Diagnostic Tools for Integrated In Situ Air Sparging Pilot Tests

In situ air sparging (IAS) pilot test procedures have been developed that provide rapid, on-site information about IAS performance. The standard pilot test consists of six activities conducted to look for indicators of infeasibility and to characterize the air distribution to the extent necessary to make design decisions about IAS well placement. In addition, safety hazards that need to be addressed prior to full-scale design are identified. Two additional pilot test activities are described in those cases where air distribution must be more precisely defined. The test activities include both chemical tests (tracking contaminant concentrations, dissolved oxygen and tracers) and physical tests (air flow rate and injection pressure, groundwater pressure response). Pilot test data from Eielson Air Force Base, Alaska illustrates implementation of the pilot test and interpretation of the data.     

Advances in In Situ Air Sparging/Biosparging

In situ air sparging (IAS) is a technology commonly used for treatment of submerged source zones and dissolved groundwater plumes. The acceptance of IAS by regulatory agencies, environmental consultants, and industry is remarkable considering the degree of skepticism initially surrounding the technology in the early 1990s. Much has been learned and reported in the literature since that time, but it appears that practice has changed little. In particular, conventional pilot testing, design, and operation practices reflect a lack of appreciation of the complex phenomena governing IAS performance and the unforgiving nature of this technology. Many systems are poorly monitored and likely to be inefficient or ineffective. Key lessons-learned since the early 1990s are reviewed and their implications for practice are discussed here. Of particular importance are issues related to: (a) the understanding of air flow distributions and the effects of geology and injection flowrate, (b) the need to characterize air flow distributions at the pilot- and field-scale, (c) how changes in operating conditions (e.g., pulsing) can affect performance improvements and reduce equipment costs, and (d) how conventional monitoring approaches are incapable of assessing if systems are performing as designed.     

A Multi-Tracer Push-Pull Diagnostic Test for In Situ Air Sparging Systems

This document describes the development and initial application of a multi-tracer push-pull test designed to provide near real-time point-specific measures of contaminant volatilization and aerobic biodegrada-tion rates during in situ air sparging (IAS) operation. Measured biodegradation and volatilization rates are specific to the tracers used, so the results provide relative measures useful for identifying spatial differences in treatment performance and changes in performance with changes in system operation and design. The diagnostic test involves injecting a solution containing multiple tracer compounds into the target treatment zone through a monitoring well, piezometer, or drive point. The injected solution is initially deoxygenated and can contain: (a) a nondegradable, non-volatile conservative tracer, (b) one or more nondegradable, volatile chemicals, (c) an aerobically biodegradable, nonvolatile compound, and (d) a visible dye. After some predetermined hold time, an excess quantity of groundwater is extracted from the same injection point and the change in the concentrations of the tracer compounds is measured. Volatilization and oxygen utilization rates are then estimated from mass balances on the tracer components. The development of this diagnostic tool was conducted in a controlled physical model study and then initial field tests were conducted at the U.S. Navy Hydrocarbon National Test Site (HNTS) in Port Hueneme, California. Spatial variations in oxygenation and volatilization rates were observed, with oxygenation rates varying from 0 to 51 mg-O₂/L-water/d, and tracer volatilization rates varying from 0 to 47%/d. Acetate and sulfur hexafluoride (SF₆) were used as tracers in the initial testing, and it was discovered that these are not ideal choices due to the potential for anaerobic acetate biodegradation and SF₆ partitioning into trapped gas in the aquifer.     

Field Validation of Helium as a Tracer Gas During Soil Vapor Sample Collection

This paper reports the results of a field study to evaluate the use of helium as a tracer gas during soil vapor sampling. The authors found that the helium tracer method recommended in regulatory guidance can detect atmospheric leakage. However, the degree of leakage can be underestimated due to: 1) losses of helium from the tracer reservoir during sampling; 2) the entry of atmospheric air from an area outside the tracer reservoir; 3) temporal variability of leakage; and 4) flow-related reduction in helium detector response. Frequent leakage was observed in soil vapor probes installed using conventional direct push techniques. Probes installed at a depth of 8 ft exhibited less leakage than those installed at 4 ft. Probes installed in glacial till exhibited greater and more frequent leakage than those installed in sandy loam. Based on the frequency of observed leakage, refinement and standardization of tracer methods, with routine incorporation of tracers and in-line pressure and flow monitoring, is recommended.     

