Soil contamination | EPFL Graph Search

Soil contamination, soil pollution, or land pollution as a part of land degradation is caused by the presence of xenobiotic (human-made) chemicals or other alteration in the natural soil environment. It is typically caused by industrial activity, agricultural chemicals or improper disposal of waste. The most common chemicals involved are petroleum hydrocarbons, polynuclear aromatic hydrocarbons (such as naphthalene and benzo(a)pyrene), solvents, pesticides, lead, and other heavy metals. Contamination is correlated with the degree of industrialization and intensity of chemical substance. The concern over soil contamination stems primarily from health risks, from direct contact with the contaminated soil, vapour from the contaminants, or from secondary contamination of water supplies within and underlying the soil. Mapping of contaminated soil sites and the resulting cleanups are time-consuming and expensive tasks, and require expertise in geology, hydrology, chemistry, computer modelin

Analysis of microbial community structures and functions in heavy metal-contaminated soils using molecular methods

Fabienne Gremion

The contamination of agricultural land and groundwater by heavy metals is essentially linked to human activities. A major problem with heavy metals is that they cannot be biodegraded and therefore reside in the environment for long periods of time if they are not removed. Thus, depending of the kind and depth of contamination, different remediation techniques were developed. One of these methods, called "phytoextraction", uses the ability of so-called hyperaccumulating plants to extract high amounts of heavy metals. Accumulation of heavy metals in the environment is a serious concern for animal and human health. At the microscopic scale, heavy metals may have also deleterious effects on bacteria which are the key-players of the different nutrient turnovers in soils. Consequently, ecosystem functioning can be seriously perturbed and the long-term soil fertility may be threatened. The recent development of molecular biology greatly contributed to the discovery of the microbial diversity and its function in the soil. However, to date only a small number of studies used molecular methods to investigate the impacts of heavy metals on the bacterial community. In this thesis, a pot experiment was conducted under controlled conditions with one hyperaccumulating plant (Thlaspi caerulescens) grown in two different soils, a long-term and an artificially heavy metal-contaminated soil. The impact of heavy metals on the microbial community was then investigated with several molecular methods. Moreover, the decrease of the bioavailable heavy metals concentrations in soil due to plant uptake allowed to study the consequences on bacterial community structure and function. Based on the 16S ribosomal RNA and the corresponding gene (16S rDNA), four clone libraries were constructed to retrieve information on the structure of the microbial community and the potentially active part of the microbial community in the rhizosphere of Thlaspi caerulescens grown during three months in a long-term contaminated soil. The data obtained with the two clone libraries (rRNA and rDNA) from the rhizosphere of Thlaspi caerulescens were compared with the bulk soil data to identify any effect of the plant on the soil microbial community structure. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, α-Proteobacteria, β-Proteobacteria, Acidobacteria and the Planctomycetales. The taxa dominating the bacterial community structure in the bulk soil also dominated the rhizosphere community, indicating that the plant did not exert a major influence on the overall bacterial diversity. However, all dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries as compared to the rDNA libraries. On the contrary, sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA. Seventy per cent of these clone sequences were related to two subgroups of the Rubrobacteria, which was an indication that this group of bacteria was probably metabolically active in heavy metal-contaminated soils. Fluorescence in situ hybridization (FISH) was used for the in situ detection and quantification of selected bacterial groups previously detected in the clone libraries from the rhizosphere of Thlaspi caerulescens. By applying the most general probe EUB338, only 20% of the total rhizosphere microbial community could be detected. Based on this result, it was difficult to conclude with certitude which were the most dominant bacteria in the rhizosphere. However, despite this low detection rate, it was possible to detect the major groups present in the rhizosphere clone libraries using group-specific oligonucleotide probes. As part of our sequences were affiliated to two emerging bacterial groups, the Acidobacteria and the Rubrobacteria, two new probes were designed, Acido228 (specific for the subgroup 1 of the Acidobacterium division) and Rubro198 (specific for the all Rubrobacteria subclass), for the detection of these microorganisms. These two probes were first checked for their specificity with pure cultures and finally applied in the rhizosphere soil allowing for the first time the detection of these organisms in situ. Finally, the impact of heavy metals and a subsequent one-year phytoextraction with Thlaspi caerulescens on the soil microbial community was investigated in an artificially heavy metal-contaminated soil. All the different molecular and culture-dependent techniques used, denaturing gradient gel electrophoresis (DGGE), community level physiological profile (CLPP) and potential ammonium-oxidation measurement showed that the heavy metal addition induced drastic changes in the bacterial community. Moreover, the analysis of the different bacterial DGGE patterns (Bacteria, β-Proteobacteria and ammonia-oxidising bacteria) obtained during this experiment showed that one-year phytoremediation was not sufficient to recover the initial community present in the non-contaminated soil. However, with the CLPP analysis, it was possible to detect a stimulating effect of the plant on a part of the microbial community in both contaminated and non-contaminated soils. The most obvious result was obtained in the contaminated soil where the number of substrates metabolised increased significantly in the presence of the plant as compared to the unplanted contaminated samples. The measurement of the potential ammonium-oxidation was used as a criterion for soil quality. Although this test showed that the ammonia-oxidising bacteria were significantly stimulated in the planted non-contaminated soil samples, the positive effect of the plant on these bacteria was not sufficient to overcome the inhibition induced by the presence of the heavy metals in the contaminated soil, even after one year phytoremediation. To conclude, molecular methods in combination with culture-dependent techniques have proven in this study to be very useful for the detection of the changes induced by the heavy metals in the structure and the function of the microbial community. Moreover, the molecular techniques contributed to the identification of bacteria which could be potentially used for the bioremediation of contaminated soils thus offering new perspectives of investigation and technology development.

