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Negligible effect of X-ray μ-CT scanning on archaea and bacteria in an agricultural soil
... Variable results have been obtained for the desirable effects of X-rays on normal flora ranging from effective [9,10] to ineffective. [11] Depending on dose, X-ray radiation with low-to-medium energy has shown no effect on some types of bacteria, [5] while the exposure of other bacteria to the same dose of X-rays revealed a significant decrease in cell number. [10] Viability of oral MOs in patients exposed to different techniques producing of X-radiation was investigated. ...
... Sensitivity of MOs toward X-rays is variable depending on many factors such as type of microbes, density of cells, nature of the environment, dose rate, and type of radiation. [4,5,11,17] Previously, several studies demonstrated that the viability of bacteria was not affected by low-to-medium energy of X-rays and also there was no mutation effect on these exposed bacteria. [5,6,11] On the other hand, X-rays were found to act as sterilizing agents upon a culture of Balantidium coli and Erythrobacillus prodigiosus [9] or even on soil MOs. ...
... [4,5,11,17] Previously, several studies demonstrated that the viability of bacteria was not affected by low-to-medium energy of X-rays and also there was no mutation effect on these exposed bacteria. [5,6,11] On the other hand, X-rays were found to act as sterilizing agents upon a culture of Balantidium coli and Erythrobacillus prodigiosus [9] or even on soil MOs. [10] In the present study, most bacteria were induced to increase their cell number after exposure to X-rays, which means that ionization energy of X-rays is an effective stimulating factor to increase the multiplication rate of bacteria after a single exposure with unknown mechanism. ...
... Unlike nuclear magnetic resonance spectroscopy, XRCT does not suffer from interference by paramagnetic ions present in the soil (Rogers and Bottomley, 1987;Metzner et al., 2015). By performing multiple scans during plant development the age, length, and density of the plant roots, and their relations with soil structure can be visualized (Bouckaert et al., 2013;Mairhofer et al., 2013;Schmidt et al., 2015;Couradeau et al., 2018;Schlüter et al., 2018). ...
... When plant-soil systems are subjected to XRCT, the radiation may affect not only the plant but also associated microorganisms, either directly or indirectly. Soil microbiome responses to XRCT have been studied at the level of microbial biomass and community structure, by phospholipid fatty acid analysis and denaturing gradient gel electrophoresis fingerprints of 16S rRNA-gene fragments amplified from total community DNA (McNamara et al., 2003;Bouckaert et al., 2013;Fischer et al., 2013;Zappala et al., 2013;Schmidt et al., 2015). With a low dose of 2.4 Gy, no effects on microbial respiration, concentration of microbial carbon, and bacterial community composition were detected in soil microcosms (Schmidt et al., 2015). ...
... Soil microbiome responses to XRCT have been studied at the level of microbial biomass and community structure, by phospholipid fatty acid analysis and denaturing gradient gel electrophoresis fingerprints of 16S rRNA-gene fragments amplified from total community DNA (McNamara et al., 2003;Bouckaert et al., 2013;Fischer et al., 2013;Zappala et al., 2013;Schmidt et al., 2015). With a low dose of 2.4 Gy, no effects on microbial respiration, concentration of microbial carbon, and bacterial community composition were detected in soil microcosms (Schmidt et al., 2015). X-ray doses up to 23 Gy also did not significantly affect the soil microbial biomass . ...
Non-invasive X-ray computed tomography (XRCT) is increasingly used in rhizosphere research to visualize development of soil-root interfaces in situ. However, exposing living systems to X-rays can potentially impact their processes and metabolites. In order to evaluate these effects, we assessed the responses of rhizosphere processes 1 h and 24 h after a low X-ray exposure (0.81 Gy). Changes in root gene expression patterns occurred 1 h after exposure with down-regulation of cell wall-, lipid metabolism-, and cell stress-related genes, but no differences remained after 24 h. At either time point, XRCT did not affect either root antioxidative enzyme activities, or the composition of rhizosphere bacterial microbiome and microbial growth parameters. The potential activities of leucine aminopeptidase and phosphomonoesterase were lower at 1 h, but did not differ from the control 24 h after exposure. A time delay of 24 h after a low X-ray exposure (0.81 Gy) was sufficient to revert any effects on the observed rhizosphere systems. Our data suggest that before implementing novel experimental designs involving XRCT, a study on its impacts on the investigated processes should be conducted.
... Variable results have been obtained for the desirable effects of X-rays on normal flora ranging from effective [9,10] to ineffective. [11] Depending on dose, X-ray radiation with low-to-medium energy has shown no effect on some types of bacteria, [5] while the exposure of other bacteria to the same dose of X-rays revealed a significant decrease in cell number. [10] Viability of oral MOs in patients exposed to different techniques producing of X-radiation was investigated. ...
... Sensitivity of MOs toward X-rays is variable depending on many factors such as type of microbes, density of cells, nature of the environment, dose rate, and type of radiation. [4,5,11,17] Previously, several studies demonstrated that the viability of bacteria was not affected by low-to-medium energy of X-rays and also there was no mutation effect on these exposed bacteria. [5,6,11] On the other hand, X-rays were found to act as sterilizing agents upon a culture of Balantidium coli and Erythrobacillus prodigiosus [9] or even on soil MOs. ...
... [4,5,11,17] Previously, several studies demonstrated that the viability of bacteria was not affected by low-to-medium energy of X-rays and also there was no mutation effect on these exposed bacteria. [5,6,11] On the other hand, X-rays were found to act as sterilizing agents upon a culture of Balantidium coli and Erythrobacillus prodigiosus [9] or even on soil MOs. [10] In the present study, most bacteria were induced to increase their cell number after exposure to X-rays, which means that ionization energy of X-rays is an effective stimulating factor to increase the multiplication rate of bacteria after a single exposure with unknown mechanism. ...
Introduction: X‑ray is the most valuable tool for diagnosis of various diseases. Its radiation energy has a
serious effect on living cells. The effects of X‑radiography and computed tomography (CT) scans on the
viability of oral microflora in the human were investigated.
Methods: A total of 432 patients in two groups exposed to X‑radiation were included in a cohort study.
Group I (215) was exposed to X‑radiography and Group II (217) to CT scan. Swab samples from the oral
cavity were collected. Viability of normal oral flora in those patients was measured by microbial counting
before and after exposure.
Results: Radiation of X‑ray techniques, especially for CT scan, showed an effect on most of bacterial
multiplication by increasing their count after exposure. Meanwhile, fungal isolates and one of the
bacteria (Staphylococcus aureus) were decreased in number after exposure. Some isolates were not affected
by radiation from X‑radiography.
Conclusion: Radiation of routinely diagnostic X‑ray found to play an important role in disturbance of microbial
counting balance among oral flora through increasing the density of most kinds of them. Limitation of
X‑ray exposure is a safety precaution that should be taken to prevent adverse effects on normal flora of
the human body.
... In addition, there was no effect on cumulative Cmin after 128 days for any of the treatments (data not shown). So, in accordance to Bouckaert et al. (2013b) and Schmidt et al. (2015) we conclude an at most minor and acceptable µCT-scanning effect on the microbial community's structure and activity. ...
... Furthermore, PLFA analysis demonstrated that the microbial community structure was not significantly affected by the applied dose of X-rays (O5). These results indicate that X-ray µCT scanning did not significantly affect the soil biota and confirms the findings of Bouckaert et al. (2013b) and Schmidt et al. (2015), who applied X-ray doses of 7.5 Gy and 2.4 Gy, respectively, which are only a small fraction of the radiation that can be tolerated by bacteria and fungi (about 5 kGy) (McNamara et al., 2003). ...
... Recently, new frontiers of pore-scale RTMs are emerging with the advances in computational power as well as with huge improvements in imaging techniques. The latter includes, for example, the static and dynamic scanning of porous structure as well as of fluids' distribution (Schlüter et al., 2019) or the detection of bacterial distributions in soil using the catalysed reporter deposition with fluorescence in situ hybridization (CARD-FISH) technique (Schmidt et al., 2015). Direct numerical models (DNMs) are becoming the nexus of the next generation of RTMs as they represent the porous structure in a fully explicit manner (directly obtained from soil samples, digitized and fed into RTMs) in addition to offering a more flexible coupling of different components of reactive transport models (Baveye et al., 2018;Raeini et al., 2012;Li et al., 2010;Yan et al., 2016). ...
The porous microenvironment of soil offers various environmental functions which are governed by physical and reactive processes. Understanding reactive transport processes in porous media is essential for many natural systems (soils, aquifers, aquatic sediments or subsurface reservoirs) or technological processes (water treatment or ceramic and fuel cell technologies). In particular, in the vadose zone of the terrestrial subsurface the spatially and temporally varying saturation of the aqueous and the gas phase leads to systems that involve complex flow and transport processes as well as reactive transformations of chemical compounds in the porous material. To describe these interacting processes and their dynamics at the pore scale requires a well-suited modelling framework accounting for the proper description of all relevant processes at a high spatial resolution. Here we present P3D-BRNS as a new open-source modelling toolbox harnessing the core libraries of OpenFOAM and coupled externally to the Biogeochemical Reaction Network Simulator (BRNS). The native OpenFOAM volume-of-fluid solver is extended to have an improved representation of the fluid–fluid interface. The solvers are further developed to couple the reaction module which can be tailored for a specific reactive transport simulation. P3D-RBNS is benchmarked against three different flow and reactive transport processes: (1) fluid–fluid configuration in a capillary corner, (2) mass transfer across the fluid–fluid interface and (3) microbial growth with a high degree of accuracy. Our model allows for simulation of the spatio-temporal distribution of all biochemical species in the porous structure (obtained from μ-CT images), for conditions that are commonly found in the laboratory and environmental systems. With our coupled computational model, we provide a reliable and efficient tool for simulating multiphase, reactive transport in porous media.
... These methods should therefore be im- multiple observations of the same object without such concerns. Moreover, X-ray computed tomography has negligible effects on root growth (Zappala et al., 2013), archaea and bacteria and their functioning in soil (Bouckaert et al., 2013;Schmidt et al., 2015). ...
The soil microhabitat is a heterogeneous and complex environment where local variations can modulate phenomena observed at the plot scale. Most of the current methods used to describe soil functioning are bulk soil analyses which do not account for fine-scale spatial variability and cannot fully account for the processes that occur under the influence of the 3D organisation of soil. A good representation of spatial heterogeneities is necessary for the parametrisation of new models, which aim to represent pore-scale processes that affect microbial activity. The visualization of soil at the scale of the microhabitat can be used to extract descriptors and reveal the nature of the relationships between the fine-scale organisation of soil's constituent parts and soil functioning.
However, soil imaging techniques tend to be under-used, possibly due to a lack of awareness of the methods or due to a lack of access to the relevant instruments. In recent years, new methods have been developed, and continuously improved, offering new possibilities to decipher and describe soil physical, chemical and biological features of the soil microhabitat in evermore exquisite detail.
This review is structured into several sections in which we consider first imaging methods that are useful for describing the distribution of microorganisms and identify them, second the methods for characterising the physical organisation and the chemical attributes of the microhabitat, including soil organic matter and, finally, methods for visualising in situ information on the activities of microorganisms are described. Special attention is given to the preparation steps that are required for the proper use of the methods, either alone or in combination.
