Phytostabilization is a remediation technology that uses plants for in-situ stabilization of contamination in soils and mine tailings. The objective of this study was to identify native plant species with potential for phytostabilization of the abandoned mine tailings in Nacozari, Sonora in northern Mexico. A flora of 42 species in 16 families of angiosperms was recorded on the tailings site and the abundance of the most common perennial species was estimated. Four of the five abundant perennial species showed evidence of regeneration: the ability to reproduce and establish new seedlings. A comparison of selected physicochemical properties of the tailings in vegetated patches with adjacent barren areas suggests that pH, electrical conductivity, texture, and concentration of potentially toxic elements do not limit plant distribution. For the most abundant species, the accumulation factor for most metals was <1, with the exception of Zn in two species. A short-term experiment on adaptation revealed limited evidence for the formation of local ecotypes in Prosopis velutina and Amaranthus watsonii . Overall, the results of this study indicate that five native plant species might have potential for phytostabilization of the Nacozari tailings and that seed could be collected locally to revegetate the site. More broadly, this study provides a methodology that can be used to identify native plants and evaluate their phytostabilization potential for similar mine tailings.

The objective of this study was to investigate the spatial and temporal trends of a phytoremediation system comprised of poplar trees designed to control groundwater flow and remove primarily polycyclic aromatic hydrocarbons (PAHs). Several lab and field studies have demonstrated the success of poplar trees in effectively decreasing concentrations of volatile hydrocarbons, but few have demonstrated effects on PAH concentrations. Thus, the focus of this report will be the response of the poplar trees in relation to hydrophobic, nonvolatile polycyclic aromatic hydrocarbons (acenapthene, acenapthylene, anthracene, chrysene, fluoranthene, naphthalene, phenanthrene, and pyrene) in a shallow, surficial aquifer. This field study was conducted on a 1.7-acre site in Oneida, Tennessee contaminated with creosote that was once used for railroad cross-tie treatment. Spatial analysis was used to divide the site into areas based on contaminant levels and a layer of coal that served as a layer of low permeability at an approximate depth of 2 feet. The semi-impermeable coal layer does have an adverse impact on tree growth, while the contamination does not appear to adversely affect tree growth. The rate of growth is also impacted by the age of the tree at planting where younger trees grow faster than the older trees. A steady decrease in PAH concentrations has occurred at the multi-level samplers surrounded by a root zone that has penetrated the contamination. PAH compounds present at relatively high concentrations in the soil and groundwater do not appear to affect tree growth to a greater or lesser extent than lower PAH concentrations. While further research is required to affirm the positive effects of poplar trees at this site, the tree stand has responded well to the high PAH levels. / Master of Science

Phytostabilization offers a less expensive alternative to traditional cap and plant methods for containing metalliferous mine tailings to prevent wind erosion and contamination of nearby communities and the environment. However, plant establishment during phytostabilization of pyritic legacy mine tailings in semiarid regions is challenging due to particularly extreme conditions including low pH, low organic carbon, low nutrients, and high toxic metal(loid) concentrations. Microorganisms drive major biogeochemical cycles in soils, however, the roles microorganisms play at the root – soil interface during phytostabilization, particularly in relation to plant health and metal immobilization, are not yet fully understood. The aims of this dissertation are to focus on bacterial communities associated with the roots of buffalo grass used in the phytostabilization of pyritic metalliferous mine tailings to: i) characterize bacterial diversity and communities of rhizosphere and bulk substrate, ii) delineate associations between rhizoplane bacterial colonization patterns and environmental and plant status parameters, and iii) develop an in situ method to visually assess associations between roots, bacteria, and metals. Key findings indicate that after addition of a compost amendment to alleviate the plant-growth inhibiting characteristics of mine tailings, rhizosphere and bulk substrate contain a diverse, plant-growth supporting bacterial community. As substrate re-acidifies due to compost erosion, an emergence of an iron (Fe)- and sulfur (S)-oxidizer and Fe-reducer dominated, less diverse community develops in the bulk and rhizosphere substrate, thus posing a threat to successful plant establishment. However, even at low pH, some plant-growth-promoting bacteria are still evident in the rhizosphere. On the rhizoplane (root surface), the relative abundance of metabolically active bacteria was positively correlated with plant health, verifying the strong association between plant health and bacteria. Furthermore, pH showed a strong association with the relative abundance of Alphaproteobacteria and Gammaproteobacteria on the rhizoplane. In relation to microbe-metal interactions on the root surface, results showed that Actinobacteria and Alphaproteobacteria colocalized with Fe-plaque and arsenic (As) contaminant on the root surface, indicating their potential role in adsorbing or cycling of these metal(loid)s. Developing a more thorough understanding of bacteria-root-metal interactions in relation to plant health and metal immobilization can help to improve phytostabilization efforts and success.

