradyrhizobium by MALDI-TOF based on the SARAMIS™ database. In the field, abundant indigenous populations 7.10 × 103 rhizobia g-1 soil, outcompeted introduced strains. As revealed by MALDI-TOF, indigenous strains clustered into six distinct groups (I, II, III, IV, V and VI), group III were most abundant occupying 80% of nodules analyzed. MALDI-TOF was rapid, affordable and reliable to identify Bradyrhizobium strains directly from nodule suspensions in competition pot assays and in the field with abundant indigenous strains thus, its suitability for future competition assays. Evaluating strain competitiveness and then symbiotic efficacy is proposed in bioprospecting for potential cowpea inoculant strains.

Cowpea (Vigna unguiculata L. Walp) hosts several widespread bradyrhizobial root nodule symbionts across contrasting agro-ecological production areas in Kenya.

Cowpea (Vigna unguiculata L. Walp.) is an important African food legume suitable for dry regions. It is the main legume in two contrasting agro-ecological regions of Kenya as an important component of crop rotations because of its relative tolerance to unpredictable drought events. This study was carried out in an effort to establish a collection of bacterial root nodule symbionts and determine their relationship to physicochemical soil parameters as well as any geographical distributional patterns. Bradyrhizobium spp. were found to be widespread in this study and several different types could be identified at each site. Unique but rare symbionts were recovered from the nodules of plants sampled in a drier in-land region, where there were also overall more different bradyrhizobia found. Plants raised in soil from uncultivated sites with a natural vegetation cover tended to also associate with more different bradyrizobia. The occurrence and abundance of different bradyrhizobia correlated with differences in soil texture and pH, but did neither with the agro-ecological origin, nor the origin from cultivated (n = 15) or uncultivated (n = 5) sites. The analytical method, protein profiling of isolated strains by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), provided higher resolution than 16S rRNA gene sequencing and was applied in this study for the first time to isolates recovered directly from field-collected cowpea root nodules. The method thus seems suitable for screening isolate collections on the presence of different groups, which, provided an appropriate reference database, can also be assigned to known species.

Cadmium Isotope Fractionation in Soil-Wheat Systems.

Analyses of stable metal isotope ratios constitute a novel tool in order to improve our understanding of biogeochemical processes in soil-plant systems. In this study, we used such measurements to assess Cd uptake and transport in wheat grown on three agricultural soils under controlled conditions. Isotope ratios of Cd were determined in the bulk C and A horizons, in the Ca(NO3)2-extractable Cd soil pool, and in roots, straw, and grains. The Ca(NO3)2-extractable Cd was isotopically heavier than the Cd in the bulk A horizon (Δ(114/110)Cdextract-Ahorizon = 0.16 to 0.45‰). The wheat plants were slightly enriched in light isotopes relative to the Ca(NO3)2-extractable Cd or showed no significant difference (Δ(114/110)Cdwheat-extract = -0.21 to 0.03‰). Among the plant parts, Cd isotopes were markedly fractionated: straw was isotopically heavier than roots (Δ(114/110)Cdstraw-root = 0.21 to 0.41‰), and grains were heavier than straw (Δ(114/110)Cdgrain-straw = 0.10 to 0.51‰). We suggest that the enrichment of heavy isotopes in the wheat grains was caused by mechanisms avoiding the accumulation of Cd in grains, such as the chelation of light Cd isotopes by thiol-containing peptides in roots and straw. These results demonstrate that Cd isotopes are significantly and systematically fractionated in soil-wheat systems, and the fractionation patterns provide information on the biogeochemical processes in these systems.

Competition and facilitation in synthetic communities of arbuscular mycorrhizal fungi.

Interactions between arbuscular mycorrhizal fungal (AMF) species cocolonizing the same host plant are still little understood in spite of major ecological significance of mycorrhizal symbiosis and widespread occurrence of these fungi in communities rather than alone. Furthermore, shifting the composition of AMF communities has demonstrated consequences for the provision of symbiotic benefits to the host as well as for the qualities of ecosystem services. Therefore, here we addressed the nature and strength of interactions between three different AMF species in all possible two-species combinations on a gradient of inoculation densities. Fungal communities were established in pots with Medicago truncatula plants, and their composition was assessed with taxon-specific real-time PCR markers. Nature of interactions between the fungi was varying from competition to facilitation and was influenced by both the identity and relative abundance of the coinoculated fungi. Plants coinoculated with Claroideoglomus and Rhizophagus grew bigger and contained more phosphorus than with any of these two fungi separately, although these fungi obviously competed for root colonization. On the other hand, plants coinoculated with Gigaspora and Rhizophagus, which facilitated each other's root colonization, grew smaller than with any of these fungi separately. Our results point to as yet little understood complexity of interactions in plant-associated symbiotic fungal communities, which, depending on their composition, can induce significant changes in plant host growth and/or phosphorus acquisition in either direction.