... A fundamental problem of radiation-based imaging is that it is ionising and penetrative, meaning that it could possibly damage both structures and microorganisms within the substrate [164]. On the other hand, low irradiation doses applied to plant-soil interfaces were reported to neither influence root growth [169], nor microbial cell number, microbial community structure, or their potential activity [165,170]. Nevertheless, even if not lethal, care should be taken for the insurgence of mutations with unpredictable consequences. Lastly, the imaging resolution chosen can limit either the range or the feature of detection [111]. ...
Due to their small size, microorganisms directly experience only a tiny portion of the environmental heterogeneity manifested in the soil. The microscale variations in soil properties constrain the distribution of fungi and bacteria, and the extent to which they can interact with each other, thereby directly influencing their behavior and ecological roles. Thus, to obtain a realistic understanding of bacterial–fungal interactions, the spatiotemporal complexity of their microenvironments must be accounted for. The objective of this review is to further raise awareness of this important aspect and to discuss an overview of possible methodologies, some of easier applicability than others, that can be implemented in the experimental design in this field of research. The experimental design can be rationalized in three different scales, namely reconstructing the physicochemical complexity of the soil matrix, identifying and locating fungi and bacteria to depict their physical interactions, and, lastly, analyzing their molecular environment to describe their activity. In the long term, only relevant experimental data at the cell-to-cell level can provide the base for any solid theory or model that may serve for accurate functional prediction at the ecosystem level. The way to this level of application is still long, but we should all start small.
... One consideration to be mindful of, however, with regard to pre-and post-decay scanning of the same individuals is the potential effect of X-radiation on the gut and other organism-associated microbiota, which may contribute to decay of the organism. Turning to the soil literature for insights on this concern yields contradictory results: several studies suggest negligible effects of X- T. SELLY AND J.D. SCHIFFBAUER 222 P A L A I O S radiation on lCT-scanned soil microbes (Bouckaert et al. 2013;Zappala et al. 2013;Schmidt et al. 2015), whereas another revealed a much more detrimental effect in microbial biomass and community structure (Fischer et al. 2013). While high-dose X-radiation indeed has been shown as a viable mechanism for food decontamination (Mahmoud 2012), for example, such doses are substantially greater than those imparted during lCT scanning. ...
Laboratory-based decay experiments have become commonly used to supplement our understanding of how organisms enter the fossil record. Differences in how these experiments are designed and evaluated, however, including dissimilarities in qualitative decay-scoring indices superimposed on variability in model organisms, renders any semblance of comparison between studies unreliable. Here, we introduce the utility of X-ray tomographic microscopy (μCT) as a means for reliable and repeatable analysis of soft-tissue decay experiment products. As proof-of-concept, we used a relatively simple experimental design with classic studies as comparators, and present our analytical protocol using μCT for capturing the entire volume of the decay subject. Segmentation software then allows for 3D volume analysis and high-resolution internal and external character identification. We describe the workflow from sample preparation, contrast-staining, and data collection to processing and analysis of the resulting data, using peppermint shrimp (Lysmata wurdemanni) as model organisms, and compare our results to previous taphonomic studies. These methods allow for improved visualization and quantification of decay and internal volume analysis with minimal handling as compared to traditional qualitative scoring methods. Using the same scoring criteria as previous studies, this study revealed similar decay results for certain features, while we were additionally able to detect other feature loss or alteration earlier—importantly without need for potentially distortive sample handling. We conclude that μCT is a more effective, straightforward, and exact means for extracting quantitative data on the progression of decay and should be adopted in future studies, where available, to streamline and standardize comparisons.
... While X-ray doses of typical industrial CT scanners have hardly any effect on physical and chemical properties of soil and water (Weon et al., 2008), studies showed the potential negative effect on living organisms. The effect of X-ray irradiation on microbial activity seems to be minor (Bouckaert et al., 2013;Schmidt et al., 2015;Ganther et al., 2020). In contrast, the impact of X-ray irradiation on higher organisms, such as plants, is more apparent (Johnson, 1936;Evans, 1965). ...
Radiation damage to plants through X-ray exposure has been reported to impair root growth. The literature on the critical dose for growth impairment is inconclusive, partly as dose measurements in soil are scarce. Here we fill this gap and show that the dose in a typical single pot scan amounts to 1.2 Gy. In addition, we demonstrate the shortcomings of estimating the dose from scan settings using the RadPro Calculator and highlight the efficient reduction of X-ray exposure by a lead shield. © 2021 The Authors. Journal of Plant Nutrition and Soil Science published by Wiley-VCH GmbH
... In particular, measurement of root exudation requires intact root systems (Oburger and Jones, 2018). For biological parameters, potential artifacts like irradiation damage caused by Xray CT scanning has to be quantified or ruled out prior the establishment of the workflow (Schmidt et al., 2015;Ganther et al., 2020). ...
The numerous feedback loops between roots, microorganisms, soil chemical and physical properties, and environmental variables result in spatial parameter patterns which are highly dynamic in time. In order to improve our understanding of the related rhizosphere processes and their relevance at the soil–plant system scale, experimental platforms are required. Those platforms should enable (1) to relate small scale observations (nm to dm) to system behaviour, (2) the integration of physical, chemical and biological sampling approaches within the same experiment, and (3) sampling at different time points during the life cycle of the system in question. Here we describe what requirements have to be met and to what extent this has been achieved in practice by the experimental platforms which were set up within the framework of DFG priority programme 2089 “Rhizosphere Spatiotemporal Organisation—a key to rhizosphere functions”. It is discussed to what extent theoretical considerations could be accommodated, in particular for the comparison across scales, i.e., from laboratory to field scale. The latter scale is of utmost importance to overcome the trade‐off between fraction of life cycle covered and the avoidance of unrealistic root length densities.
... While cells overlaid by minerals and MPs were not detectable and did not affect mineral and MP signals, cells laying onto mineral and MP surfaces fluoresced at intermediate wavelengths; thus, this may be another explanation for the pixels falling between the two confidence limits. Overlaying can be quantified using the microtome technique [155], micro-computed tomography scanning [228,268,297], and focused ion beam nanotomography [289]; however, aggregates have to be fixed in a solid matrix for sampling and may experience substantial perturbation during sample preparation, and thus, coupling these techniques to the OMCEC method requires further consideration. ...
Transport of suspended particulate matter (SPM) plays a vital role in controlling large-scale processes related to geophysical flows such as dispersal and sinking of organic matter and contaminants to offshore and deep waters, nutrient cycles, food web stability, morphodynamics and sedimentation in both limnetic and pelagic ecosystems. Although it has been recognized that small-scale microbial processes can introduce substantial differences to the way in which SPM moves in natural waters, the extent to which the attached biological matter affects SPM dynamics is still not well characterized. This thesis focuses on quantifying the attached biomass fraction on SPM aggregates and investigating its contribution to SPM flocculation dynamics, which consequently control SPM aggregate geometrical properties and transport. A novel laboratory-based Optical Measurement of Cell Colonization (OMCEC) system and a microbiological-physical model (BFLOC2) are the main achievements of this thesis that allow the analyses of the correlations between environmental conditions, aggregate-attached biomass fraction, cell colonization patterns, aggregate size, fractal dimension and settling velocity.
OMCEC is an experimental system that can simultaneously measure the material composition, geometric properties, and motion of individual suspended aggregates in a non-invasive and non-destructive way. OMCEC consists of a full-color high-resolution optical system and real-time algorithms for (i) material segmentation based on light spectra emission analysis, (ii) quantification of various geometrical properties, and (iii) motion detection with micro particle tracking velocimetry (μPTV). OMCEC was applied herein on three types of aggregates: cell-associated minerals, cell-associated microplastics, and three-phase aggregates made of minerals, microplastics, and biological matter.
OMCEC application on Saccharomyces cerevisiae-colonized minerals at four sucrose concentrations showed the likelihood of cell colonization to increase with increasing nutrient concentration. The attached biomass fraction was found to increase nonlinearly regarding an increase of aggregate size but almost constant with fractal dimension variation. Cell distribution on mineral surfaces was then analyzed and classified into three colonization patterns: (i) scattered, (ii) well-touched, and (iii) poorly-touched, with the second being predominant. Cell clusters in the well-touched pattern were found to have lower fractal dimension than those in the other patterns. A strong correlation of colonization patterns with aggregate biomass fraction and properties suggests dynamic colonization mechanisms from cell attachment to minerals, to joining of isolated cell clusters, and finally cell growth over the entire aggregate.
OMCEC application on microplastics (MPs) being colonized by natural biological matter from Hawkesbury River, NSW, Australia demonstrated that the biomass fraction of MP aggregates has substantial control over their size, shape and, most importantly, their settling velocity. Polyurethane MP aggregates made of 80% biological matter had an average size almost double that of MP aggregates containing 5% biological matter and sank two times slower. Based on our experimental data, we introduce a settling velocity equation that accounts for the shape irregularity and fractal structure of MP aggregates. This equation can capture the settling velocity of both virgin MPs and cell-associated MP aggregates with 7% error and can be applied widely to predict the settling flux of MP aggregates made of different polymers and various types of biological matter.
To consider the complex genesis of cell-associated mineral aggregates, the BFLOC2 model was introduced to predict aggregate geometry and settling velocity under simultaneous effects of hydrodynamic and biological processes. While minerals can contribute to aggregate dynamics through collision, aggregation, and breakup, living microorganisms can colonize and establish food web interactions that involve growth and grazing, and modify the aggregate structure. Modeling of cell-associated mineral aggregate dynamics over a wide range of environmental conditions showed that maximum aggregate size, biomass fraction, and settling velocity could occur at different optimal environmental conditions. Unlike mineral aggregates, which have maximum size when shear rates tend to zero, a relative maximum size of cell-associated mineral aggregates can be reached at intermediate shear rates as a result of microbiological processes. The settling velocity was ultimately controlled by aggregate size, fractal dimension, and biomass fraction.
The innovative aspect of this thesis is the simultaneous quantification of composition, architecture, and settling velocity of individual aggregates. Therefore, it puts forth the analysis and prediction of cell colonization impacts on dynamics and transport of suspended particulate matter in natural waters. The output of this thesis can be used in natural water monitoring programs to estimate the biological content based on SPM size, capacity dimension, and settling velocity, which can be measured using in-situ methods. Furthermore, the evidence and tools to quantify the sinking and floating of microplastic subjected to bio-fouling can be implemented in microplastics transport models to enable the three-dimension modeling of both low- and high-density microplastics. The BFLOC2 model can be coupled to traditional sediment transport models to better describe the sediment formation dynamics, thus giving a more precise prediction of sedimentation and carbon flux to deep waters and offshore.
... Kravchenko et al. (2014a) successfully used geostatistical parameters of POM fragments as indicators of the presence of POM in intact soil samples. This approach has advantages over POM identification via Os staining, since, unlike Os staining, CT scanning has minimal effect on soil microorganisms (Bouckaert et al., 2013b;Kravchenko et al., 2014b;Schmidt et al., 2015). Thus, the samples can be used for exploring the decomposition of the identified POM fragments in a sequence of initial CT scanning, incubation, and post-incubation CT scanning activities, as done by Kravchenko et al. (2015). ...