Master of Science / Department of Agronomy / Ganga M. Hettiarachchi / Mine waste materials from the Tri-State mining region in Kansas, Missouri, and Oklahoma pose environmental hazards. The area is contaminated with trace elements, such as Pb, Zn and Cd, which are transported to surrounding areas through water, wind erosion, and runoff. Phytostabilization or establishing healthy vegetative cover could be used to reduce or control these contaminated materials from further spreading with wind and water. However, further research is needed to monitor the long-term sustainability and assess if high applications of compost amendments could help to facilitate soil reclamation. The overall focus of this thesis was to monitor long-term effects of compost or lime additions at two different rates, with or without other soil amendments, on soil properties, plants, and soil biota. We used the earthworm avoidance test as a screening tool for testing effects of soil amendments on ecotoxicity. In the first field study, pelletized manure compost additions of 448 Mg ha⁻¹ significantly decreased the bioavailable Pb, Zn, and Cd while increasing plant nutrients, vegetative cover, and plant biomass as compared to the contaminated control and the low addition of compost (224 Mg ha⁻¹) over 2.5 years. Plant tissue metal concentrations with compost addition did not show any phytotoxicity in this study. Lime additions did not show any significant effect on any of the measurements. Results from the first study suggest that one time addition of large quantities of compost at 224 to 448 Mg ha⁻¹ can support establishing and maintaining healthy vegetative cover at least for a 2.5 year period. In the second field study, long-term monitoring of the effectiveness of the amendments was studied. Compost was applied at two different rates (45 or 269 Mg ha⁻¹) in 2006. Various chemical properties, microbial activities, and vegetative growth or plant biomass were measured approximately for 4.5 years to evaluate long-term changes in soil quality and sustainability of phytostabilization efforts, when combined or assisted with soil amendments to improve the quality of trace element-contaminated mine waste materials. Plants grown with compost additions of 269 Mg ha⁻¹ showed higher nutrients, biomass, and enzyme activities as compared to plants grown on the contaminated control and with the low addition of compost (45 Mg ha⁻¹) over 4.5 years. Decrease in plant biomass and enzyme activities seen in the high compost treatments by the end of the study period suggested that long-term sustainability of these efforts may require repeated addition of soil amendments every 4 to 5 years. Additionally, a laboratory study was conducted to assess the long-term effects of treatments used in the second field study on ecotoxicity using the avoidance behavior responses of the earthworm Eisenia fetida. There was no mortality of earthworms after a 48 h exposure period of any of these treated and untreated mine waste materials. Avoidance was clear for the contaminated control and the low compost treatment (45 Mg ha⁻¹). Moreover, the contaminated control did show habitat limitation (< 20% of earthworms was found in test soil). Current research studies provide evidence that, high rates of compost applications can be used to stabilize and reduce the bioavailability of trace elements in mine waste materials.

