Cd and Pb removal from contaminated environment by metal resistant bacterium Cupriavidus metallidurans CH34: importance of the complexation and competition effects

The present study aims to improve the understanding of the role of complexation and competition effects on Cd and Pb accumulation by the metal resistant bacterium Cupriavidus metallidurans largely used in bioremediation. Adsorbed and intracellular metal content in bacteria were determined in model exposure medium within a concentration range spanning from 10(-9) to 5 x 10(-5) M of Cd or Pb and water extracts from soils. In parallel, the free metal ion concentrations ([M2+]) were measured by an ion exchange technique. Obtained results demonstrated that Cd and Pb accumulation by C. metallidurans was related to [M2+] in the solution. The adsorbed and intracellular M fractions were significantly reduced by nitrilotriacetic acid, Elliot or Pahokee Peat humic acids, as well as by a large excess of Ca, Mg and Zn. No effect on Cd and Pb bioaccumulation was observed in the presence of Mn, Cu or Co at a 10-fold excess for bacteria exposed to 10(-6) M of Cd or Pb. Adsorbed and intracellular metal determined when bacteria were exposed to water extracts of soil were in the same order as expected from the model experiments when complexation and competition effects are considered. The study emphasises the necessity of taking into account chemical site-specificity of soil solutions and water, including dissolved organic ligands, pH and the presence of other metals when developing metal removal technologies by living bacteria.

Behaviour of carbon-14 containing low molecular weight organic compounds in contaminated groundwater under aerobic conditions

Aislinn Ann Boylan

Short chain carbon-14 (C-14) containing organic compounds can be formed by abiotic oxidation of carbides and impurities within nuclear fuel cladding. During fuel reprocessing and subsequent waste storage there is potential for these organic compounds to enter shallow subsurface environments due to accidental discharges. Currently there is little data on the persistence of these compounds in such environments. Four C-14-labelled compounds (acetate; formate; formaldehyde and methanol) were added to aerobic microcosm experiments that contained glacial outwash sediments and groundwater simulant representative of the Sellafield nuclear reprocessing site, UK. Two concentrations of each electron donor were used, low concentration (10(-5) M) to replicate predicted concentrations from an accidental release and high concentration (10(-2) M) to study the impact of the individual electron donor on the indigenous microbial community in the sediment. In the low concentration system only similar to 5% of initial C-14 remained in solution at the end of experiments in contact with atmosphere (250-350 h). The production of (CO2)-C-14(g) (measured after 48 h) suggests microbially mediated breakdown is the primary removal mechanism for these organic compounds, although methanol loss may have been partially by volatilisation. Highest retention of C-14 by the solid fractions was found in the acetate experiment, with 12% being associated with the inorganic fraction, suggesting modest precipitation as solid carbonate. In the high concentration systems only similar to 5% of initial C-14 remains in solution at the end of the experiments for acetate, formate and methanol. In the formaldehyde experiment only limited loss from solution was observed (76% remained in solution). The microbial populations of unaltered sediment and those in the low concentration experiments were broadly similar, with highly diverse bacterial phyla present. Under high concentrations of the organic compounds the abundance of common operational taxonomic units was reduced by 66% and the community structure was dominated by Proteobacteria (particularly Betaproteobacteria) signifying a shift in community structure in response to the electron donor available. The results of this study suggest that many bacterial phyla that are ubiquitous in near surface soils are able to utilise a range of C-14-containing low molecular weight organic substances very rapidly, and thus such substances are unlikely to persist In aerobic shallow subsurface environments.

