Protein markers for identification of Yersinia pestis and their variation related to culture.

The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C-30 °C) and therefore are more likely to be used for bulk production. Analysis of Y. pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.

RhizoMAP: a comprehensive, nondestructive, and sensitive platform for metabolic imaging of the rhizosphere.

BACKGROUND: Elucidating the intricate structural organization and spatial gradients of biomolecular composition within the rhizosphere is critical to understanding important biogeochemical processes, which include the mechanisms of root-microbe interactions for maintaining sustainable plant ecosystem services. While various analytical methods have been developed to assess the spatial heterogeneity within the rhizosphere, a comprehensive view of the fine distribution of metabolites within the root-soil interface has remained a significant challenge. This is primarily due to the difficulty of maintaining the original spatial organization during sample preparation without compromising its molecular content. RESULTS: In this study, we present a novel approach, RhizoMAP, in which the rhizosphere molecules are imprinted on selected polymer membranes and then spatially profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). We enhanced the performance of RhizoMAP by combining the use of two thin (< 20 µm) membranes (polyester and polycarbonate) with distinct MALDI sample preparations. This optimization allowed us to gain insight into the distribution of over 500 different molecules within the rhizosphere of poplar (Populus trichocarpa) grown in rhizoboxes filled with mycorrhizae soil. These two membranes, coupled with three different sample preparation conditions, enabled us to capture the distribution of a wide variety of molecules that included phytohormones, amino acids, sugars, sugar glycosides, polycarboxylic acids components of the Krebs cycle, fatty acids, short aldehydes and ketones, terpenes, volatile organic compounds, fertilizers from the soil, and others. Their spatial distribution varies greatly, with some following root traces, others showing diffusion from roots, some associated with soil particles, and many having distinct hot spots along the plant root or surrounding soil. Moreover, we showed how RhizoMAP can be used to localize the origin of the molecules and molecular transformation during root growth. Finally, we demonstrated the power of RhizoMAP to capture molecular distributions of key metabolites throughout a 20 cm deep rhizosphere. CONCLUSIONS: RhizoMAP is a method that provides nondestructive, untargeted, broad, and sensitive metabolite imaging of root-associated molecules, exudates, and soil organic matter throughout the rhizosphere, as demonstrated in a lab-controlled native soil environment.

Experimental study of crossover from capillary to viscous fingering for supercritical CO2-water displacement in a homogeneous pore network.

Carbon sequestration in saline aquifers involves displacing brine from the pore space by supercritical CO(2) (scCO(2)). The displacement process is considered unstable due to the unfavorable viscosity ratio between the invading scCO(2) and the resident brine. The mechanisms that affect scCO(2)-water displacement under reservoir conditions (41 °C, 9 MPa) were investigated in a homogeneous micromodel. A large range of injection rates, expressed as the dimensionless capillary number (Ca), was studied in two sets of experiments: discontinuous-rate injection, where the micromodel was saturated with water before each injection rate was imposed, and continuous-rate injection, where the rate was increased after quasi-steady conditions were reached for a certain rate. For the discontinuous-rate experiments, capillary fingering and viscous fingering are the dominant mechanisms for low (logCa ≤ -6.61) and high injection rates (logCa ≥ -5.21), respectively. Crossover from capillary to viscous fingering was observed for logCa = -5.91 to -5.21, resulting in a large decrease in scCO(2) saturation. The discontinuous-rate experimental results confirmed the decrease in nonwetting fluid saturation during crossover from capillary to viscous fingering predicted by numerical simulations by Lenormand et al. (J. Fluid Mech.1988, 189, 165-187). Capillary fingering was the dominant mechanism for all injection rates in the continuous-rate experiment, resulting in monotonic increase in scCO(2) saturation.

Correlation of oil-water and air-water contact angles of diverse silanized surfaces and relationship to fluid interfacial tensions.