EPFL2003

Insights into the microbial reduction of pentavalent and hexavalent uranium species by Shewanella oneidensis MR-1

Margaux Camille Andréa Molinas

Decades of uranium-related activities such as ore mining, nuclear power generation, weapon manufacture, storage of nuclear wastes, have contributed to the release of U in the environment. U occurrence is concerning, since in surface environments, U is typically found in the U(VI) oxidation state, as uranyl, complexed with various ligands, and is highly soluble. Therefore, this toxic metal is likely to leech into soils and contaminate surface waters and groundwaters. In addition, owing to the long half-lives of the predominant isotopes 238U and 235U, U is considered as a persistent contaminant over geological timescales. For several decades, efforts have focused on the development of in situ remediation solutions to tackle U pollution in the subsurface, and limit its mobility.With this aim, interest in the metabolic potential for U(VI)-respiration by dissimilatory metal-reducing bacteria (DMRB), such as Shewanella oneidensis MR-1, has seen a significant increase. This metabolically-versatile bacterium has been reported to reduce mobile U(VI) to typically insoluble crystalline and amorphous U(IV). The reduction of U(VI) in S. oneidensis MR-1 is coupled to the oxidation of an electron donor, which feeds electrons into an electron transport chain, extending from the cytoplasm to the outer-membrane of the cells. The microbially mediated reduction of U(VI) to U(IV) is the result of a two-step process. It is assumed that one electron is first transferred to U(VI) to form a pentavalent U(V) intermediate, followed by the abiotic disproportionation of two U(V) atoms into U(IV) and U(VI). However, evidence for this mechanism is limited to experimental systems rich in carbonate, which permits the rapid disproportionation of U(V). Thus, it remains unclear whether a second, biologically-mediated, electron transfer to U(V) is possible under conditions in which disproportionation is limited. To explore this, a novel U(V)-dpaea complex, that is stable in water at pH 7, was utilized to investigate the second step of the reduction mechanism. Here, we observed that U(V) can be biologically reduced by an additional one-electron transfer, resulting in the accumulation of U(IV) without the need for disproportionation. To improve our understanding of the molecular mechanism of U-dpaea reduction, we incubated mutant strains of S. oneidensis MR-1, lacking (i) only outer-membrane c-type cytochromes or (ii) all c-type cytochromes, with solid phase U(VI)-dpaea and aqueous U(V)-dpaea. We determined that U(VI)-dpaea reduction proceeds via the initial dissolution of the solid phase and that U(V)-dpaea reduction is mediated by outer-membrane c-type cytochromes. In particular, in vitro reactions between the purified outer-membrane c-type cytochrome MtrC and U(V)-dpaea demonstrated that MtrC can directly transfer electrons to U(V)-dpaea.Finally, we sought to determine the factors that influence electron transfer kinetics between DMRB and U. To this end, we reacted U(VI) coordinated by various aminocarboxylate ligands with purified MtrC. Here, U speciation significantly impacted reduction rates and appeared to be related to the binding strength of the U-MtrC interaction, i.e., hydrogen bonding versus electrostatic.All together, these findings provide further insights in the reduction mechanism of U by DMRB, and underline the importance of U speciation in controlling the pathway and rate of electron transfer.

EPFL2021

Assessment of microbial community changes and limiting factors during bioremediation of hydrocarbon-polluted soil with new miniaturized physiological methods