Over the last 60 years, soil microbiologists have accumulated a wealth of experimental data showing that the bulk, macroscopic parameters (e.g., granulometry, pH, soil organic matter, and biomass contents) commonly used to characterize soils provide insufficient information to describe quantitatively the activity of soil microorganisms and some of its outcomes, like the emission of greenhouse gasses. Clearly, new, more appropriate macroscopic parameters are needed, which reflect better the spatial heterogeneity of soils at the microscale (i.e., the pore scale) that is commensurate with the habitat of many microorganisms. For a long time, spectroscopic and microscopic tools were lacking to quantify processes at that scale, but major technological advances over the last 15 years have made suitable equipment available to researchers. In this context, the objective of the present article is to review progress achieved to date in the significant research program that has ensued. This program can be rationalized as a sequence of steps, namely the quantification and modeling of the physical-, (bio)chemical-, and microbiological properties of soils, the integration of these different perspectives into a unified theory, its upscaling to the macroscopic scale, and, eventually, the development of new approaches to measure macroscopic soil characteristics. At this stage, significant progress has been achieved on the physical front, and to a lesser extent on the (bio)chemical one as well, both in terms of experiments and modeling. With regard to the microbial aspects, although a lot of work has been devoted to the modeling of bacterial and fungal activity in soils at the pore scale, the appropriateness of model assumptions cannot be readily assessed because of the scarcity of relevant experimental data. For significant progress to be made, it is crucial to make sure that research on the microbial components of soil systems does not keep lagging behind the work on the physical and (bio)chemical characteristics. Concerning the subsequent steps in the program, very little integration of the various disciplinary perspectives has occurred so far, and, as a result, researchers have not yet been able to tackle the scaling up to the macroscopic level. Many challenges, some of them daunting, remain on the path ahead. Fortunately, a number of these challenges may be resolved by brand new measuring equipment that will become commercially available in the very near future.
Introduction
... In addition, there was no effect on cumulative Cmin after 128 days for any of the treatments (data not shown). So, in accordance to Bouckaert et al. (2013) and Schmidt et al. (2015) we conclude an at most minor and acceptable µCT-scanning effect on the microbial community's structure and activity. ...
Mineral nitrogen (N) availability to heterotrophic micro-organisms is known to impact organic matter (OM) decomposition. Different pathways determining the N accessibility depend to a large extent on soil structure. Contact between soil mineral and OM substrate particles can facilitate N transport toward decomposition hot spots. However, the impact of soil structure on N availability to microbes and thus heterotrophic microbial activity and community structure is not yet fully understood. We hypothesized that carbon mineralization (Cmin) from low-N substrate would be stimulated by increased N availability caused by closer contact with soil particles or by a higher moisture level, enhancing potential for N-diffusion. Under opposite conditions retarded heterotrophic activity and a dominance of fungi were expected. A 128-days incubation experiment with CO2 emission monitoring from artificially reconstructed miniature soil cores with contrasting soil structures, viz. high or low degree of contact between soil particles, was conducted to study impacts on heterotrophic activity. The soil cores were subjected to different water filled pore space percentages (25 or 50% WFPS) and amended with either easily degradable OM high in N (grass) or more resistant OM low in N (sawdust). X-ray μCT image processing allowed to quantify the pore space in 350 μm around OM substrates, i.e., the microbial habitat of involved decomposers. A lower local porosity surrounding sawdust particles in soils with stonger contact was confirmed, at least at 25% WFPS. Mineral N addition to sawdust amended soils with small particle contact at 25% WFPS resulted in a stimulated respiration. Cmin in the latter soils was lower than in case of high particle contact. This was not observed for grass substrate particles or at 50% WFPS. The interactive effect of substrate type and soil structure suggests that the latter controls Cmin through mediation of N diffusion and in turn N availability. Phospholipid fatty acid did not reveal promotion of fungal over bacterial biomarkers in treatments with N-limited substrate decomposition. Combining X-ray μCT with tailoring soil structure allows for more reliable investigation of effects on the soil microbial community, because as also found here, the established soil pore network structure can strongly deviate from the intended one.
... Another point to take into account when considering applying this technique to microbial ecology in general is that even though nominally non destructive, the use of hard X-rays may damage the sample (in particular its biological component). It was shown for instance that it can induce death of large population of selected groups of organisms directly after the scan (Fischer et al., 2013) or have negligible effect depending the dose of X-ray and scan duration (Schmidt et al., 2015). In our case it prevented the cyanobacterium to migrate to the biocrust surface upon rewetting after the experiment (data not shown), which they invariably do when healthy (Pringault and Garcia-Pichel, 2004) suggesting that the imaging process may have impaired their physiology. ...
Biological soil crusts (biocrusts) are millimeter-sized microbial communities developing on the topsoils of arid lands that cover some 12% of Earth's continental area. Biocrusts consist of an assemblage of mineral soil particles consolidated into a crust by microbial organic polymeric substances that are mainly produced by filamentous bundle-forming cyanobacteria, among which Microcoleus vaginatus is perhaps the most widespread. This cyanobacterium is the primary producer for, and main architect of biocrusts in many arid soils, sustaining the development of a diverse microbial community. Biocrusts are only active when wet, and spend most of their time in a state of desiccated quiescence, from which they can quickly recover upon wetting. Despite their ecological importance for arid ecosystems, little is known about the mechanisms that allow biocrust organisms to endure long periods of dryness while remaining viable for rapid resuscitation upon wetting. We had previously observed the persistence of significant rates of light-dependent carbon fixation in apparently dry biocrusts dominated by M. vaginatus, indicating that it may be able to remain hydrated against a background soil of very low water potential. One potential explanation for this may be that the abundant exopolysaccharide sheaths of M. vaginatus can preferentially retain moisture thus slowing the water equilibration with the surrounding soil allowing for extended activity periods. In order to test this hypothesis we aimed to develop methodologies to visualize and quantify the water dynamics within an undisturbed biocrust undergoing desiccation. We used synchrotron based X-ray microtomography and were able to resolve the distribution of air, liquid water, mineral particles and cyanobacterial bundles at the microscale. We could demonstrate the formation of steep, decreasing gradients of water content from the cyanobacterial bundle surface outward, while the bundle volume remained stable, as the local bulk water content decreased linearly, hence demonstrating a preferential retention of water in the microbes. Our data also suggest a transfer of hydration water from the EPS sheath material into the cyanobacterial filament as desiccation progresses. This work demonstrates the value of X-ray tomography as a tool to study microbe-scale water redistribution in biocrusts.
... While cells overlaid by minerals were not detectable and did not affect the mineral fluorescence, cells laying onto mineral surfaces fluoresced at wavelengths between those of stained minerals and cells; thus, this may be another explanation for the pixels falling between the two confidence limits. Overlaying can be quantified using the microtome technique (e.g., Li & Ganczarczyk, 1990), micro-computed tomography scanning (e.g., Schmidt et al., 2015;Tisato et al., 2015), and focused ion beam nanotomography (Wheatland, Bushby, & Spencer, 2017); however, aggregates have to be fixed in a solid matrix for sampling and may experience substantial perturbation during sample preparation, and thus, coupling these techniques to the OMCEC method requires further consideration. Additionally, an aggregate image obtained in this OMCEC method is a 2-D projection of a complex 3-D aggregate, and thus, a potential development of OMCEC could include the use of the stereoscopic PIV and tomographic PIV methods (e.g., Elsinga et al., 2006;Jeon & Sung, 2012;Prasad, 2000). ...
Microbial processes can make substantial differences to the way in which particles settle in aquatic environments. A novel method (OMCEC, optical measurement of cell colonization) is introduced to systematically map the biological spatial distribution over individual suspended sediment aggregates settling through a water column. OMCEC was used to investigate (1) whether a carbon source concentration has an impact on cell colonization, (2) how cells colonize minerals, and (3) if a correlation between colonization patterns and aggregate geometry exists. Incubations of Saccharomyces cerevisiae and stained montmorillonite at four sucrose concentrations were tested in a settling column equipped with a full-color microparticle image velocimetry system. The acquired high-resolution images were processed to map the cell distribution on aggregates based on emission spectra separation. The likelihood of cells colonizing minerals increased with increasing sucrose concentration. Colonization patterns were classified into (i) scattered, (ii) well touched, and (iii) poorly touched, with the second being predominant. Cell clusters in well-touched patterns were found to have lower capacity dimension than those in other patterns, while the capacity dimension of the corresponding aggregates was relatively high. A strong correlation of colonization patterns with aggregate biomass fraction and properties suggests dynamic colonization mechanisms from cell attachment to minerals, to joining of isolated cell clusters, and finally cell growth over the entire aggregate. This paper introduces a widely applicable method for analyses of microbial-affected sediment dynamics and highlights the microbial control on aggregate geometry, which can improve the prediction of large-scale morphodynamics processes.
... However, the abiotic ferric leach cannot mimic the reaction environment of a biotic system fully, in terms of ore surface characteristics (absence of attached microbes), localised leaching reagent concentrations and redox values. A number of studies in agricultural soils found that X-ray μCT scanning at low energy doses (80-120 kV) has negligible influence on archaea and bacteria [9]. Due to the importance of microbial activity in the mineral bioleaching system, here we investigate the integration of X-ray μCT into leaching experiments, allowing combined monitoring of leaching, microbial population and activity to address the knowledge gap with regard to influence of operating parameters on leaching of non-surface mineral grains. ...
In heap bioleaching, a process in which microorganisms are required for the regeneration of leach reagents and control of reaction products, inaccessibility of non-surface mineral grains is a key cause of low recovery and long extraction times. High resolution, non-destructive 3D X-ray micro-computed tomography (μCT) is an imaging technique that has been successfully demonstrated for the study of abiotic leaching of non-surface minerals. For this technique to be applied to biotic leaching, it is required that the iron and sulphur oxidizing abilities of the microorganisms are not affected by the irradiation experienced. In the current study, the feasibility of investigating biotic leaching by X-ray μCT is explored by examining the relative energies required to achieve the high image resolution needed for mineral grain mapping while avoiding microbial deactivation. A mixed mesophilic and moderately thermophilic culture in solution was used and exposed to various X-ray energy doses. Direct microscopic cell counting and redox potential were measured to quantify the microbial activity and growth. The results showed that exposure to X-ray does not affect microbial activity at 35-90 kV, 200-280 μA and a distance of 7.2 cm between energy source and sample, however, it has an influence at 120 and 150 kV. This indicates that while X-ray μCT does influence the microbial cultures, it can be used for bioleaching studies at lower energy doses.
... Further they found a clear gradient of effects from the outer to the inner portions of the mesocosm cross-sections, due to the attenuation of X-rays by the soil as the beam traveled through the 20 cm diameter samples (dose was not provided). More detailed studies have been conducted with a total dose in the range of 2.5-7.5 Gy; Bouckaert et al. (2013) and Schmidt et al. (2015) showed no significant impact on any of the key microbial parameters (respiration, enzyme activity, microbial biomass, abundance, community structure) with the exception of archaeal cell numbers. Nevertheless, it is important to determine how repeated scanning influences rhizosphere processes, since one needs to determine a tradeoff between image quality versus the number of scans per lifecycle of the sample. ...