Diagnosis of In Situ Air Sparging Performance Using Transient Groundwater Pressure Changes during Startup and Shutdown

Groundwater pressure measurements during startup and shutdown of in situ air sparging (IAS) systems are used to diagnose air flow behavior below the water table. The magnitude of the pressure response provides insight into the permeability of the zone into which the air is flowing. The duration of elevated pressures during startup and reduced pressures during shutdown indicate the extent to which air is being trapped below the water table by lower-permeability layers. The pressure measurements can be easily and quickly repeated and as a result are useful for both pilot tests and for optimizing operating conditions of existing IAS systems. Whether used alone or in conjunction with other diagnostic tools, pressure measurements are an important tool for assessing IAS performance.     

A Practical Approach for the Selection, Pilot Testing, Design, and Monitoring of In Situ Air Sparging/Biosparging Systems

The use of in situ air sparging (IAS) has increased rapidly since the early 1990s, and it is now likely to be the most practiced engineered in situ remediation option when targeting the treatment of hydrocarbon-impacted aquifers. To date, IAS system design has remained largely empirical, with significant variability in approaches and results. Here, the valuable knowledge gained from IAS studies and applications over the past decade has been integrated into a new paradigm for feasibility assessment, pilot testing, design, and operation. The basis for this Design Paradigm, the initial feasibility assessment, monitoring, and the overall design approach are discussed in detail here; other referenced documents contain the details of specific recommended activities. The proposed design approach is unique in that it contains two design routes; the first is a non-site-specific approach requiring minimal site characterization and testing (Standard Design Approach), while the second is a more site-specific approach (Site-Specific Design Approach).     

An Immunoblot Study of Neurofilament Degradation In Situ and During Calcium-Activated Proteolysis

The degradation of neurofilament (NF) proteins was examined by immunoblot methods to identify, characterize, and monitor the appearance of immunoreactive breakdown products during the loss of NF triplet proteins. Individual NF proteins and their breakdown products were identified using polyclonal and monoclonal antibodies to NF proteins. NF degradation was compared during calcium-activated proteolysis of isolated rat NF, during an experimental influx of calcium into excised rat spinal nerve roots, and during NF breakdown in transected rat peripheral nerve. These different experimental conditions produced similar patterns of NF fragmentation, including the transient appearance of NF immunobands between Mr 150,000-200,000 and 110,000-120,000 as well as the appearance and accumulation of NF immunobands between Mr 45,000 and 65,000. Most immunoreactive NF fragments remained Triton-insoluble. Low levels of the same immunoreactive fragments were present in control neural tissues, suggesting that calcium-activated proteolysis may be operative in the turnover and/or processing of NF proteins in vivo. Very similar patterns of NF degradation during experimental calcium influxes into different CNS and PNS tissues are indicative of the widespread distribution of calcium-activated NF protease in neural tissues.     

In Situ Speciation and Distribution of Toxic Selenium in Hydrated Roots of Cowpea