The molecular environment of phosphorus in sewage sludge ash: implications for bioavailability.

Producing a P fertilizer from sewage sludge ash (SSA) is a strategy to efficiently recycle P from a secondary raw material. The P speciation in four SSAs was characterized before and after the removal of heavy metals by a thermo-chemical treatment that involved CaCl addition. We chose complementary techniques to determine the direct P speciation, including X-ray powder diffraction, solid-state P direct-polarization magic-angle spinning nuclear magnetic resonance, and X-ray absorption near edge structure. Results from these techniques were compared with operational and functional speciation information obtained from a sequential P extraction and a plant biotest with Italian ryegrass grown on a soil-sand mixture with little available P. The speciation of P in untreated and thermo-chemically treated SSAs depended on their elemental composition. At a molar ratio of Ca:P ≤ 2, SSAs contained combinations of polymorphs of AlPO, ß-tricalcium phosphate, and apatite-like P species. In SSAs with a molar ratio of Ca:P > 2, an apatite-like molecular environment was predominant. The thermo-chemical process induced an increase in crystalline phases and enhanced the crystallinity of the P species. The structural order of the bulk sample was the most decisive parameter in controlling the P availability of the studied SSAs to plants. We conclude that, to produce a high-quality fertilizer and despite of the successful heavy metal removal, the thermo-chemical process requires further development toward enhanced P bioavailability.

Fertilization strategies affect phosphorus forms and release from soils and suspended solids.

The release of phosphorus from soils in surface runoff is strongly influenced by fertilizer inputs and contributes significantly to agriculturally driven eutrophication. This work evaluated the forms and availability of P in bulk soils and suspended solids (SS) produced by a water dispersion test that mimics the action of rain events and/or irrigation. This test was applied on soils cultivated with maize and fertilized with mineral N, P, and K (NPK); mineral P and K (PK); bovine slurry and P (S); or manure and P (M) for 15 yr. The P surplus in the treated soils was in the order NPK < PK < S < M. Forms and availability of P were analyzed in bulk soils, and their respective SS (<20 µm) by the Hedley sequential P fractionation method and the isotopic exchange kinetics. The labile forms increased according to P surplus and represented up to 15 and 25% of total P in the bulk soil and in the SS, respectively, indicating a selective enrichment of the more labile P forms in the erodible particles. Exchangeability of P from SS was rapid and intense as a result of a shift of P solution equilibrium at the increased water/solid ratio and a larger accumulation of more labile P in the detached particles than in the bulk soil. Phosphorus saturation of iron and aluminum oxides and the enrichment of fertilizer-derived P salts in the suspended solids control P forms and exchangeability for mineral fertilizer treatments, whereas in M soil carbon content assumed a key role.

Recycling nutrients from organic waste for growing higher plants in the Micro Ecological Life Support System Alternative (MELiSSA) loop during long-term space missions.

Space agencies are developing Bioregenerative Life Support Systems (BLSS) in view of upcoming long-term crewed space missions. Most of these BLSS plan to include various crops to produce different types of foods, clean water, and O2 while capturing CO2 from the atmosphere. However, growing these plants will require the appropriate addition of nutrients in forms that are available. As shipping fertilizers from Earth would be too costly, it will be necessary to use waste-derived nutrients. Using the example of the MELiSSA (Micro-Ecological Life Support System Alternative) loop of the European Space Agency, this paper reviews what should be considered so that nutrients recycled from waste streams could be used by plants grown in a hydroponic system. Whereas substantial research has been conducted on nitrogen and phosphorus recovery from human urine, much work remains to be done on recovering nutrients from other liquid and solid organic waste. It is essential to continue to study ways to efficiently remove sodium and chloride from urine and other organic waste to prevent the spread of these elements to the rest of the MELiSSA loop. A full nitrogen balance at habitat level will have to be achieved; on one hand, sufficient N2 will be needed to maintain atmospheric pressure at a proper level and on the other, enough mineral nitrogen will have to be provided to the plants to ensure biomass production. From a plant nutrition point of view, we will need to evaluate whether the flux of nutrients reaching the hydroponic system will enable the production of nutrient solutions able to sustain a wide variety of crops. We will also have to assess the nutrient use efficiency of these crops and how that efficiency might be increased. Techniques and sensors will have to be developed to grow the plants, considering low levels or the total absence of gravity, the limited volume available to plant growth systems, variations in plant needs, the recycling of nutrient solutions, and eventually the ultimate disposal of waste that can no longer be used.