The use of air-water, θ(wa), or air-liquid contact angles is customary in surface science, while oil-water contact angles, θ(ow), are of paramount importance in subsurface multiphase flow phenomena including petroleum recovery, nonaqueous phase liquid fate and transport, and geological carbon sequestration. In this paper we determine both the air-water and oil-water contact angles of silica surfaces modified with a diverse selection of silanes, using hexadecane as the oil. The silanes included alkylsilanes, alkylarylsilanes, and silanes with alkyl or aryl groups that are functionalized with heteroatoms such as N, O, and S. These silanes yielded surfaces with wettabilities from water wet to oil wet, including specific silanized surfaces functionalized with heteroatoms that yield intermediate wet surfaces. The oil-water contact angles for clean and silanized surfaces, excluding one partially fluorinated surface, correlate linearly with air-water contact angles with a slope of 1.41 (R = 0.981, n = 13). These data were used to examine a previously untested theoretical treatment relating air-water and oil-water contact angles in terms of fluid interfacial energies. Plotting the cosines of these contact angles against one another, we obtain the relationship cos θ(wa) = 0.667 cos θ(ow) + 0.384 (R = 0.981, n = 13), intercepting cos θ(ow) = -1 at -0.284, which is in excellent agreement with the linear assumption of the theory. The theoretical slope, based on the fluid interfacial tensions σ(wa), σ(ow), and σ(oa), is 0.67. We also demonstrate how silanes can be used to alter the wettability of the interior of a pore network micromodel device constructed in silicon/silica with a glass cover plate. Such micromodels are used to study multiphase flow phenomena. The contact angle of the resulting interior was determined in situ. An intermediate wet micromodel gave a contact angle in excellent agreement with that obtained on an open planar silica surface using the same silane.

Liquid CO2 displacement of water in a dual-permeability pore network micromodel.

Permeability contrasts exist in multilayer geological formations under consideration for carbon sequestration. To improve our understanding of heterogeneous pore-scale displacements, liquid CO(2) (LCO(2))-water displacement was evaluated in a pore network micromodel with two distinct permeability zones. Due to the low viscosity ratio (logM = -1.1), unstable displacement occurred at all injection rates over 2 orders of magnitude. LCO(2) displaced water only in the high permeability zone at low injection rates with the mechanism shifting from capillary fingering to viscous fingering with increasing flow rate. At high injection rates, LCO(2) displaced water in the low permeability zone with capillary fingering as the dominant mechanism. LCO(2) saturation (S(LCO2)) as a function of injection rate was quantified using fluorescent microscopy. In all experiments, more than 50% of LCO(2) resided in the active flowpaths, and this fraction increased as displacement transitioned from capillary to viscous fingering. A continuum-scale two-phase flow model with independently determined fluid and hydraulic parameters was used to predict S(LCO2) in the dual-permeability field. Agreement with the micromodel experiments was obtained for low injection rates. However, the numerical model does not account for the unstable viscous fingering processes observed experimentally at higher rates and hence overestimated S(LCO2).

Pore-scale study of transverse mixing induced CaCO3 precipitation and permeability reduction in a model subsurface sedimentary system.

A microfluidic pore structure etched into a silicon wafer was used as a two-dimensional model subsurface sedimentary system (i.e., micromodel) to study mineral precipitation and permeability reduction relevant to groundwater remediation and geological carbon sequestration. Solutions containing CaCl(2) and Na(2)CO(3) at four different saturation states (Ω = [Ca(2+)][CO(3)(2-)]/K(spCaCO(3))) were introduced through two separate inlets, and they mixed by diffusion transverse to the main flow direction along the center of the micromodel resulting in CaCO(3) precipitation. Precipitation rates increased and the total amount of precipitates decreased with increasing saturation state, and only vaterite and calcite crystals were formed (no aragonite). The relative amount of vaterite increased from 80% at the lowest saturation state (Ω(v) = 2.8 for vaterite) to 95% at the highest saturation state (Ω(v) = 4.5). Fluorescent tracer tests conducted before and after CaCO(3) precipitation indicate that pore spaces were occluded by CaCO(3) precipitates along the transverse mixing zone, thus substantially reducing porosity and permeability, and potentially limiting transformation from vaterite to the more stable calcite. The results suggest that mineral precipitation along plume margins can decrease both reactant mixing during groundwater remediation, and injection and storage efficiency during CO(2) sequestration.

Dynamics of Global Gene Expression and Regulatory Elements in Growing Brachypodium Root System.