Background
Plant-soil interaction is central to human food production and ecosystem function. Thus, it is essential to not only understand, but also to develop predictive mathematical models which can be used to assess how climate and soil management practices will affect these interactions. ScopeIn this paper we review the current developments in structural and chemical imaging of rhizosphere processes within the context of multiscale mathematical image based modeling. We outline areas that need more research and areas which would benefit from more detailed understanding. Conclusions
We conclude that the combination of structural and chemical imaging with modeling is an incredibly powerful tool which is fundamental for understanding how plant roots interact with soil. We emphasize the need for more researchers to be attracted to this area that is so fertile for future discoveries. Finally, model building must go hand in hand with experiments. In particular, there is a real need to integrate rhizosphere structural and chemical imaging with modeling for better understanding of the rhizosphere processes leading to models which explicitly account for pore scale processes.
... These techniques could potentially be combined with X-ray CT as most studies to date appear to show that X-rays do not harm microbes significantly at doses commonly used (e.g. Schmidt et al., 2015), although older research suggests that various forms of radiation and dose can influence microbial populations (reviewed in Zappala et al., 2013). Soil fauna are also known to influence the rhizosphere, principally via direct effects upon roots by parasitism (nematodes) or grazing. ...
Despite often being conceptualized as a thin layer of soil around roots, the rhizosphere is actually a dynamic system of interacting processes. Hiltner originally defined the rhizosphere as the soil influenced by plant roots. However, soil physicists, chemists, microbiologists, and plant physiologists have studied the rhizosphere independently, and therefore conceptualized the rhizosphere in different ways and using contrasting terminology. Rather than research-specific conceptions of the rhizosphere, the authors propose a holistic rhizosphere encapsulating the following components: microbial community gradients, macroorganisms, mucigel, volumes of soil structure modification, and depletion or accumulation zones of nutrients, water, root exudates, volatiles, and gases. These rhizosphere components are the result of dynamic processes and understanding the integration of these processes will be necessary for future contributions to rhizosphere science based upon interdisciplinary collaborations. In this review, current knowledge of the rhizosphere is synthesized using this holistic perspective with a focus on integrating traditionally separated rhizosphere studies. The temporal dynamics of rhizosphere activities will also be considered, from annual fine root turnover to diurnal fluctuations of water and nutrient uptake. The latest empirical and computational methods are discussed in the context of rhizosphere integration. Clarification of rhizosphere semantics, a holistic model of the rhizosphere, examples of integration of rhizosphere studies across disciplines, and review of the latest rhizosphere methods will empower rhizosphere scientists from different disciplines to engage in the interdisciplinary collaborations needed to break new ground in truly understanding the rhizosphere and to apply this knowledge for practical guidance.
... Consequently, X-ray mCT has recently been used to (i) monitor the growth of mature root systems ( Figure 1H) [112,113], (ii) study root-soil and rootroot interactions [114][115][116], and (iii) gain insights into hydraulic properties of the rhizosphere [117][118][119]. Low irradiation doses applied to plant-soil interfaces were reported to neither influence root growth [120] nor microbial cell number, community structure, or potential activity [121,122]. Thus, X-ray mCT may be well-compatible with studies of rhizosphere ecology. The potential inherent in such combinatorial approaches was shown very recently by visualizing plant-pathogen interactions in situ at high spatial resolution for the first time [123]. ...
Root-triggered processes (growth, uptake and release of solutes) vary in space and time, and interact with heterogeneous soil microenvironments that provide habitats for (micro)biota on various scales. Despite tremendous progress in method development in the past decades, finding a suitable experimental set-up to investigate processes occurring at the dynamic conjunction of biosphere, hydrosphere, and pedosphere in the close vicinity of active plant roots still represents a major challenge. We discuss recent methodological developments in rhizosphere research with a focus on imaging techniques. We further review established concepts that have been updated with novel techniques, highlighting the need for combinatorial approaches to disentangle rhizosphere processes on relevant scales.
... After being placed in the syringes the samples were subjected to μ CT scanning. The samples were scanned while air-dry, since it has been demonstrated that scanning has minimal effect on soil microorganisms in dry conditions 40 . Our previous assessments of the effect of scanning on the microbial activities and microbial community structure in intact soil samples of the same size scanned at the same settings demonstrated minimal interference of scanning with microorganisms 23 . ...
Soil contains almost twice as much carbon (C) as the atmosphere and 5-15% of soil C is stored in a form of particulate organic matter (POM). Particulate organic matter C is regarded as one of the most labile components of the soil C, such that can be easily lost under right environmental settings. Conceptually, micro-environmental conditions are understood to be responsible for protection of soil C. However, quantitative knowledge of the specific mechanisms driving micro-environmental effects is still lacking. Here we combined CO 2 respiration measurements of intact soil samples with X-ray computed micro-tomography imaging and investigated how micro-environmental conditions, represented by soil pores, influence decomposition of POM. We found that atmosphere-connected soil pores influenced soil Câ (tm) s, and especially POM's, decomposition. In presence of such pores losses in POM were 3-15 times higher than in their absence. Moreover, we demonstrated the presence of a feed-forward relationship between soil C decomposition and pore connections that enhance it. Since soil hydrology and soil pores are likely to be affected by future climate changes, our findings indicate that not-accounting for the influence of soil pores can add another sizable source of uncertainty to estimates of future soil C losses.
... (Dufour et al., 2005). Continual refinement of the technique means that it can now be used to observe and quantify microscale structural heterogeneity in samples with negligible effect on both soil and bacteria (Schmidt et al., 2015), and at resolutions below 1 mm (Van den Bulcke et al., 2009;Helliwell et al., 2013). Methods have been developed for retrieval of undisturbed soil samples from most soil types and conditions (Rogasik et al., 1997). ...
Decay experiments are ever increasing in complexity to better understand taphonomic processes. However, adding new variables, such as sediment, can create methodological biases, such as artificial anatomical character loss during exhumation. Non-invasive in situ imaging techniques such as X-ray computed tomography (XCT scanning) could mitigate this, but the consequences of exposing carcasses to X-rays are not fully understood, and evidence regarding the impact of X-rays on internal microbial faunas that drive decay is conflicting. Here, we test whether XCT scanning impacts the decay of Danio rerio carcasses within a substrate. Our control experiments show that quartz sand sediment physically stabilises the carcass throughout decay and the sequence of anatomical character loss remains constant, however, both the onset and rate of decay of soft tissues are initially accelerated. Our XCT data show that exposure to X-rays does not cause a deviation from the normal sequence of decay, validating XCT as a non-destructive visualisation method for decay experiments. Furthermore, when accompanied with traditional exhumation and dissection, XCT provides decay data with higher accuracy of character analysis than traditional methods, and allows novel quantitative techniques to monitor physical changes in the decaying carcass (e.g., total volume, build-up of gases, collapse of the body cavity etc.). We also underline limitations with the technique, but our experiment acts as an important 'stepping stone' for progression toward non-invasive designs of decay experiments.
X-ray computed tomography (X-ray CT) is a non-destructive method of soil analysis which can provide three-dimensional (3D) view, quantitative information of the internal organization of the soil. In this paper, we discuss the potential application of X-ray CT in characterization of soil properties like porosity and pore size distribution (PSD), root architecture, soil phase classification, water and solute transport in soil, and highlight the research during last 10–15 years. Here, we review the recent development of X-ray CT in soil science, use of artificial intelligence and machine learning in image analysis, point out the major challenges associated with its use, discuss few improvements to overcome these difficulties and elaborate the possible future technological developments for non-invasive/destructive soil characterization by integrating X-ray CT with recently available complementary techniques.
Growing roots interact with soil, and its structure, across a range of spatial and temporal scales, and thus adapt to the local environment (Downie et al., 2015). Several factors influence the development of roots, and as such, shape the root system architecture (RSA). Well-known examples of these are water stress, mechanical impedance, pore connectivity and porosity (Bengough et al., 2011; Correa et al., 2019; Lucas et al., 2019a). However, it is not just physical but also biological and chemical factors such as the availability of nitrogen that can change the growth of roots and their RSA (Flavel et al., 2014; Gao, Blaser, et al., 2019b; Nwankwo et al., 2018). Due to all these factors, perhaps not surprisingly root growth in artificial soil, such as gel media and indeed sieved soil, shows significantly different root growth when compared to roots developed in undisturbed soil (Hargreaves et al., 2009). In this chapter we review how rhizosphere research with X-ray CT has developed within the last three decades and show X-ray imaging is an excellent tool to investigate changes in root architecture in soil over time.
The output of CT imaging (after reconstruction) of soil samples are grayscale images with a series of gray values corresponding to soil components with different densities. Most current techniques to quantify the soil pore system rely on binary images, with the target objects or foreground pixels (e.g. pores) labelled as 1 and the background (e.g. soil) labelled as 0. This means the grayscale images need to be segmented to binary images before further quantification. However, because of the partial volume effect and noise in the images, in most cases the histogram of the grayscale image does not exhibit clear, distinct peaks, which makes image segmentation a very challenging task. Many filtering techniques have been developed to smooth image noise and enhance the contrast between the target and background, which can greatly help image segmentation if correctly used. Numerous image segmentation techniques have also been developed to deal with specific problems in a variety of disciplines. In soil science, a range of global and local segmentation methods have been used and their performances compared and summarized in previous studies. Recently, artificial intelligence (AI) has been used to segment soil CT images and showed great potential in extracting not only soil pore space, but also other soil components. In this chapter, we briefly summarize the widely used traditional global and local segmentation methods and then present a new segmentation protocol that combines machine learning and deep learning strategies to segment pore space in soil aggregate images. The results of the AI-based method are very promising, and we propose the AI techniques will soon be extensively used in the processing of the soil CT images.
Since C. Darwin (1809–1882), V. Dokuchaev (1846–1903) and H. Jenny (1899–1992), soil organisms have been well-known as the key element of soil formation (Schaetzl & Anderson, 2005), particularly important for development and properties of soil structure (Meurer et al., 2020). Yet, the opaque nature of the soil and near impossibility of observing soil micro- and macro-fauna activities in their natural habitat, without destroying the habitat itself, have made studies of the soil organisms’ behaviours and their subsequent influence on the soil extremely challenging. The development of X-ray Computed Tomography (CT) provided a long-needed capability to look inside soil samples, even though of relatively small (<10–20 cm) sizes, while keeping them intact. X-ray CT enables visualization of intact soil structure with organic objects, such as plant roots or animals, located within it. Thus, it is particularly useful in assessing the role of soil biota, including microorganisms, on soil structure formation and properties and in informing studies of biology and behaviour of soil meso- and macro-fauna, especially earthworms and larger insects. X-ray CT has been readily employed by soil scientists, generating a large body of knowledge concerning interactions between soil physical (e.g. structural) components and soil organisms. Improvements in CT image quality and resolution over the past couple of decades have expanded its implementation in soil studies from viewing relatively large organisms, such as insects, to exploring micro-habitats relevant for functioning of soil microorganisms, to visualizing soil organic matter.