The speciation and spatial distribution of selenium (Se) in hydrated plant tissues is not well understood. Using synchrotron-based x-ray absorption spectroscopy and x-ray fluorescence microscopy (two-dimensional scanning [and associated mathematical model] and computed tomography), the speciation and distribution of toxic Se were examined within hydrated roots of cowpea (Vigna unguiculata) exposed to either 20 µm selenite or selenate. Based upon bulk solution concentrations, selenate was 9-fold more toxic to the roots than selenite, most likely due to increased accumulation of organoselenium (e.g. selenomethionine) in selenate-treated roots. Specifically, uptake of selenate (probably by sulfate transporters) occurred at a much higher rate than for selenite (apparently by both passive diffusion and phosphate transporters), with bulk root tissue Se concentrations approximately 18-fold higher in the selenate treatment. Although the proportion of Se converted to organic forms was higher for selenite (100%) than for selenate (26%), the absolute concentration of organoselenium was actually approximately 5-fold higher for selenate-treated roots. In addition, the longitudinal and radial distribution of Se in roots differed markedly: the highest tissue concentrations were in the endodermis and cortex approximately 4 mm or more behind the apex when exposed to selenate but in the meristem (approximately 1 mm from the apex) when exposed to selenite. The examination of the distribution and speciation of Se in hydrated roots provides valuable data in understanding Se uptake, transport, and toxicity.Selenium (Se) is an essential micronutrient for humans and other animals (Rayman, 2008). At elevated concentrations, however, it is toxic, and the concentration range from deficiency to lethality is unusually narrow (Terry et al., 2000). Plants represent a direct entrance to the wider food chain as the main sources of dietary Se (Rayman, 2008). The uptake and accumulation of Se by plants is an important process in controlling the health risks resulting from Se deficiency or toxicity. Se toxicity to plants has been observed in arid and semiarid soils derived from seleniferous rocks and shales, although anthropogenic contamination is also of concern (Terry et al., 2000). Therefore, it is important that the mechanisms of Se uptake, transformation, and toxicity in plants are understood in order to reduce health risks.Selenite (Se[IV]) and selenate (Se[VI]) are the two dominant inorganic species in soils depending upon the redox potential and pH (Elrashidi et al., 1987). The mechanism of Se[VI] uptake is well known: it is taken up by plant roots via the high-affinity sulfate transporters (Terry et al., 2000) due to the similarity between Se[VI] and sulfate. By contrast, little is known about the uptake mechanism involved in Se[IV] in plant roots. Some studies suggested that Se[IV] is taken up via passive diffusion (Shrift and Ulrich, 1969; Arvy, 1993). Recently, Zhao et al. (2010) reported that the uptake of Se[IV] is mediated by the silicon (Si) influx transporter Lsi1 (OsNIP2;1) in rice (Oryza sativa). Furthermore, Se[IV] uptake was found to occur via both passive diffusion and phosphate transporters in the marine coccolithophore Emiliania huxleyi (Araie et al., 2011). Apart from the difference in their mechanisms of uptake, they also differ in their mobility within plants (Li et al., 2008). Se[VI] is relatively easily translocated from roots to shoots, whereas Se[IV] tends to accumulate within the roots (Arvy, 1993). Despite this important progress, much less is known about the sites of uptake of Se[IV]/Se[VI] and their possible chemical transformations in hydrated plant roots. This information regarding the in situ distribution and chemical forms of Se would be helpful in elucidating the mechanism(s) responsible for Se uptake, transformation, and toxicity in plants.Recent advances in synchrotron-based techniques allow in situ measurement of the distribution of metal(loid)s in hydrated fresh plant tissues (Kopittke et al., 2011, 2012; Lombi et al., 2011a). In particular, the prototype Maia detector system, jointly developed by the Australian Synchrotron, the Commonwealth Scientific and Industrial Research Organization, and the Brookhaven National Laboratory, represents a new-generation x-ray fluorescence detector and real-time processing approach that provides unprecedented capabilities in in situ element imaging and measurement (Lombi et al., 2011b). The Maia uses an annular array of 384 silicon-diode detectors positioned in a backscatter geometry to subtend a large solid angle (approximately 1.3 steradian) and to achieve high count-rate capacity (Kirkham et al., 2010). Data acquisition times are approximately 10 to 100 times faster in the Maia than for other detectors, thereby allowing analysis of highly hydrated biological specimens (e.g. roots) without observable damage (Lombi et al., 2011a). This has allowed us to overcome the analytical challenges of examining the two-dimensional and virtual three-dimensional distribution of low-concentration metal(loid)s in hydrated and fresh plant tissues (Kopittke et al., 2011, 2012; Lombi et al., 2011a, 2011c).In this study, we investigated the speciation and quantified the longitudinal and radial distribution of Se in hydrated roots of cowpea (Vigna unguiculata) exposed to either Se[IV] or Se[VI]. Cowpea is a model species of rhizotoxicity and is also one of the most important food legume crops in the semiarid tropics, where Se toxicity is often a concern. The chemical forms of Se in cowpea roots were first examined using x-ray absorption spectroscopy (XAS). Second, with x-ray fluorescence microscopy (µ-XRF), we used two-dimensional imaging (coupled with an associated mathematical model to calculate concentrations of Se within various tissues of the root cylinder) to determine the spatially resolved distribution of Se within root tissues. Additionally, sequential tomography was used to provide virtual three-dimensional reconstructions of Se distribution in roots, enabling comparison of computed tomography with the mathematical model.     