Root systems are dynamic and adaptable organs that play critical roles in plant development. However, how roots grow and accumulate biomass during plant life cycle and in relation to shoot growth phenology remains understudied. A comprehensive time-dependent root morphological analysis integrated with molecular signatures is then required to advance our understanding of root growth and development. Here we studied Brachypodium distachyon rooting process by monitoring root morphology, biomass production, and C/N ratios during developmental stages. To provide insight into gene regulation that accompanies root growth, we generated comprehensive transcript profiles of Brachypodium whole-root system at four developmental stages. Our data analysis revealed that multiple biological processes including trehalose metabolism and various families of transcription factors (TFs) were differentially expressed in root system during plant development. In particular, the AUX/IAA, ERFs, WRKY, NAC, and MADS TF family members were upregulated as plant entered the booting/heading stage, while ARFs and GRFs were downregulated suggesting these TF families as important factors involved in specific phases of rooting, and possibly in regulation of transition to plant reproductive stages. We identified several Brachypodium candidate root biomass-promoting genes and cis-regulatory elements for further functional validations and root growth improvements in grasses.

Visualizing Microbial Community Dynamics via a Controllable Soil Environment.

Understanding the basic biology that underpins soil microbiome interactions is required to predict the metaphenomic response to environmental shifts. A significant knowledge gap remains in how such changes affect microbial community dynamics and their metabolic landscape at microbially relevant spatial scales. Using a custom-built SoilBox system, here we demonstrated changes in microbial community growth and composition in different soil environments (14%, 24%, and 34% soil moisture), contingent upon access to reservoirs of nutrient sources. The SoilBox emulates the probing depth of a common soil core and enables determination of both the spatial organization of the microbial communities and their metabolites, as shown by confocal microscopy in combination with mass spectrometry imaging (MSI). Using chitin as a nutrient source, we used the SoilBox system to observe increased adhesion of microbial biomass on chitin islands resulting in degradation of chitin into N-acetylglucosamine (NAG) and chitobiose. With matrix-assisted laser desorption/ionization (MALDI)-MSI, we also observed several phospholipid families that are functional biomarkers for microbial growth on the chitin islands. Fungal hyphal networks bridging different chitin islands over distances of 27 mm were observed only in the 14% soil moisture regime, indicating that such bridges may act as nutrient highways under drought conditions. In total, these results illustrate a system that can provide unprecedented spatial information about interactions within soil microbial communities as a function of changing environments. We anticipate that this platform will be invaluable in spatially probing specific intra- and interkingdom functional relationships of microbiomes within soil.IMPORTANCE Microbial communities are key components of the soil ecosystem. Recent advances in metagenomics and other omics capabilities have expanded our ability to characterize the composition and function of the soil microbiome. However, characterizing the spatial metabolic and morphological diversity of microbial communities remains a challenge due to the dynamic and complex nature of soil microenvironments. The SoilBox system, demonstrated in this work, simulates an ∼12-cm soil depth, similar to a typical soil core, and provides a platform that facilitates imaging the molecular and topographical landscape of soil microbial communities as a function of environmental gradients. Moreover, the nondestructive harvesting of soil microbial communities for the imaging experiments can enable simultaneous multiomics analysis throughout the depth of the SoilBox. Our results show that by correlating molecular and optical imaging data obtained using the SoilBox platform, deeper insights into the nature of specific soil microbial interactions can be achieved.

Calcareous organic matter coatings sequester siderophores in alkaline soils.