Artificial Intelligence (AI) is expected to play a crucial role in the field of phytoremediation and its effective management in monitoring the growth of the plant in different contaminated soils and their phenotype characteristic such as the biomass of plants. This review focuses on recent applications of various AI techniques and remote sensing approaches in the field of phytoremediation to monitor plant growth with relevant morphological parameters using novel sensors, cameras, and associated modern technologies. Novel sensing and various measurement techniques are highlighted.
Input parameters are used to develop futuristic models utilizing AI and statistical approaches. Additionally, a brief discussion has been presented on the use of AI techniques to detect metal hyperaccumulation in all parts of the plant, carbon capture, and sequestration along with its effect on food production to ensure food safety and security. This article highlights the application, limitation, and future perspectives of phytoremediation in monitoring the mobility, bioavailability, seasonal variation, effect of temperature on plant growth, and plant response to the heavy metals in soil by using the AI technique. Suggestions are made for future research in this area to analyze which
would help to enhance plant growth and improve food security in long run.
The porous microenvironment of soil offers various environmental functions which are governed by physical and reactive processes. Understanding reactive transport processes in porous media is essential for many natural systems (soils, aquifers, aquatic sediments or subsurface reservoirs) or technological processes (water treatment, or ceramic and fuel cell technologies). In particular, in the vadose zone of the terrestrial subsurface the spatially and temporally varying saturation of the aqueous and the gas phase leads to systems that involve complex flow and transport processes as well as reactive transformations of chemical compounds in the porous material. To describe these interacting processes and their dynamics at the pore scale requires a well-suited modelling framework accounting for the proper description of all relevant processes at a high spatial resolution. Here we present P3D-BRNS as a new open-source modelling toolbox harnessing the core libraries of OpenFOAM and coupled externally to the Biogeochemical Reaction Network Simulator (BRNS). The native OpenFOAM Volume of Fluid solver is extended to have an improved representation of the fluid-fluid interface. The solvers are further developed to couple the reaction module which can be tailored for a specific reactive transport simulation. P3D-RBNS is benchmarked against three different flow and reactive transport processes; 1) fluid-fluid configuration in a capillary corner, 2) mass transfer across the fluid-fluid interface and 3) microbial growth with a high degree of accuracy. Our model allows for simulation of the spatio-temporal distribution of all bio-chemical species in the porous structure (obtained from µ-CT images), for conditions that are commonly found in the laboratory and environmental systems. With our coupled computational model, we provide a reliable and efficient tool for simulating multiphase, reactive transport in porous media.
The study of below-ground features of roots and soil and their interactions is essential for understanding the configuration and diversities in such a dynamic environment. X-ray computed tomography is recognized as a tool for visualizing the physical interactions in the soil. In many studies, it has been used as a stand-alone tool to describe soil and root parameters. However, in recent times, attempts to couple it with other complementary tools are gaining rapid interest among researchers. The paper therefore provides an overview of the major application of combining X-ray computed tomography with other relevant methods in analyzing the structural characteristics of roots and soil media. The relevance of using this multidisciplinary approach for unraveling the mysteries surrounding root–soil dynamic interactions is stressed. The current and future trends of such studies are also pointed out. © 2015 Jiangsu University of the People's Republic of China http://www.gov.cn/.
Biological and physical interactions in unsaturated soil, the vadose zone, have received a surge of research interest over the past several years. This article reviews recent research, focusing on the limitations imposed by the complexity of soil, the use of model systems to understand processes, new technologies, and the understanding of how biology changes soil structure. Research using model systems to mimic natural structure, such as rough planar surfaces or packed columns, has made it possible to demonstrate and quantify microbial interactions at very small spatial scales, including the coexistence of competing microbes and the invasion of soil pores by organisms that should be too large to fit. It is now possible to see inside soil at micrometer resolution in three dimensions, either by the use of noninvasive imaging techniques on intact soils or a model transparent soil with the same refractive index as water. Soil biology also changes soil structure. Techniques from engineering such as fracture mechanics and rheology have measured enhanced particle bonding, dispersion, and aggregation caused by root and microbial derived exudates. Models of soil structure dynamics are beginning to use these data. Concurrent research on naturally structured soil is essential, but using model systems that allow for the application of material science approaches or the detection and modeling of specific processes will enable the building of complexity by piecing together simpler systems. A major challenge for future research is gaining a quantitative understanding of how soil biology changes structure and incorporating this knowledge with studies of soil biodiversity, microbial functions, and root-soil interactions. Upscaling from microbial processes at micrometer resolution to the whole plant, field or catchment presents an even greater challenge.
It is known that variability in the characteristics of soil physical microenvi- ronments, e.g., locations and characteristics of soil pores, can have a major influence on microorganisms. This study compared the characteristics of intra-aggregate pores and their relationships with bacterial community composition in 4- to 6-mm soil macroaggregates from two contrasting agri- cultural systems, namely a corn (Zea mays L.)–soybean [Glycine max (L.) Merr.]–wheat (Triticum aestivum L.) rotation (i) with conventional chemi- cal inputs and (ii) without chemical inputs but with legume cover crops. Characteristics of intra-aggregate pores and particulate organic matter (POM) were obtained from three-dimensional X-ray computed microtomog- raphy aggregate images at 13-�m resolution. Microbial community analyses were conducted using 16S rRNA pyrosequencing. We found that the aggre- gates from long-term (>20 yr) organic management with cover crops differed from the aggregates from conventional management in terms of intra-aggregate pore-size distribution, intra-aggregate pore variability, POM contents, as well as microbial community compositions. Relative abundanc- es of Actinobacteria, Proteobacteria, and Firmicutes in the aggregates from cover-crop-based management were positively correlated with the presence of large (>110 �m) pores, while the presence of medium-sized pores (32–84 �m) was significantly correlated with 30 to 40% of the top 100 bacterial operational taxonomic units identified during sequencing. The results sug- gested that the legacy of rhizosphere presence in terms of intra-aggregate pore structure and microbial community composition can last in the studied soil for at least 4 to 9 mo. Our study demonstrated that working with bulk sieved soil samples can mask substantial differences present within distinct soil aggregate units.
A comprehensive, but simple-to-use software package for executing a range of standard numerical analysis and operations used in quantitative paleontology has been developed. The program, called PAST (PAleontological STatistics), runs on standard Windows computers and is available free of charge. PAST integrates spreadsheettype data entry with univariate and multivariate statistics, curve fitting, time-series analysis, data plotting, and simple phylogenetic analysis. Many of the functions are specific to paleontology and ecology, and these functions are not found in standard, more extensive, statistical packages. PAST also includes fourteen case studies (data files and exercises) illustrating use of the program for paleontological problems, making it a complete educational package for courses in quantitative methods.
X-ray Computed Tomography (CT) is a non-destructive imaging technique originally designed for diagnostic medicine, which was adopted for rhizosphere and soil science applications in the early 1980s. X-ray CT enables researchers to simultaneously visualise and quantify the heterogeneous soil matrix of mineral grains, organic matter, air-filled pores and water-filled pores. Additionally, X-ray CT allows visualisation of plant roots in situ without the need for traditional invasive methods such as root washing. However, one routinely unreported aspect of X-ray CT is the potential effect of X-ray dose on the soil-borne microorganisms and plants in rhizosphere investigations. Here we aimed to i) highlight the need for more consistent reporting of X-ray CT parameters for dose to sample, ii) to provide an overview of previously reported impacts of X-rays on soil microorganisms and plant roots and iii) present new data investigating the response of plant roots and microbial communities to X-ray exposure. Fewer than 5% of the 126 publications included in the literature review contained sufficient information to calculate dose and only 2.4% of the publications explicitly state an estimate of dose received by each sample. We conducted a study involving rice roots growing in soil, observing no significant difference between the numbers of root tips, root volume and total root length in scanned versus unscanned samples. In parallel, a soil microbe experiment scanning samples over a total of 24 weeks observed no significant difference between the scanned and unscanned microbial biomass values. We conclude from the literature review and our own experiments that X-ray CT does not impact plant growth or soil microbial populations when employing a low level of dose (<30 Gy). However, the call for higher throughput X-ray CT means that doses that biological samples receive are likely to increase and thus should be closely monitored.
Aims
Roots need to be in good contact with the soil to take up water and nutrients. However, when the soil dries and roots shrink, air-filled gaps form at the root-soil interface. Do gaps actually limit the root water uptake, or do they form after water flow in soil is already limiting?
Methods
Four white lupins were grown in cylinders of 20 cm height and 8 cm diameter. The dynamics of root and soil structure were recorded using X-ray CT at regular intervals during one drying/wetting cycle. Tensiometers were inserted at 5 and 18 cm depth to measure soil matric potential. Transpiration rate was monitored by continuously weighing the columns and gas exchange measurements.
Results
Transpiration started to decrease at soil matric potential ψ between −5 kPa and −10 kPa. Air-filled gaps appeared along tap roots between ψ = −10 kPa and ψ = −20 kPa. As ψ decreased below −40 kPa, roots further shrank and gaps expanded to 0.1 to 0.35 mm. Gaps around lateral roots were smaller, but a higher resolution is required to estimate their size.
Conclusions
Gaps formed after the transpiration rate decreased. We conclude that gaps are not the cause but a consequence of reduced water availability for lupins.
Background
X-ray micro-Computed Tomography (μCT) offers the ability to visualise the three-dimensional structure of plant roots growing in their natural environment – soil. Recovery of root architecture descriptions from X-ray CT data is, however, challenging. The X-ray attenuation values of roots and soil overlap, and the attenuation values of root material vary. Any successful root identification method must both explicitly target root material and be able to adapt to local changes in root properties.
RooTrak meets these requirements by combining the level set method with a visual tracking framework and has been shown to be capable of segmenting a variety of plant roots from soil in X-ray μCT images. The approach provides high quality root descriptions, but tracks root systems top to bottom and so omits upward-growing (plagiotropic) branches.
Results
We present an extension to RooTrak which allows it to extract plagiotropic roots. An additional backward-looking step revisits the previous image, marking possible upward-growing roots. These are then tracked, leading to efficient and more complete recovery of the root system. Results show clear improvement in root extraction, without which key architectural traits would be underestimated.
Conclusions
The visual tracking framework adopted in RooTrak provides the focus and flexibility needed to separate roots from soil in X-ray CT imagery and can be extended to detect plagiotropic roots. The extended software tool produces more complete descriptions of plant root structure and supports more accurate computation of architectural traits.