<p>Mercury (Hg) pollution is a global environmental problem. Numerous Hg-contaminated sites exist in the world and new techniques for remediation are urgently needed. Phytoremediation, use of plants to remove pollutants from the environment or to render them harmless, is considered as an environment-friendly method to remediate contaminated soil <i>in-situ</i> and has been applied for some other heavy metals. Whether this approach is suitable for remediation of Hg-contaminated soil is, however, an open question. The aim of this thesis was to study the fate of Hg in terrestrial plants (particularly the high biomass producing willow, <i>Salix spp</i>.) and thus to clarify the potential use of plants to remediate Hg-contaminated soils.</p><p>Plants used for phytoremediation of Hg must tolerate Hg. A large variation (up to 30-fold difference) was detected among the six investigated clones of willow in their sensitivity to Hg as reflected in their empirical toxicity threshold (TT_95b), the maximum unit toxicity (UTmax) and EC50 levels. This gives us a possibility to select Hg-tolerant willow clones to successfully grow in Hgcontaminated soils for phytoremediation.</p><p>Release of Hg into air by plants is a concern when using phytoremediation in practice. No evidence was found in this study that Hg was released to the air via shoots of willow, garden pea (<i>Pisum sativum</i> L. cv Faenomen), spring wheat (<i>Triticum aestivum</i> L. cv Dragon), sugar beet (<i>Beta vulgaris </i>L. cv Monohill), oil-seed rape (<i>Brassica napus</i> L. cv Paroll) and white clover (<i>Trifolium repens</i> L.). Thus, we conclude that the Hg burden to the atmosphere via phytoremediation is not increased.</p><p>Phytoremediation processes are based on the ability of plant roots to accumulate Hg and to translocate it to the shoots. Willow roots were shown to be able to efficiently accumulate Hg in hydroponics, however, no variation in the ability to accumulate was found among the eight willow clones using CVAAS to analyze Hg content in plants. The majority of the Hg accumulated remained in the roots and only 0.5-0.6% of the Hg accumulation was translocated to the shoots. Similar results were found for the five common cultivated plant species mentioned above. Moreover, the accumulation of Hg in willow was higher when being cultivated in methyl-Hg solution than in inorganic Hg solution, whereas the translocation of Hg to the shoots did not differ.</p><p>The low bioavailability of Hg in contaminated soil is a restricting factor for the phytoextraction of Hg. A selected tolerant willow clone was used to study whether iodide addition could increase the plant-accumulation of Hg from contaminated soil. Both pot tests and field trials were carried out. Potassium iodide (KI) addition was found to mobilize Hg in contaminated soil and thus increase the bioavailability of Hg in soils. Addition of KI (0.2–1 mM) increased the Hg concentrations up to about 5, 3 and 8 times in the leaves, branches and roots, respectively. However, too high concentrations of KI were toxic to plants. As the majority of the Hg accumulated in the roots, it might be unrealistic to use willow for phytoextraction of Hg in practice, even though iodide could enhance the phytoextraction efficiency.</p><p>In order to study the effect of willow on various soil fractions of Hg-contaminated soil, a 5-step sequential soil extraction method was used. Both the largest Hg-contaminated fractions, i.e. the Hg bound to residual organic matter (53%) and sulphides (43%), and the residual fraction (2.5%), were found to remain stable during cultivations of willow. The exchangeable Hg (0.1%) and the Hg bound to humic and fulvic acids (1.1%) decreased in the rhizospheric soil, whereas the plant accumulation of Hg increased with the cultivation time. The sum of the decrease of the two Hg fractions in soils was approximately equal to the amount of the Hg accumulated in plants. Consequently, plants may be suitable for phytostabilization of aged Hg-contaminated soil, in which root systems trap the bioavailable Hg and reduce the leakage of Hg from contaminated soils.</p>

