Microbial community evolution in a marine shore porphyry tailings deposit throughout wetland remediation

Background Two porphyry copper mines located in southern Peru initiated the discharge of mine wastes (tailings) for a final shore deposition in the Bay of Ite (Pacific Ocean). 780 millions metric tons of tailings were deposited, which formed a delta with a surface of ca. 16 km2. A low-pH oxidation zone characterized by the presence of iron hydroxides developed on the surface, along with a strong build-up of efflorescent salts rich in heavy metals. Objectives A 3 years investigation was carried out in order to understand the biogeochemical processes resulting from the remediation of the oxidizing tailings by the implementation of a wetland cover1. Methods Analysis of the bacterial communities was carried out by molecular techniques (T-RFLP and cloning-sequencing). A combination of physical, chemical and mineralogical methods was applied for the characterization of solid tailings and water samples. Results The non-remediated tailings was characterized by a low-pH oxidation zone (pH 3 – 4), in which the oxidative dissolution of sulphide was identified as the dominant process, carried out mainly by Leptospirillum spp, Acidithiobacillus spp and Sulfobacillus spp. This dissolution led to an increase in metal concentrations in the pore water. Below the oxidation zone, the neutrophilic primary zone was characterized by SO42- and Fe reductions induced by sulphate- and iron-reducing bacteria (SRB and FeRB). Drastic changes were observed along the remediation process, as the heavy metal concentrations decreased progressively close or below the detection limits together with the gradual installation of a near neutral pH and more reducing conditions. SO42- and Fe reductions were still observed in the deep section of the remediated tailings. Close to the surface, the presence of lithoautotrophic neutrophilic iron oxidizing bacteria such as Mariprofundus ferroxidans, Galionella spp, Leptotrix mobilis suggested that iron oxidation played an important role in the iron cycle. Other ubiquitous bacteria related to fresh water and marine environment were also detected in the wetland. Conclusions The implementation of a wetland promoted neutralization of the acid mine drainage and induced favourable geochemical conditions for the development of both SRB and FeRB, which lead to the metal removal from solution. References Diaby, N. 2009. Biogeochemical evolution of a marine shore tailings deposit during bioremediation. Ph. D. Thesis, University of Lausanne, Switzerland. 217 pp.

Temporal evolution of bacterial communities associated with the in situ wetland-based remediation of a marine shore porphyry copper tailings deposit

Nouhou Diaby, Christof Holliger, Emmanuelle Rohrbach, Pierre Rossi

Mine tailings are a serious threat to the environment and public health. Remediation of these residues can be carried out effectively by the activation of specific microbial processes. This article presents detailed information about temporal changes in bacterial community composition during the remediation of a section of porphyry copper tailings deposited on the Bahía de Ite shoreline (Peru). An experimental remediation cell was flooded and transformed into a wetland in order to prevent oxidation processes, immobilizing metals. Initially, the top oxidation zone of the tailings deposit displayed a low pH (3.1) and high concentrations of metals, sulfate, and chloride, in a sandy grain size geological matrix. This habitatwas dominated by sulfur- and iron-oxidizing bacteria, such as Leptospirillumspp., Acidithiobacillus spp., and Sulfobacillus spp., in a microbial community which structure resembled acid mine drainage environments. After wetland implementation, the cell was water-saturated, the acidity was consumed and metals dropped to a fraction of their initial respective concentrations. Bacterial communities analyzed by massive sequencing showed time-dependent changes both in composition and cell numbers. The final remediation stage was characterized by the highest bacterial diversity and evenness. Aside from classical sulfate reducers from the phyla δ-Proteobacteria and Firmicutes, community structure comprised taxa derived fromvery diverse habitats. The community was also characterized by an elevated proportion of rare phyla and unaffiliated sequences. Numerical ecology analysis confirmed that the temporal population evolution was driven by pH, redox, and K. Results of this study demonstrated the usefulness of a detailed follow-up of the remediation process, not only for the elucidation of the communities gradually switching from autotrophic, oxidizing to heterotrophic and reducing living conditions, but also for the long term management of the remediation wetlands.

Elsevier2015

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

Study of the microbial communities of core and water samples in a chloroethenes contaminated aquifer

Guillaume Laulan

Widely used in industrial activities, chloroethenes have become common groundwater contaminants. Dehalorespiration is an anaerobic bacterial respiration coupling their sequential degradation to energy production. The interactions between the dehalorespiring bacteria and the other guilds and environmental variables are presumed to influence the dehalorespiration efficiency. Here, water and core samples were extracted from the PCE-contaminated site of Lyss (BE, Switzerland). Bacterial DNA was extracted and the 16S rRNA gene amplified by PCR. Terminal restriction fragment length polymorphism (T-RFLP) was used to assess the diversity of the bacterial communities. Core and groundwater environmental variables were analyzed as well. The T-RFLP profiles and the environmental data obtained were processed using statistical multivariate analysis. Within a period of two months, the bacterial communities remained relatively specific to their respective sampling origins. Communities from the oxic layer shared a similar set of bacterial species whereas piezometers from the anoxic layer had a specific one. The T-RFLP profiles obtained from single depth and multi-level piezometers were clearly distinct, probably due to differences in the pumping flow rates and their associated transport regimes, which can have an impact on i) the sediments extracted from the aquifer, ii) the hydraulic zone reached and iii) a potential selective bacterial adsorption onto the pumping tube wall. Environmental factors related to the groundwater redox state appeared as the major variables influencing the bacterial populations. Bacterial communities from core samples clustered into two distinct sets: i) samples from the strongly reductive silt layer, and ii) samples from the coarser and less reductive layer. Grain size distribution and redox state both had a major influence on the bacterial communities of core samples. Finally, it was shown that microbial populations extracted from groundwater differed from the ones extracted from core material, and thus were not representative of the overall aquifer microbial diversity.

2009