Soil organic matter (SOM) is heterogeneous in structure and has been considered to consist of various pools with different intrinsic turnover rates. Although those pools have been conceptually expressed in models and analyzed according to soil physical and chemical properties, separation of SOM into component pools is still challenging. In this study, we conducted inverse analyses with data from a long-term (385 days) incubation experiment with two types of soil (from plant interspace and from underneath plants) to deconvolute soil carbon (C) efflux into different source pools. We analyzed the two datasets with one-, two- and three-pool models and used probability density functions as a criterion to judge the best model to fit the datasets. Our results indicated that soil C release trajectories over the 385 days of the incubation study were best modeled with a two-pool C model. For both soil types, released C within the first 10 days of the incubation study originated from the labile pool. Decomposition of C in the recalcitrant pool was modeled to contribute to the total CO(2) efflux by 9-11 % at the beginning of the incubation. At the end of the experiment, 75-85 % of the initial soil organic carbon (SOC) was modeled to be released over the incubation period. Our modeling analysis also indicated that the labile C-pool in the soil underneath plants was larger than that in soil from interspace. This deconvolution analysis was based on information contained in incubation data to separate carbon pools and can facilitate integration of results from incubation experiments into ecosystem models with improved parameterization.
The effects of fumigation on organic C extractable by 0.5 M K2SO4 were examined in a contrasting range of soils. EC (the difference between organic C extracted by 0.5 M K2SO4 from fumigated and non-fumigated soil) was about 70% of FC (the flush of CO2-C caused by fumigation during a 10 day incubation), meaned for ten soils. There was a close relationship between microbial biomass C, measured by fumigation-incubation (from the relationship Biomass C = FC/0.45) and EC given by the equation: Biomass C = (2.64 ± 0.060) EC that accounted for 99.2% of the variance in the data. This relationship held over a wide range of soil pH (3.9–8.0).ATP and microbial biomass N concentrations were measured in four of the soils. The ratios were very similar in the four soils, suggesting that both ATP and the organic C rendered decomposable by CHCl3 came from the soil microbial biomass. The C:N ratio of the biomass in a strongly acid (pH 4.2) soil was greater (9.4) than in the three less-acid soils (mean C:N ratio 5.1).We propose that the organic C rendered extractable to 0.5 m K2SO4 after a 24 h CHCl3-fumigation (EC) comes from the cells of the microbial biomass and can be used to estimate soil microbial biomass C in both neutral and acid soils.
One key constraint to further understanding plant root development is the inability to observe root growth in situ due to the opaque nature of soil. Of the present non-destructive techniques, computed tomography (CT) is best able to capture
the complexities of the edaphic environment. This study compared the accuracy and impact of X-ray CT measurement of in situ root systems with standard technology (soil core washing and WinRhizo analysis) in the context of treatments that differed
in the vertical placement of phosphorus fertilizers within the soil profile. Although root lengths quantified using WinRhizo
were 8% higher than that observed in the same plants using CT, measurements of root length by the two methodologies were highly
correlated. Comparison of scanned and unscanned plants revealed no effect of repeated scanning on plant growth and CT was
not able to detect any changes in roots between phosphorus treatments that was observed using WinRhizo. Overall, the CT technique
was found to be fast, safe, and able to detect roots at high spatial resolutions. The potential drawbacks of CT relate to
the software to digitally segment roots from soil and air, which will improve significantly as automated segmentation algorithms
are developed. The combination of very fast scans and automated segmentation will allow CT methodology to realize its potential
as a high-throughput technique for the quantification of roots in soils.
Changes in chemical properties in soil around plant roots influence many microbial processes, including those having an impact on greenhouse gas emissions. To potentially mitigate these emissions according to the Kyoto protocol, knowledge about how and where these gases are produced and consumed in soils is required. In this review, we focus on the greenhouse gases nitrous oxide and methane, which are produced by nitrifying and denitrifying prokaryotes and methanogenic archaea, respectively. After describing the microbial processes involved in
production and consumption of nitrous oxide and methane and how they can be affected in the rhizosphere, we give an overview of nitrous oxide and methane emissions from the rhizosphere and soils and sediments with plants. We also discuss strategies to mitigate emissions from the rhizosphere and consider possibilities for carbon sequestration.
Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genus Thiomicrospira. To identify these 'phylotypes' in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three described Thiomicrospira species, viz., Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate, Thiomicrospira sp. strain MA2-6. All Thiomicrospira species except Tms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria. Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated with Thiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivision Thiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence of Tms. crunogena. Identifying a dominant molecular isolate as Tms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another 'phylotype' represented a new Thiomicrospira species, phylogenetically in an intermediate position between Tms. crunogena and Tms. pelophila. The third 'phylotype' was identified as a Desulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.
The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.
Our understanding of thermophile diversity is based predominantly on PCR studies of community DNA. "Universal" and domain-specific rRNA gene PCR primers have historically been used for the assessment of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic groups. In a reassessment of the published primers commonly used for "universal" and archaeal 16 S rDNA sequence amplification we note that substantial variations in specificity exist. An unconsidered choice of primers may therefore lead to significant bias in determination of microbial community composition. In particular, Archaea-specific primer sequences typically lack specificity for the Korarchaeota and Nanoarchaea and are often biased towards certain clades. New primer pairs specifically designed for "universal" archaeal 16 S rDNA sequence amplification, with homology to all four archaeal groups, have been designed. Here we present the application of these new primers for preparation of 16 S libraries from thermophile communities.
Soils contain the greatest reservoir of biodiversity on Earth, and the functionality of the soil ecosystem sustains the rest of the terrestrial biosphere. This functionality results from complex interactions between biological and physical processes that are strongly modulated by the soil physical structure. Using a novel combination of biochemical and biophysical indicators and synchrotron microtomography, we have discovered that soil microbes and plant roots microengineer their habitats by changing the porosity and clustering properties (i.e., spatial correlation) of the soil pores. Our results indicate that biota act to significantly alter their habitat toward a more porous, ordered, and aggregated structure that has important consequences for functional properties, including transport processes. These observations support the hypothesis that the soil-plant-microbe complex is self-organized.
The introduction of new approaches for characterizing microbial communities and imaging soil environments has benefited soil microbiology by providing new ways of detecting and locating microorganisms. Consequently, soil microbiology is poised to progress from simply cataloguing microbial complexity to becoming a systems science. A systems approach will enable the structures of microbial communities to be characterized and will inform how microbial communities affect soil function. Systems approaches require accurate analyses of the spatio-temporal properties of the different microenvironments present in soil. In this Review we advocate the need for the convergence of the experimental and theoretical approaches that are used to characterize and model the development of microbial communities in soils.
Soil deformation is a perpetual process in the pedosphere where besides physicochemical stresses primarily alternating hydraulic and mechanical stresses continuously re-arrange the configuration of solid particles. In this study we present a local strain analysis and changes in soil structure resulting from hydraulic and mechanical stresses based on X-ray microtomography data. Digital image reconstructions were used to quantify local structural pore space characteristics and local soil deformation by 3D morphological and correlation analysis of grayscale tomograms. Swelling and shrinkage resulted in a complex heterogeneous soil structure which proofed to be very stable when mechanical loads were applied. The mechanism of soil deformation for both structure formation by internal hydraulic stresses and structure degradation by external mechanical stresses were in both cases controlled by pre-existing
(micro)-structures. Especially during wetting such structures served as a nucleus for subsequent structure evolution. The results demonstrate the potential of more detailed non-invasive micro-mechanical analysis of soil deformation processes which could improve the conceptual understanding of the physical behavior of soil systems.
The majority of microbial mediated soil processes depend on availability of organic matter (OM), water and air. Because of its ability to visualize the 3D architecture of soil non-destructively, X-ray computed tomography (CT) is becoming a widespread tool for studying soil pore network structure. However, phase determination of pore space, soil OM, soil mineral matter (MM) and water is often limited even with the latest technological and software advances, allowing high resolution and better quality imaging. Contrast agents commonly used in histology enable enhancement of X-ray attenuation of targeted structures or compounds. Here we report on the first systematic investigation of the use of such X-ray contrast agents for soil research. An evaluation procedure as well as a method to apply the agents to soil samples was developed and applied on reference soil samples. The effectiveness and selectivity of the contrast agents was evaluated for soil organic matter (SOM), MM and water. Several products were found to selectively increase the attenuation of water or SOM. The four agents with the best OM-staining capabilities (phosphomolybdenic acid (PMA), silver nitrate, lead nitrate and lead acetate) were further tested on an OM-MM mixture and all showed an increased of the SOM attenuation coefficient above the MM values.
Root system architecture and associated root-soil interactions exhibit large changes over time. Nondestructive methods for the quantification of root systems and their temporal development are needed to improve our understanding of root activity in natural soils. X-ray computed tomography (X-ray CT) was used to visualize and quantify growth of a single Vicia faba L. root system during a drying period. The plant was grown under controlled conditions in a sandy soil mixture and imaged every second day. Minkowski functionals and Euclidean distance transform were used to quantify root architectural traits. We were able to image the root system with water content decreasing from 29.6 to 6.75%. Root length was slightly underestimated compared with destructive measurements. Based on repeated measurements over time it was possible to quantify the dynamics of root growth and the demography of roots along soil depth. Measurement of Euclidean distances from any point within the soil to the nearest root surface yielded a frequency distribution of travel distances for water and nutrients towards roots. Our results demonstrate that a meaningful quantitative characterization of root systems and their temporal dynamics is possible.
Combining X-ray micro-Computed Tomography (X-ray micro-CT) analysis with measures of soil microbial functioning would provide a powerful tool for revealing the influence of soil pore structure on soil organic matter (SOM) decomposition. We investigated the impact of X-ray micro-CT scanning on soil microbial functioning for the first time by assessing C-mineralization, enzyme activities and microbial community structure by phospholipid fatty acid (PLFA) analysis 1 and 22 days after irradiation. There was no evidence for a disturbance of soil biological functioning following the X-ray micro-CT scanning, except for a small (but significant) decrease in dehydrogenase activity and a small (but significant) increase in the concentration of actinomycetes PLFA biomarkers. We conclude that X-ray micro-CT is fully compatible with soil biological experiments and that the combination of both may result in exciting new insights in the controls of soil pore architecture on soil biological experiments.
We tested the accuracy of the chloroform fumigation–extraction method, which is commonly used to determine soil biomass C concentrations. Accurate and precise determination of total microbial biomass is important in order to characterize soil properties and to develop predictive metal transport models for soils. Two natural soils, and individual soil components, including silica sand, montmorillonite, kaolinite, a humic acid, and Bacillus subtilis bacterial cells, were fumigated for 24h. Following the fumigation, C from fumigated and unfumigated samples was extracted using a 0.5M K2SO4 solution. The difference between the C content in the fumigated and unfumigated samples ideally represents C due to biomass because the fumigation procedure should lyse cells and release biomass C. We observed increased C release upon fumigation for bacteria-only samples, confirming the ability of fumigation to lyse cells. There was no difference in extracted C concentration between fumigated and unfumigated samples of silica sand and of humic acid, confirming that the fumigation process does not introduce additional organic C to samples of these soil components. However, the fumigated clay samples both showed increased C release relative to the unfumigated controls, indicating that significant concentrations of the fumigant, chloroform, adsorbed onto the clay minerals studied here. Additionally, we found significant chloroform remaining in the extracts from two fumigated natural soils. Attempts to remove the chloroform from the soils or soil components prior to extraction by increasing the evacuation time, or to remove chloroform in the extracts by sparging them vigorously with nitrogen gas, both failed. This research reveals that chloroform gas may adsorb significantly to clays and the clay fraction of natural soils. Thus, the fumigation–extraction method must be corrected to account for the added chloroform C and accurately assess the concentration of biomass C in soils that contain significant concentrations of clays.