Systematic Modeling Approach to Selective Plugging Using In Situ Bacterial Biopolymer Production and Its Potential for Microbial-enhanced Oil Recovery

This study presents a systematic modeling approach for examining the efficiency of the MEOR process based on in situ selective plugging by bacterial biopolymer production and optimization of the nutrient injection strategy to yield the maximum oil recovery. This study focuses on modeling in situ selective plugging by the bacterial biopolymer dextran that is generated by Leuconostoc mesenteroides. Bacterial growth and dextran generation were described by a stoichiometric equation and kinetic reactions using batch model simulation. Based on the parameters for permeability reduction obtained from the sandpack model, the MEOR process was implemented in a pilot-scale system that included a highly permeable thief zone in a low-permeability reservoir. The base MEOR design yielded a 61.5% improvement of the recovery factor compared to that obtained with waterflooding. The parametric simulations revealed that the recovery efficiency was influenced by the amount of dextran, as well as the distribution of dextran, and thus, the injection strategy is critical for controlling the dextran distribution. By incorporating the results from the sensitivity analysis and optimization to determine the optimal design parameters, a 36.7% improvement of the oil recovery was achieved with the optimized MEOR process in comparison with the base case.     

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background

Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption.

Methodology/Principal Findings

In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues.

Conclusions/Significance

These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.     

Structural Evolution During Perovskite Crystal Formation and Degradation: In Situ and Operando X‐Ray Diffraction Studies

This review provides an update on the progress in understanding formation and degradation mechanisms in halide perovskites for photovoltaic applications, as supported by in situ and operando X‐ray scattering techniques. The value of these real‐time analyses is particularly high for gaining insights into the structural evolution during crystal formation and decomposition upon exposure to external stress factors. This type of analysis reveals the pathways between starting and end points of a process rather than being limited to comparing states before and after the process. Special attention is put on the successful efforts toward upscaling including deposition techniques that are compatible to roll‐to‐roll processing. These processes are realized using fast annealing procedures. The development of these processes strongly benefited from in situ studies exploring the direct transition from precursor to perovskite without going through observable crystalline intermediate phases. A particular focus of this review is the benefit of using in situ and operando X‐ray scattering techniques to better understand and ultimately improve device stability. The difference between structural stability of thin films and structural stability under device operation is highlighted, convincingly demonstrating the indispensability of operando studies.     

Vertical Distribution of Archaea and Bacteria in a Meromictic Lake as Determined by Fluorescent In Situ Hybridization

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A “red-water” layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem.     

In Situ Prebiotics for Weaning Piglets: In Vitro Production and Fermentation of Potato Galacto-Rhamnogalacturonan

Postweaning diarrhea (PWD) in pigs is a leading cause of economic loss in pork production worldwide. The current practice of using antibiotics and zinc to treat PWD is unsustainable due to the potential of antibiotic resistance and ecological disturbance, and novel methods are required. In this study, an in vitro model was used to test the possibility of producing prebiotic fiber in situ in the gastrointestinal (GI) tract of the piglet and the prebiotic activity of the resulting fiber in the terminal ileum. Soluble fiber was successfully produced from potato pulp, an industrial waste product, with the minimal enzyme dose in a simulated upper GI tract model extracting 26.9% of the initial dry matter. The fiber was rich in galactose and galacturonic acid and was fermented at 2.5, 5, or 10 g/liter in a glucose-free medium inoculated with the gut contents of piglet terminal ileum. Fermentations of 5 g/liter inulin or 5 g/liter of a purified potato fiber were used as controls. The fibers showed high fermentability, evident by a dose-dependent drop in pH and an increase in the organic acid content, with lactate in particular being increased. Deep sequencing showed a significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fiber being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. For animal studies, a dosage corresponding to the 5-g/liter treatment is suggested.     