ELSEVIER SCI LTD2018

The effect of seasonal soil frost on the alpine groundwater recharge including climate change aspects

Daniel Bayard

In alpine areas, the snow cover plays an important role as a water reservoir. Water is stored as snow over the winter and released in spring, recharging mountain aquifers through infiltration. These aquifers are essential, especially for supplying water for human activities during dry seasons. Numerous studies have shown that locally soil frost can drastically reduce the water infiltration. However, we know much less about the hydrological impact of soil frost at a larger scale, in particular with regard to groundwater recharge. This was our motivation for initiating an extensive field experiment in the southern Swiss Alps. Several methods at different spatial scales were adapted to explore (a) the local effect of a partially frozen ground on the snowmelt discharge in an alpine area, (b) the key processes influencing groundwater recharge during snowmelt periods, such as snow cover and soil frost evolution, snowmelt water runoff types, (c) the large-scale effect of soil frost on the aquifer recharge, and (d) winter situations that are critical with respect to flooding. In order to take into account spatial, altitudinal and climatic differences, the field study was run for two winter seasons at Gd St Bernard (2500 m), a site with very high winter precipitation and strong winds, and at Hannigalp above Grächen (2100 m), a ski resort with a rather dry winter climate. The different components of the soil water balance (lateral runoff, deep percolation, liquid soil water content) were measured on delimited plots of 5 m2. Additionally, a dye tracer experiment visualized the snowmelt infiltration patterns into the frozen, respectively non-frozen ground. At Gd St Bernard, investigations of the liquid water content and the soil temperature were additionally conducted at two secondary sites, differing in their orientation, so as to gain a better insight of the spatial soil frost expansion. Finally, an analysis of the water-table variation at the village of Grächen (some 500 m below Hannigalp) during the snowmelt was carried out for a 10-year time period, including our experimental seasons. We complemented our field investigations by using a numerical soil-snow-atmosphere transfer model that simulates the seasonal development of the soil frost and the snow cover, as well as lateral and vertical water flow in the top soil. This model was used as an upper boundary condition to a 2-D groundwater model, to calculate the outlet flow at the base of a simplified alpine aquifer. The two winters investigated had contrasting meteorological conditions, which resulted in intriguing differences and similarities with respect to the soil physical conditions: the first one having a thick snowpack and hardly any soil frost, and the second one with hardly any snow until January and a deep and persistent soil frost. In spite of a very thick snowpack, the water balance measurements from winter 2000/2001 showed for both sites zero to low surface and subsurface flow due to the fact that soil frost was local and the soil permeability high. During the next winter, approximately 25% of the total meltwater run off laterally. The soil infiltration capacity was mainly reduced by the presence of a basal ice sheet, caused by the freezing of meltwater after a mid-winter snowmelt event. The field data were used to calibrate and validate a physically-based one-dimensional model. To allow an accurate parameter setting for the water repellent soil of Hannigalp, laboratory infiltration experiments on soil columns were additionally carried out. Using the resultant information, the model predicted correctly most measured soil parameters, in particular good matching was observed between measured and simulated snowmelt discharge. At Gd St Bernard, the simulated results were less satisfactory, especially during the first winter, when large discrepancies were noted between measured and simulated lateral runoff. Apart from the shortage in the soil parameter characterization, the deviation was a result of the simplified description of the experimental field. The model was, for example, not able to simulate surface runoff generated by a steep slope under unfrozen conditions, as the soil surface is