Samples of Rifle peat were irradiated with gamma rays from a Co ⁶⁰ source at doses of 2, 8, 32, 64, 128, and 250 kiloroentgens (kr.). The irradiated samples were incubated for 54 days, and periodic determinations were made on the rate of CO 2 evolution, P and N mineralization, pH changes, and development of bacterial and fungal populations.
The radiation doses employed had no significant effect on development of the bacterial population, pH, mineralization of P, and evolution of CO 2 . Growth of the fungal population and ammonia utilization was increasingly inhibited by doses of 8 to 250 kr.
Gross changes in the metabolism of the soil microflora did not take place. Higher dose levels than those used in this study apparently are required for the sterilization of organic soils.
The kEN value (= extractable part of microbial biomass N after fumigation) of the fumigation-extraction method was assessed using the C-to-N ratio of the organic matter which was rendered extractable by CHCl3 fumigation. The data for this calibration approach was obtained from 51 arable and 23 grassland soils. The second calibration approach was to compare the relationship between N rendered extractable by CHCl3 fumigation and the C-to-N ratio measured in the flush of the fumigation-incubation method by recalculating data obtained from the literature. On the basis of these two approaches, we recommend using a kEN value of 0.54 as originally proposed by Brookes et al. (Soil Biology & Biochemistry17, 837–842, 1985).
The puddled layer of paddy soils represents a highly dynamic environment regarding the spatio-temporal variability of biogeochemical conditions. To study these effects on the abundance and community structure of microbial populations, a rhizotron experiment was conducted throughout an entire growing season of wetland rice. Soil samples were taken from selected areas of the puddled layer (bulk soil, oxidized layer, rhizosphere) at main plant developmental stages such as (i) the initial stage, (ii) tillering, (iii) panicle initiation, (iv) flowering, and (v) maturity. Cell numbers of archaea, bacteria, and selected phyla were assessed by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The structure and diversity of microbial communities was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) along with sequencing of selected bands. Following submergence of the paddy soil, shifts of bacterial community structure were observed in the oxidized layer and the rhizosphere. Members of the β-Proteobacteria became predominant in the rhizosphere at tillering stage and were affiliated with aerobic, iron-oxidizing bacteria of the genus Sideroxydans. Seasonal effects were mainly visible in the rhizosphere, as several phylogenetic subgroups including methanotrophic bacteria showed increased cell numbers at flowering stage. Cell numbers of methanogenic archaea were also highest at flowering stage (bulk soil, rhizosphere) and members of the Methanocellales were identified as predominant archaeal populations in areas of oxic and anoxic conditions. In contrast to bacteria, the communities of archaea in the puddled layer of the studied paddy soil were less influenced by spatio-temporal variations of biogeochemical conditions.
Thirty‐gram soil samples, in plastic tissue culture dishes (60 × 15 mm), were irradiated with gamma rays (8000 curie Co‐60 source) at doses ranging from 0 to 3 million rads. Fungal and bacterial plus actinomycetal counts were made by plating soil suspensions on rose bengal‐streptomycin agar and egg albumin agar, respectively. Plates were incubated for 4 to 8 days before counting. The numbers of fungal and bacterial units reflected differences in survival among the microbial populations from soil samples taken at three horizons within field profiles. Microbial counts in the soils at 30% water content (by weight) were generally reduced to zero at lower radiation doses than those in the air‐dry soils. One million rads were required to kill all fungi, but 2 to 3 million rads were required to kill all bacteria in a 30‐g soil sample. At a given water content, samples with the higher initial microbial populations required greater radiation doses for sterilization than samples with lower populations. For small soil samples, exposure to gamma radiation proved a rapid and efficient method of sterilization.
Adequate contact with the soil is essential for water and nutrient adsorption by plant roots, but the determination of root–soil contact is a challenging task because it is difficult to visualize roots in situ and quantify their interactions with the soil at the scale of micrometres. A method to determine root–soil contact using X-ray microtomography was developed. Contact areas were determined from 3D volumetric images using segmentation and iso-surface determination tools. The accuracy of the method was tested with physical model systems of contact between two objects (phantoms). Volumes, surface areas and contact areas calculated from the measured phantoms were compared with those estimated from image analysis. The volume was accurate to within 0.3%, the surface area to within 2–4%, and the contact area to within 2.5%. Maize and lupin roots were grown in soil (<2 mm) and vermiculite at matric potentials of −0.03 and −1.6 MPa and in aggregate fractions of 4–2, 2–1, 1–0.5 and < 0.5 mm at a matric potential of −0.03 MPa. The contact of the roots with their growth medium was determined from 3D volumetric images. Macroporosity (>70 µm) of the soil sieved to different aggregate fractions was calculated from binarized data. Root-soil contact was greater in soil than in vermiculite and increased with decreasing aggregate or particle size. The differences in root–soil contact could not be explained solely by the decrease in porosity with decreasing aggregate size but may also result from changes in particle and aggregate packing around the root.
In this study the influence of X-ray computed tomography (XRCT) on the microbial community structure and function in soils has been investigated. Our results clearly indicate that XRCT of soil samples has a strong impact on microbial communities and changes structure and function significantly due to the death of selected microbial groups as a result of the treatment.
Soil systems are characterized by the spatial and temporal distribution of organic and mineral particles, water and air within a soil profile. Investigations into the complex interactions between soil constituents have greatly benefited from the advent of non‐invasive techniques for structural analysis. In this paper we present a review of the application of one such technique, X‐ray computed tomography (CT), for studies of undisturbed soil systems, focusing on research during the last 10 years in particular. The ability to undertake three‐dimensional imaging has provided valuable insights regarding the quantitative assessment of soil features, in a way previously unachievable because of the opaque nature of soil. A dynamic approach to the evaluation of soil pore networks, hydro‐physical characteristics and soil faunal behaviour has seen numerous scanning methodologies employed and a diverse range of image analysis protocols used. This has shed light on functional processes across multiple scales whilst also bringing its own challenges. In particular, much work has been carried out to link a soil's porous architecture with hydraulic function, although new technical improvements allowing the characterization of organic matter and the influence of soil biota on structural development are showing great promise. Here we summarize the development of X‐ray CT in soil science, highlight the major issues relating to its use, outline some of the applications for overcoming these challenges and describe the potential of future technological advances for non‐invasive soil characterization through integration with other complementary techniques.
MicroCT is increasingly being used to observe soft animal and plant tissues. Conventional electron and light microscope staining protocols used to enhance the contrast of soft tissues have the potential to be adapted for use in microCT. This would increase the versatility of the microCT beyond improving qualitative observations to facilitating quantitative analysis of soft tissues. This paper describes the development of a culture system and staining protocol which has successfully been used to obtain three dimensional (3-D) quantitative data of filamentous and zoosporic soil fungi. The fungi were grown in an artificial matrix that was developed to simulate the particulate nature of soil. The combination of high contrast staining protocol and use of an x-ray translucent matrix allowed for 3-D qualitative and quantitative analysis of fungal growth. A salient point raised by this study is that the effectiveness of a protocol is reliant on the tissue or cell culture system which includes sample, the sampling vessel, the depth of a sample and the combination of stains used. The potential use of this method extends to other fields where distribution and growth patterns in 3-D need to be quantified.
The community response of soil microbes exposed to acute gamma (γ)-irradiation at 0, 1, 5 and 10 kGy doses were examined for 56 days after irradiation. Physiological activity was determined by community substrate utilisation potentials (BIOLOG™) and a glucose substrate assay. Culture dependant analysis was used to track the changes in fungal and bacteria numbers while culture independent analyses were carried out to assess changes in bacterial community composition. As a consequence of irradiation, bacterial populations initially decreased and then exceeded control populations at the higher radiation doses, while fungal populations did not recover substantially after irradiation. These data suggest that either changes in niche competition between bacteria and fungi or a change in nutrient availability has led to the re-establishment of bacterial populations. The recovery in bacterial populations was mirrored by substrate utilisation potentials increasing in both the rate of utilisation as well as the number of substrates being used. Molecular profiling (DGGE) at later time points showed discrete dominant bands in profiles from the 5 and 10 kGy treatments suggesting outgrowth of certain taxa above background levels. The identities of these taxa were typical of soil environments. In conclusion microbial functioning and physiology can recover from acute doses of γ-irradiation, yet higher doses may lead to changes in the underlying community structure, presumably due to competition for recently opened niches.
For the investigation of many geometrical features of soils, computer-assisted image analysis has become a method of choice over the last few decades. This analysis involves numerous steps, regarding which subjective decisions have to be made by the individuals conducting the research. This is particularly the case with the thresholding step, required to transform the original (color or greyscale) images into the type of binary representation (e.g., pores in white, solids in black) needed for fractal analysis or simulation with Lattice–Boltzmann models. Limited information exists at present on whether different observers, analyzing the same soil, would be likely to obtain similar results. In this general context, the first objective of the research reported in this article was to determine, through a so-called “round-robin” test, how much variation exists among the outcomes of various image thresholding strategies (including any image pre-treatment deemed appropriate), routinely adopted by soil scientists. Three test images – of a field soil, a soil thin section, and a virtual section through a 3-dimensional CT data set – were thresholded by 13 experts, worldwide. At the same time, variability of the outcomes of a set of automatic thresholding algorithms, applied to portions of the test images, was also investigated. The experimental results obtained illustrate the fact that experts rely on very different approaches to threshold images of soils, and that there is considerable observer influence associated with this thresholding. This observer dependence is not likely to be alleviated by adoption of one of the many existing automatic thresholding algorithms, many of which produce thresholded images that are equally, or even more, variable than those of the experts. These observations suggest that, at this point, analysis of the same image of a soil, be it a simple photograph or 3-dimensional X-ray CT data, by different individuals can lead to very different results, without any assurance that any of them would be even approximately “correct” or best suited to the objective at hand. Different strategies are proposed to cope with this situation, including the use of physical “standards”, adoption of procedures to assess the accuracy of thresholding, benchmarking with physical measurements, or the development of computational methods that do not require binary images.
We investigated the utility of combining micro-scale computed tomography (micro-CT), image analysis and geostatistics to quantify pore geometry at spatial scales ranging from 4.4 μm to 2 mm. To facilitate this, we investigated soil taken from an old permanent upland pasture known to support very high levels of microbial diversity and that had not been cultivated for many centuries. Aggregates (< 3 mm in diameter) from three treatments (control, sewage sludge amended and biocide treated) derived from the site were imaged using synchrotron-based computed microtomography. Image analysis was used to determine aggregate porosity and pore shape parameters, and semivariance analysis was used to measure the spatial correlation of pore space within the three land treatments. For all plots high porosities were observed (c. 30%) at scales below 3 mm. High variations of porosity were also observed, ranging from 22% to 47%, but no significant differences among treatments were found. No significant differences among treatments were found in the distribution of pores within aggregates, as revealed by semivariance analysis, or in pore shape parameters. No treatment effects were observed. However, the work presented here shows that the combination of approaches adopted has great potential for quantifying the soil microbial physical habitat. Future work, should investigate the use of these novel techniques in more controlled soil ecosystems to provide an exciting new way of understanding the soil-microbe interactions at appropriate scales.