Improved Flotation Technique for Microscopy of In Situ Soil and Sediment Microorganisms

An improved flotation method for microscopy of in situ soil and sediment microorganisms was developed. Microbial cells were released into gellike flotation films that were stripped from soil and sediment aggregates as these aggregates were submerged in 0.5% solutions of polyvinylpyrrolidone. The use of polyvinylpyrrolidone solutions instead of water facilitated the release of films from saturated samples such as aquifer sediments as well as from typical surface soils. In situ microbial morphological characteristics could then be surveyed rapidly by light microscopy of films stained with acridine orange. This method effectively determined the ranges of morphological diversity in a variety of sample types. It also detected microcolonies and other spatial relationships among microbial cells. Only a small fraction (3.4 to 10.1%) of the microflora was released into the flotation films, but plating and direct evaluations by microscopy showed that this fraction was representative of the total population.     

Multi-Scale Imaging and Informatics Pipeline for In Situ Pluripotent Stem Cell Analysis

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured in vitro exhibit a high degree of heterogeneity, presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures, necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool''s utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.     

In Situ Optical Extinction Measurement for Locally Control of Surface Plasmon Resonance During Nanosecond Laser Irradiation of Silver Ion Exchanged Silicate Glass

Online UV/visible extinction measurement have been achieved during nanosecond Nd:YAG laser irradiation at 532 nm of a silver-exchanged silicate glass after each shot. We have explained the evolution of the integrated spectral evolution with the help of a few observed spots after the laser/glass interaction and completed them by optical and surface measurements. This optical method allows to in situ follow silver ions precipitation in nanoparticles (NPs) and the consequently surface plasmon resonance evolution (SPR). In this study, we focus on the interest of this method for one silver-exchanged soda-lime glass by direct observation of the sample surface. Scanning electron microscopy measurement and optical microscopy were used to identify the various ablation mechanisms. Profilometry was used to evaluate the material distribution and the surface roughness evolution (Rms parameter). Thus, we explain the silver NPs effect on glass matrix at various micrometric scales according to the deposited fluence and silver concentration.     

Distribution and Evolution of Repeated Sequences in Genomes of Triatominae (Hemiptera-Reduviidae) Inferred from Genomic In Situ Hybridization

The subfamily Triatominae, vectors of Chagas disease, comprises 140 species characterized by a highly homogeneous chromosome number. We analyzed the chromosomal distribution and evolution of repeated sequences in Triatominae genomes by Genomic in situ Hybridization using Triatoma delpontei and Triatoma infestans genomic DNAs as probes. Hybridizations were performed on their own chromosomes and on nine species included in six genera from the two main tribes: Triatomini and Rhodniini. Genomic probes clearly generate two different hybridization patterns, dispersed or accumulated in specific regions or chromosomes. The three used probes generate the same hybridization pattern in each species. However, these patterns are species-specific. In closely related species, the probes strongly hybridized in the autosomal heterochromatic regions, resembling C-banding and DAPI patterns. However, in more distant species these co-localizations are not observed. The heterochromatic Y chromosome is constituted by highly repeated sequences, which is conserved among 10 species of Triatomini tribe suggesting be an ancestral character for this group. However, the Y chromosome in Rhodniini tribe is markedly different, supporting the early evolutionary dichotomy between both tribes. In some species, sex chromosomes and autosomes shared repeated sequences, suggesting meiotic chromatin exchanges among these heterologous chromosomes. Our GISH analyses enabled us to acquire not only reliable information about autosomal repeated sequences distribution but also an insight into sex chromosome evolution in Triatominae. Furthermore, the differentiation obtained by GISH might be a valuable marker to establish phylogenetic relationships and to test the controversial origin of the Triatominae subfamily.