considered by the model to be flat. Nevertheless, results were accurate enough to reproduce the occurrences of snowmelt events and the accumulated values of snow-melt discharge pathways under frozen conditions. Long-term weather data were used to simulate the soil frost depth at both sites. The simulation results showed that the probability of the occurrence of frozen soils was lowest on slopes with south or north exposure. On southerly exposed plots, the heat stored in the soil during the summer inhibited the development of deep and persistent soil frost, while most pore ice thawed due to underneath heating. On north plots, the building of soil frost was rare, as early snowfall insulated the ground from the atmosphere. However, during snow-poor winters, the frost penetrated deep into the ground, and the low underneath heat flux at that location hardly affected the frost depth until snowmelt. The snow depth and average soil temperature were hence the two components affecting the frost depth extent. The altitude soil frost variation was related to the spatial extent of these two components. The higher the altitude, the higher the precipitation and the lower the mean air/surface temperature. Simulating the whole Grächen recharge area, seasonal soil frost was rare between 1800 m and 2000 m, as the snowpack was thick enough to insulate the ground, and the soil was warm enough to inhibit deep soil frost. In lower areas, the shallow snowpack enabled the soil to freeze during most winters, whereas at higher areas, deep and persistent soil frost was simulated due to the low underneath heating. A 10-year water-table depth record was used to examine possible large-scale effects of seasonal soil frost. We noted that the water-table rise at snowmelt was lowest at the end of winters with extensive soil frost. However, such winters were characterized by low precipitation, and reduced snow cover. In contrast, winters with little soil frost were mostly snow rich. Consequently, only a reduced number of winters were directly comparable. Considering these winters, the reduction in the water-table rise at snowmelt due to seasonal soil frost varied only between 10 and 30%, as, during snowmelt, most meltwater was able to re-infiltrate into the very permeable ground in lower unfrozen areas. We may assume that a soil with a smaller infiltration capacity can increase the influence of the soil frost on the aquifer recharge. Finally, we investigated the effect of a changing climate on the hydraulic and thermic regimes at different altitudes in the area of Grächen. We assume an increase in the air temperature of 2°C generates deeper soil frost on lower areas, as the resultant smaller snow cover does not insulate the soil from the atmosphere any more. However, it does reduce the soil frost on higher, snow rich areas, due to the warmer air temperature. As a whole, minor changes were simulated on the mean groundwater recharge, as more/less meltwater was able to infiltrate in higher/lower areas. By increasing additionally the precipitation by 15%, the soil frost diminished slightly over the whole catchment. By combining both climate change scenarios, we may expect an increase in extreme events, as, on the one hand, the rainfall intensity increases, and, on the other hand, rain on snow events over frozen soils occurs more often in lower areas. In practice, this work shows the importance of better investigating the hydrothermal behaviour of alpine regions. Such a knowledge is necessary, as more and more efforts are put into managing efficiently, durably and globally the available water resources. This work stresses also the necessity to consider seasonally soil frost in alpine risk management. As an example, flooding is mostly a consequence of strong rain precipitation at high altitudes combined with heavy snowmelt. Obviously, the risk of extreme runoff events increases during frozen winters, when the soil infiltration capacity is further reduced by the soil ice. Also, it has been shown that unstable slopes are mostly related to specific hydrogeological situations, like strong precipitation, which induces a sharp increase in the underneath water circulation. The melt water in spring is hence a potential activating factor, and a frozen soil may reduce the risk of sliding, as less water infiltrates into the ground.