18O enrichment in phosphorus pools extracted from soybean leaves.

The objective of this study was to investigate the isotopic composition of oxygen bound to phosphate (δ(18)O-PO(4)) in different phosphorus (P) pools in plant leaves. As a model plant we used soybean (Glycine max cv Toliman) grown in the presence of ample P in hydroponic cultures. The leaf blades were extracted with 0.3 M trichloroacetic acid (TCA) and with 10 M nitric acid. These extractions allowed measurement of the TCA-soluble reactive P (TCA P) that is rapidly cycled within the cell and the total leaf P. The difference between total leaf P and TCA P yielded the structural P which includes organic P compounds not extractable by TCA. P uptake and its translocation and transformation within the soybean plants lead to an (18)O enrichment of TCA P (δ(18)O-PO(4) between 16.9 and 27.5‰) and structural P (δ(18)O-PO(4) between 42.6 and 68.0 ‰) compared with 12.4‰ in the phosphate in the nutrient solution. δ(18)O values of phosphate extracted from soybean leaves grown under optimal conditions are greater than the δ(18)O-PO(4) values of the provided P source. Furthermore, the δ(18)O-PO(4) of TCA P seems to be controlled by the δ(18)O of leaf water and the activity of inorganic pyrophosphatase or other pyrophosphatases.

Oxygen isotopes unravel the role of microorganisms in phosphate cycling in soils.

Phosphorus (P) is considered the ultimate limiting nutrient for plants in most natural systems and changes in the distribution of inorganic and organic P forms during soil development have been well documented. In particular, microbial activity has been shown to be an important control on P cycling but its contribution in building up the pool of plant-available P during soil development is still poorly quantified. To determine the importance of different biological processes on P cycling, we analyzed the isotopic composition of oxygen in phosphate (δ(18)O-Pi) from the parent material, soil microorganisms, the available P pool, and from the vegetation along a 150-year soil chronosequence of a glacier forefield. Our results show that at all sites, δ(18)O-Pi of microbial Pi is within the range expected for the temperature-dependent equilibrium between phosphate and water. In addition, the isotopic signature of available Pi is close to the signature of microbial Pi, independently of the contribution of parent material Pi, vegetation Pi or Pi released from organic matter mineralization. Thus, we show that phosphate is cycled through soil microorganisms before being released to the available pool. This isotopic approach demonstrates for the first time in the field and over long time scales, and not only through controlled experiments, the role of the microbial activity in cycling of P in soils.

Understanding soil selenium accumulation and bioavailability through size resolved and elemental characterization of soil extracts.

Dietary deficiency of selenium is a global health threat related to low selenium concentrations in crops. Despite the chemical similarity of selenium to the two more abundantly studied elements sulfur and arsenic, the understanding of its accumulation in soils and availability for plants is limited. The lack of understanding of soil selenium cycling is largely due to the unavailability of methods to characterize selenium species in soils, especially the organic ones. Here we develop a size-resolved multi-elemental method using liquid chromatography and elemental mass spectrometry, which enables an advanced characterization of selenium, sulfur, and arsenic species in soil extracts. We apply the analytical approach to soils sampled along the Kohala rainfall gradient on Big Island (Hawaii), which cover a large range of organic carbon and (oxy)hydroxides contents. Similarly to sulfur but contrarily to arsenic, a large fraction of selenium is found associated with organic matter in these soils. However, while sulfur and arsenic are predominantly found as oxyanions in water extracts, selenium mainly exists as small hydrophilic organic compounds. Combining Kohala soil speciation data with concentrations in parent rock and plants further suggests that selenium association with organic matter limits its mobility in soils and availability for plants.