Karin Kaufmann

Due to human activities, organic pollutants are spilled to the environment where they threaten public health, often as contaminants of soil or groundwater. Living organisms are able to transform or mineralize many organic pollutants, and bioremediation techniques have been developed to remove pollutants from a contaminated site. However, fast and easy methods to document both the efficacy of bioremediation and the changes in soil microbial communities during bioremediation are not well developed. The major aim of this thesis was to develop miniaturized methods targeting the physiology of microbial communities during pollutant degradation in soil and to assess pollution-induced community changes. Furthermore, the methods should identify factors limiting efficient pollutant biodegradation. Petroleum hydrocarbons have been chosen as pollutants because these compounds are frequently spilled, readily water-soluble, partly toxic and carcinogenic, and therefore undesirable in soils and drinking water supplies. The influence of petroleum hydrocarbons on a microbial community in the vadose zone was assessed under field conditions. An artificial hydrocarbon mixture consisting of volatile and semi-volatile compounds similar to jet-fuel was emplaced in a previously uncontaminated vadose zone in nutrient-poor glacial melt water sand. The experiment included monitoring of microbial parameters and CO2 concentrations in soil gas over 3 months, in- and outside the hydrocarbon vapor plume that formed around the buried petroleum. Microbial and chemical analyses of vadose zone samples were performed on material from 9 cores drilled on 3 days at 3 distances from the buried petroleum mass to 3.3 m depth. Significantly elevated CO2 concentrations were observed after contamination. Total cell numbers as determined by fluorescence microscopy were strongly correlated with soil organic carbon and nitrogen content but varied little with contamination. Redundancy analysis (RDA) allowed direct analysis of effects of selected environmental variables or the artificial contamination on microbiological parameters. Variation in biomass and CO2 production was to 46 % explained by soil parameters and to 39.8 % by the duration of contamination. The microbial community structure was assessed by community-level physiological profile analysis using BiologTM EcoPlates. Only 35.9 % of variation in BiologTM data could be attributed to soil parameters and contamination, however, the samples with greatest exposure to hydrocarbons grouped together on RDA plots. It is concluded that at this nutrient-poor site, the microbial community was dominated by the natural heterogeneity and that the influence of petroleum hydrocarbon vapor was weak. BiologTM Eco-Plates combined with RDA were able to distinguish between samples with high and low hydrocarbon exposure. However, the method was not sensitive enough to produce consistent patterns in three replicates when extracts from subsurface soils with low cell numbers were inoculated. A more sensitive, MPN-based system was therefore developed and its ability to provide information about specific pollutant degraders was tested in alluvial sand. Eight dilutions of sand extracts were inoculated on medium- and substrate-containing polypropylene deep-well plates. To keep the concentrations of the volatile substrates constant over several days of incubation without intoxicating the soil community, a substrate-containing organic carrier phase was applied to each vial. The biochemically inert 2,2,4,4,6,8,8-heptamethylnonane (HMN) was appropriate for this purpose. Thus, the volatile substrates partition from HMN into the microorganism-containing soil water where they achieve relatively low concentrations. The sensitive watersoluble tetrazolium dye (WST-1) was added to each well after 7 days incubation to detect dehydrogenase activity. A dark yellow signal was developed for the substrates toluene, n-octane, n-dodecane, 1,2,4-trimethylbenzene and methylcyclohexane but not for isooctane, being strongest in n-alkane amended wells. With this method, more hydrocarbon degrading bacteria were detected in an alluvial sand exposed to kerosene for 72 hours compared to the community in the pristine sand. Both communities were mainly composed of n-alkane degraders. Although encouraging results were achieved, we noticed that the tetrazolium reduction was inhibited in some cases. Wells with high cell numbers, in which obvious growth occurred, did not produce a signal. In a next step, the two-liquid phase system with HMN was applied to modify a miniaturized respiration detection system using whole soil samples instead of soil extracts (Campbell et al., AEM 69, p. 3593). Soil was incubated in deep-well plates in presence of single volatile organic hydrocarbons. The carrier phase was added to the interstitial space in a bead layer on the bottom of the well (below the soil sample), and the hydrocarbons diffused into the soil-water. The soil activity was determined by means of an agar plate containing a pH indicator, which changes color as a function of the produced CO2. Physiological profiles specific to petroleum hydrocarbons in pristine and contaminated soils were assessed. The substrate concentrations, which induced highest microbial activity, as well as concentrations causing inhibition, were determined with effect-concentration curves. Community-level physiological profiles based on hydrocarbon degradation were obtained by applying multivariate analysis on the CO2 yields. The first measurement period of 0-6 hours after addition of relatively high hydrocarbon concentrations to the system yields values, which best separated soil types as well as pristine and contaminated soils. If low concentrations were applied, we obtained only significant separations after 24 hours incubation, probably after a growth step. The same micro-respiration system equipped with a 14CO2 detection plate was applied to study N and P limitation of aerobic mineralization of 14C-toluene in a soil previously exposed to petroleum. Significant nutrient limitation during short-term tests (24-48 hours) was identified and the optimized combination of nutrient addition was determined. Conclusively, miniaturized physiological methods have been proven to detect soil microbial community changes in petroleum hydrocarbon-contaminated soils. Adding an organic carrier phase to the miniaturized systems allowed assuring a volatile organic hydrocarbon supply in non-toxic concentrations in the soil water or the medium for several days. Thus simple, cost-effective systems to investigate the soil community during bioremediation and to address questions of nutrient shortage in soils have been provided with this thesis.

EPFL2004