The aim of the present study was to estimate the relative contribution of cytochrome P450 isoforms (P450s), including P450s of the CYP2C subfamily, to the metabolism of caffeine in human liver. The experiments were carried out in vitro using cDNA-expressed P450s, liver microsomes and specific P450 inhibitors. The obtained results show that (1) apart from the 3-N-demethylation of caffeine – a CYP1A2 marker reaction and the main oxidation pathway of caffeine in man – 1-N-demethylation is also specifically catalyzed by CYP1A2 (not reported previously); (2) 7-N-demethylation is catalyzed non-specifically, mainly by CYP1A2 and, to a smaller extent, by CYP2C8/9 and CYP3A4 (and not by CYP2E1, as suggested previously); (3) C-8-hydroxylation preferentially involves CYP1A2 and CYP3A4 and, to a smaller degree, CYP2C8/9 and CYP2E1 (and not only CYP3A, as suggested previously) at a concentration of 100 μM corresponding to the maximum therapeutic concentration in humans. At a higher caffeine concentration, the contribution of CYP1A2 to this reaction decreases in favour of CYP2C8/9. The obtained data show for the first time the contribution of CYP2C isoforms to the metabolism of caffeine in human liver and suggest that apart from 3-N-demethylation, 1-N-demethylation may also be used for testing CYP1A2 activity. Moreover, they indicate that the C-8-hydroxylation is not exclusively catalyzed by CYP3A4.
The use of gamma (γ-) irradiation as a method for soil sterilisation for laboratory experiments has been recommended over other sterilisation techniques. We reviewed literature dating back over 50 years to investigate the chemical and biological effects on γ-irradiated soils and to determine its practicality for sterilising soils which will subsequently be used for experimental purposes. Typically, γ-irradiation at 10 kGy will eliminate actinomycetes, fungi and invertebrates in most soils. The majority of soil bacteria are eliminated by 20 kGy, however, a dose higher than 70 kGy may be required to kill certain radio-resistant bacteria. We recommend prior to experimentation that the radiosensitivity of soils are determined so as to ensure the desired chemical and biological effects are achieved. γ-Irradiation may not be an appropriate method for all experiments as it can influence soil chemical properties, in particular soil nitrate and ammonium levels. Where chemical stability is required we recommend sterilising soils air-dry rather than moist.
In recent years, methods of molecular microbiology have been used for the investigation of soil microbial diversity. Fluorescence in situ hybridization (FISH) represents a method which allows a specific staining and enumeration of soil microorganisms by using fluorescent-labelled oligonucleotide probes. However, the detection of FISH-stained cells is often affected by strong autofluorescence of the background, especially in samples of the top soils.In this study a more efficient FISH-approach coupled with catalyzed reporter deposition (CARD) was adapted to soils. Due to tyramide signal amplification (TSA) the fluorescence intensity has been considerably increased at the target binding site of a probe.Six different soils were investigated to evaluate the effect of sample preparation and pre-treatments, TSA, and the procedure of detection. The results show that both cell permeabilization and TSA are two important factors which improve in situ hybridization of soil microorganisms. Soils with higher clay contents have shown better results when prepared on polycarbonate filters rather than on glass slides.Using specific fluorescence filter systems and dye combinations the detection of hybridized cells was extensively increased compared with the application of monolabelled oligonucleotide probes in regular FISH-analysis. As a result, CARD-FISH-stained cells were suitable for automated counting using digital image analysis. Nevertheless, the counterstain with DAPI had to be analyzed manually as it was strongly affected by autofluorescence.
The kEC value (=extractable part of microbial biomass C) of the fumigation-extraction (FE) method was assessed on the basis of 153 soils (94 arable, 46 grassland and 13 forest soils) by indirect calibration using the fumigation-incubation (FI) method. Sixty-six soils were investigated for the first time and the data on a further 87 soils were obtained from the literature. The single kEC values ranged from 0.23 to 0.84. A split according to the form of land use resulted in a significantly (Scheffé, P = 0.05) lower kEC value for the arable soils (0.42; n = 94) in comparison to those for the grassland (0.49; n = 46) and the forest soils (0.51; n = 13). This difference is mainly due to the significant effects of the respiration rate measured in non-fumigated control samples of the FI method which was used for calibration of the kEC value. For that reason, I investigated the effects of incubation temperature (22°, 25° and 28°C) on biomass C data obtained by the FI method, and thus on the kEC value of the FE method, and discuss further problems of direct and indirect calibration. Based on experimental and literature data, I conclude that the kEC values of Vance et al. (Soil Biology & Biochemistry19, 703–707, 1987) and Wu et al. (Soil Biology & Biochemistry22, 1167–1169, 1990) remain valid. A kEC value of 0.38 can be recommended for C analysis by dichromate consumption and a kEC value of 0.45 for that by UV-persulfate or oven oxidation.
For many analyses, grey scale images from X-ray tomography and other sources need to be segmented into objects and background which often is a difficult task and afflicted by an arbitrary and subjective choice of threshold values. This is especially true if the volume fraction of objects is small and the histogram becomes unimodal. Bi-level segmentation based on region growing is a promising approach to cope with the fuzzy transition zone between object and background due to the partial volume effect, but until now there is no method to properly determine the required thresholds in case of unimodality. We propose an automatic and robust technique for threshold selection based on edge detection. The method uses gradient masks which are defined as regions of interest for the determination of threshold values. Its robustness is analysed by a systematic performance test and finally demonstrated for the segmentation of pores in different soils using images from X-ray tomography.
A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100mM arsenate and 10mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.
The bacterial community composition of the active layer (0-45 cm) of a permafrost-affected tundra soil was analysed by fluorescence in situ hybridisation (FISH). Arctic tundra soils contain large amounts of organic carbon, accumulated in thick soil layers and are known as a major sink of atmospheric CO(2). These soils are totally frozen throughout the year and only a thin active layer is unfrozen and shows biological activity during the short summer. To improve the understanding of how the carbon fluxes in the active layer are controlled, detailed analysis of composition, functionality and interaction of soil microorganisms was done. The FISH analyses of the active layer showed large variations in absolute cell numbers and in the composition of the active microbial community between the different horizons, which is caused by the different environmental conditions (e.g., soil temperature, amount of organic matter, aeration) in this vertically structured ecosystem. Universal protein stain 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) showed an exponential decrease of total cell counts from the top to the bottom of the active layer (2.3 x 10(9)-1.2 x 10(8) cells per gram dry soil). Using FISH, up to 59% of the DTAF-detected cells could be detected in the surface horizon, and up to 84% of these FISH-detected cells could be affiliated to a known phylogenetic group. The amount of FISH-detectable cells decreased with increasing depth and so did the diversity of ascertained phylogenetic groups.
Archaea (archaebacteria) are a phenotypically diverse group of microorganisms that share a common evolutionary history. There are four general phenotypic groups of archaea: the methanogens, the extreme halophiles, the sulfate-reducing archaea, and the extreme thermophiles. In the marine environment, archaeal habitats are generally limited to shallow or deep-sea anaerobic sediments (free-living and endosymbiotic methanogens), hot springs or deep-sea hydrothermal vents (methanogens, sulfate reducers, and extreme thermophiles), and highly saline land-locked seas (halophiles). This report provides evidence for the widespread occurrence of unusual archaea in oxygenated coastal surface waters of North America. Quantitative estimates indicated that up to 2% of the total ribosomal RNA extracted from coastal bacterioplankton assemblages was archaeal. Archaeal small-subunit ribosomal RNA-encoding DNAs (rDNAs) were cloned from mixed bacterioplankton populations collected at geographically distant sampling sites. Phylogenetic and nucleotide signature analyses of these cloned rDNAs revealed the presence of two lineages of archaea, each sharing the diagnostic signatures and structural features previously established for the domain Archaea. Both of these lineages were found in bacterioplankton populations collected off the east and west coasts of North America. The abundance and distribution of these archaea in oxic coastal surface waters suggests that these microorganisms represent undescribed physiological types of archaea, which reside and compete with aerobic, mesophilic eubacteria in marine coastal environments.
Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.
In situ hybridization with rRNA-targeted oligonucleotide probes has become a widely applied tool for direct analysis of microbial population structures of complex natural and engineered systems. In such studies probe EUB338 (AMANN et al., 1990) is routinely used to quantify members of the domain Bacteria with a sufficiently high cellular ribosome content. Recent reevaluations of probe EUB338 coverage based on all publicly available 16S rRNA sequences, however, indicated that important bacterial phyla, most notably the Planctomycetales and Verrucomicrobia, are missed by this probe. We therefore designed and evaluated two supplementary versions (EUB338-II and EUB338-III) of probe EUB338 for in situ detection of most of those phyla not detected with probe EUB338. In situ dissociation curves with target and non-target organisms were recorded under increasing stringency to optimize hybridization conditions. For that purpose a digital image software routine was developed. In situ hybridization of a complex biofilm community with the three EUB338 probes demonstrated the presence of significant numbers of probe EUB338-II and EUB338-III target organisms. The application of EUB338, EUB338-II and EUB338-III should allow a more accurate quantification of members of the domain Bacteria in future molecular ecological studies.
Bacterial communities of four arable soils--pelosol, gley, para brown soil, and podsol brown soil--were analysed by fingerprinting of 16S rRNA gene fragments amplified from total DNA of four replicate samples for each soil type. Fingerprints were generated in parallel by denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP) to test whether these commonly applied techniques are interchangeable. PCR amplicons could be separated with all three methods resulting in complex ribotype patterns. Although the fragments amplified comprised different variable regions and lengths, DGGE, T-RFLP and SSCP analyses led to similar findings: (a) a clustering of fingerprints which correlated with soil physico-chemical properties, (b) little variability between the four replicates of the same soil, (c) the patterns of the two brown soils were more similar to each other than to those of the other two soils, and (d) the fingerprints of the different soil types revealed significant differences in a permutation test, which was recently developed for this purpose.
Methods in Applied Soil Microbiology and Biochem-istry Uncertainties in determining microbial biomass C using the chloroform fumigation-extraction method
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- P Nannipieri
Alef, K., Nannipieri, P., 1995. Methods in Applied Soil Microbiology and Biochem-istry. Academic Press, London. Alessi, D.S., Walsh, D.M., Fein, J.B., 2011. Uncertainties in determining microbial biomass C using the chloroform fumigation-extraction method. Chemical Ge-ology 280, 58e64.
Biophysics of the vadose zone: from reality to model systems and back again PAST: paleontological statistics soft-ware package for education and data analysis
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Rad Pro X-ray Device Dose-rate Calculator Online. Massachusetts Institute of Technology
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McGinnis, R., 2009. Rad Pro X-ray Device Dose-rate Calculator Online. Massachusetts Institute of Technology.
Investigating microbial micro-habitat structure using X-ray computed tomography Visu-alization, modelling and prediction in soil microbiology Non-invasive 3D analysis of local soil deformation under mechanical and hydraulic stresses by mCT and digital image correlation. Soil and Tillage Research 111
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- J D Crawford
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- J Nellesen
- G Fischer
- R Horn
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