Although most studies of organic matter (OM) stabilization in soils have focused on adsorption to aluminosilicate and iron-oxide minerals due to their strong interactions with organic nucleophiles, stabilization within alkaline soils has been empirically correlated with exchangeable Ca. Yet the extent of competing processes within natural soils remains unclear because of inadequate characterization of soil mineralogy and OM distribution within the soil in relation to minerals, particularly in C poor alkaline soils. In this study, we employed bulk and surface-sensitive spectroscopic methods including X-ray diffraction, 57Fe-Mössbauer, and X-ray photoemission spectroscopy (XPS), and transmission electron microscopy (TEM) methods to investigate the minerology and soil organic C and N distribution on individual fine particles within an alkaline soil. Microscopy and XPS analyses demonstrated preferential sorption of Ca-containing OM onto surfaces of Fe-oxides and calcite. This result was unexpected given that the bulk combined amounts of quartz and Fe-containing feldspars of the soil constitute ~90% of total minerals and the surface atomic composition was largely Fe and Al (>10% combined) compared to Ca (4.2%). Soil sorption experiments were conducted with two siderophores, pyoverdine and enterobactin, to evaluate the adsorption of organic molecules with functional groups that strongly and preferentially bind Fe. A greater fraction of pyoverdine was adsorbed compared to enterobactin, which is smaller, less polar, and has a lower aqueous solubility. Using NanoSIMS to map the distribution of isotopically-labeled siderophores, we observed correlations with Ca and Fe, along with strong isotopic dilution with native C, indicating associations with OM coatings rather than with bare mineral surfaces. We propose a mechanism of adsorption by which organics aggregate within alkaline soils via cation bridging, favoring the stabilization of larger molecules with a greater number of nucleophilic functional groups.

Metabolic shifts associated with drought-induced senescence in Brachypodium.

The metabolic underpinnings of plant survival under severe drought-induced senescence conditions are poorly understood. In this study, we assessed the morphological, physiological and metabolic responses to sustained water deficit in Brachypodium distachyon, a model organism for research on temperate grasses. Relative to control plants, fresh biomass, leaf water potential, and chlorophyll levels decreased rapidly in plants grown under drought conditions, demonstrating an early onset of senescence. The leaf C/N ratio and protein content showed an increase in plants subjected to drought stress. The concentrations of several small molecule carbohydrates and amino acid-derived metabolites previously implicated in osmotic protection increased rapidly in plants experiencing water deficit. Malic acid, a low molecular weight organic acid with demonstrated roles in stomatal closure, also increased rapidly as a response to drought treatment. The concentrations of prenyl lipids, such as phytol and α-tocopherol, increased early during the drought treatment but then dropped dramatically. Surprisingly, continued changes in the quantities of metabolites were observed, even in samples harvested from visibly senesced plants. The data presented here provide insights into the processes underlying persistent metabolic activity during sustained water deficit and can aid in identifying mechanisms of drought tolerance in plants.

Numerical and experimental investigation of DNAPL removal mechanisms in a layered porous medium by means of soil vapor extraction.

The purpose of this work is to identify the mechanisms that govern the removal of carbon tetrachloride (CT) during soil vapor extraction (SVE) by comparing numerical and analytical model simulations with a detailed data set from a well-defined intermediate-scale flow cell experiment. The flow cell was packed with a fine-grained sand layer embedded in a coarse-grained sand matrix. A total of 499 mL CT was injected at the top of the flow cell and allowed to redistribute in the variably saturated system. A dual-energy gamma radiation system was used to determine the initial NAPL saturation profile in the fine-grained sand layer. Gas concentrations at the outlet of the flow cell and 15 sampling ports inside the flow cell were measured during subsequent CT removal using SVE. Results show that CT mass was removed quickly in coarse-grained sand, followed by a slow removal from the fine-grained sand layer. Consequently, effluent gas concentrations decreased quickly at first, and then started to decrease gradually, resulting in long-term tailing. The long-term tailing was mainly due to diffusion from the fine-grained sand layer to the coarse-grained sand zone. An analytical solution for a one-dimensional advection and a first-order mass transfer model matched the tailing well with two fitting parameters. Given detailed knowledge of the permeability field and initial CT distribution, we were also able to predict the effluent concentration tailing and gas concentration profiles at sampling ports using a numerical simulator assuming equilibrium CT evaporation. The numerical model predictions were accurate within the uncertainty of independently measured or literature derived parameters. This study demonstrates that proper numerical modeling of CT removal through SVE can be achieved using equilibrium evaporation of NAPL if detailed fine-scale knowledge of the CT distribution and physical heterogeneity is incorporated into the model. However, CT removal could also be fit by a first-order mass transfer analytical model, potentially leading to an erroneous conclusion that the long-term tailing in the experiment was kinetically controlled due to rate-limited NAPL evaporation.