EPFL2003

Molecular analysis of the biodiversity of protists in hydrocarbon-polluted and pristine soils

Enrique Lara

Eukaryotic microorganisms are an essential component of the Earth's global diversity. It is believed that the number of protistan taxa far outnumbers the one of viruses and bacteria. The taxonomic classification of these organisms, which also represent the major part of the total eukaryotic diversity, is still not very well known. It is only lately that the main phylogenetically consistent eukaryotic super assemblages (also called "kingdoms") have been defined. Inside these kingdoms, lateral branches represent the main multicellular taxa, the plants, animals and fungi. Protists are present in virtually all environments; in this thesis, attention was focused on the soil as habitat for the eukaryotic microorganisms. The communities inhabiting soil are characterized by adaptations to drought (the capacity to form cysts). Moreover, certain groups are found exclusively in this environment, to which they are morphologically adapted, whereas others are absent and exclusively found in aquatic systems. Detailed studies on the ecology of certain groups have been performed, and it is possible to define amongst them typical fast reproducing, early colonizers for new environments (r-specialists), whereas others can be found only in stable and highly productive habitats (K-specialists). All these organisms are distributed in a heterogeneous manner in the soil, being usually more frequent in the top horizon and around hotspots such as, for instance, root tips. Prokaryotes have drawn most of the attention of microbial ecologists, who almost neglected the eukaryotic microbes with the exception of fungi. Ecologists usually define four categories ("guilds") of protists in soil: Flagellates, naked amoebae, testate amoebae and ciliates. The importance of protistan activity in soil ecosystems has been shown with the concept of microbial loop: nutrients, produced by autotrophic organisms, are taken up by decomposers (bacteria and fungi) to produce their own biomass. Heterotrophic protists, by preying on them, release these compounds in a form which can be directly used by the autotrophs ("mineralization"), thus closing the loop. This phenomenon is of major significance, because protistan grazing controls the composition and activity of the prokaryotic community, and also the growth of the autotrophs. In soil, this phenomenon takes mainly place at the tip of the roots of vascular plants, where exudates are released. When soils are exposed to an organic pollution, the protistan communities and thus also their impact on the microbial loop, are influenced in two different ways: either by the direct toxic effects of the pollutant and or the indirect effects on their bacterial preys. A detailed study on the negative effects of environmental pollution, however, requires reliable methods to study protists. Molecular approaches offer the possibility to study protists in situ without the limitations inherent to cultivation dependent approaches. The aim of this thesis was to develop molecular methods and to apply these in combination with classical approaches for the characterization of protistan communities in a polycyclic aromatic hydrocarbon contamined and a non-contaminated control soil (PAH). The eukaryotic communities in a PAH polluted and a non-polluted control soil were first compared by amplifying total soil DNA with eukaryotic domain-specific primers targeting the 18S rRNA genes, cloning and sequencing the resulting PCR products. Using non-specific eukaryotic primers proved to be non-applicable for a reliable analysis of the 18S rRNA gene pools in soil, since protistan 18S rRNA gene sequences were (with the exception of the genus Acanthamoeba) underrepresented in the clone libraries obtained.. The biased picture obtained in the first chapter of the thesis was clearly shown when protist were cultivated from both soils: enrichment and cultivation of protists confirmed that members of the genus Acanthamoeba were indeed the most common protists in polluted soils, whereas they were completely absent from the control soil. Additionally, a diverse protistan community was recovered, which could not be detected by the molecular 18S rRNA-based approach. Twenty-two isolates of flagellates and naked amoebas were obtained, and characterised morphologically as well as by 18S rRNA gene sequence analysis. The diversity of cultivated organisms from the polluted soil was lower as compared to the control soil; however, the total number of protists was higher. Some of the cultures obtained in this cultivation dependant approach were used for further detailed studies; a jakobid isolate was described using light and electron microscopy, thus providing new insights into the knowledge of a relatively unknown group of excavates, i.e. the group of Jakoba incarcerata. Other cultures belonging to the kineoplastea were used to design a set of 18S rRNA-targeting fluorescently labelled oligonucleotide probes for fluorescence in situ hybridization. These probes are specific for the detection of the class Kinetoplastea and four genera within the class, i.e. Bodo, Parabodo, Rhynchomonas and Dimastigella and were applied to pure cultures and to environmental samples obtained by the so-called coverslip method. The effect of the PAH-pollution on a selected soil protistan guild, the ciliates, was further analyzed by applying ciliate-specific PCR based techniques. A specific primer set was used to amplify ciliate 18S rRNA genes from the environment and to conduct comparative sequence analysis of environmental clone libraries. This study indicated that the ciliate 18S rRNA gene pools present in the polluted soil were less diverse than those of the control soil. They contained mainly representatives of the class Colpodea, a clade of r-selected ciliates, typical for unstable environments. The control soil was characterized by a variety of trophic specialists, including carnivores, cyanobacteria and diatom feeders, in contrast to the polluted soil, which harboured mainly sequences affiliating to bacterivores and generalists. Based on these results, we could deduce that the trophic webs are less complex in a PAH polluted soil in comparison to a pristine control. This observation was further confirmed when we studied a larger number of samples using a fingerprinting technique, i.e. denaturing gradient gel electrophoresis (DGGE) with ciliate specific PCR primers. The profiles obtained showed again a dominance of the members of the class Colpodea in polluted soils.

EPFL2005