Mercury (Hg) pollution is a global environmental problem. Numerous Hg-contaminated sites exist in the world and new techniques for remediation are urgently needed. Phytoremediation, use of plants to remove pollutants from the environment or to render them harmless, is considered as an environment-friendly method to remediate contaminated soil in-situ and has been applied for some other heavy metals. Whether this approach is suitable for remediation of Hg-contaminated soil is, however, an open question. The aim of this thesis was to study the fate of Hg in terrestrial plants (particularly the high biomass producing willow, Salix spp.) and thus to clarify the potential use of plants to remediate Hg-contaminated soils. Plants used for phytoremediation of Hg must tolerate Hg. A large variation (up to 30-fold difference) was detected among the six investigated clones of willow in their sensitivity to Hg as reflected in their empirical toxicity threshold (TT95b), the maximum unit toxicity (UTmax) and EC50 levels. This gives us a possibility to select Hg-tolerant willow clones to successfully grow in Hgcontaminated soils for phytoremediation. Release of Hg into air by plants is a concern when using phytoremediation in practice. No evidence was found in this study that Hg was released to the air via shoots of willow, garden pea (Pisum sativum L. cv Faenomen), spring wheat (Triticum aestivum L. cv Dragon), sugar beet (Beta vulgaris L. cv Monohill), oil-seed rape (Brassica napus L. cv Paroll) and white clover (Trifolium repens L.). Thus, we conclude that the Hg burden to the atmosphere via phytoremediation is not increased. Phytoremediation processes are based on the ability of plant roots to accumulate Hg and to translocate it to the shoots. Willow roots were shown to be able to efficiently accumulate Hg in hydroponics, however, no variation in the ability to accumulate was found among the eight willow clones using CVAAS to analyze Hg content in plants. The majority of the Hg accumulated remained in the roots and only 0.5-0.6% of the Hg accumulation was translocated to the shoots. Similar results were found for the five common cultivated plant species mentioned above. Moreover, the accumulation of Hg in willow was higher when being cultivated in methyl-Hg solution than in inorganic Hg solution, whereas the translocation of Hg to the shoots did not differ. The low bioavailability of Hg in contaminated soil is a restricting factor for the phytoextraction of Hg. A selected tolerant willow clone was used to study whether iodide addition could increase the plant-accumulation of Hg from contaminated soil. Both pot tests and field trials were carried out. Potassium iodide (KI) addition was found to mobilize Hg in contaminated soil and thus increase the bioavailability of Hg in soils. Addition of KI (0.2–1 mM) increased the Hg concentrations up to about 5, 3 and 8 times in the leaves, branches and roots, respectively. However, too high concentrations of KI were toxic to plants. As the majority of the Hg accumulated in the roots, it might be unrealistic to use willow for phytoextraction of Hg in practice, even though iodide could enhance the phytoextraction efficiency. In order to study the effect of willow on various soil fractions of Hg-contaminated soil, a 5-step sequential soil extraction method was used. Both the largest Hg-contaminated fractions, i.e. the Hg bound to residual organic matter (53%) and sulphides (43%), and the residual fraction (2.5%), were found to remain stable during cultivations of willow. The exchangeable Hg (0.1%) and the Hg bound to humic and fulvic acids (1.1%) decreased in the rhizospheric soil, whereas the plant accumulation of Hg increased with the cultivation time. The sum of the decrease of the two Hg fractions in soils was approximately equal to the amount of the Hg accumulated in plants. Consequently, plants may be suitable for phytostabilization of aged Hg-contaminated soil, in which root systems trap the bioavailable Hg and reduce the leakage of Hg from contaminated soils.

Mine tailings pose a health risk for populations and ecosystems in the Southwest; this is why effective, and low-cost solutions for the long term are needed. This work is groundbreaking since little information is available with regards to applying greenhouse studies of phytostabilization to the field for mine tailing remediation. Mine tailings from Iron King Mine and Humboldt Smelter Superfund (IKMHSS) site can be considered one of the worst scenarios due to the extreme conditions which prevent the growth of a vegetation cap. The high concentration of metals, such as arsenic and lead, highly acidic, lack of the nutrients carbon and nitrogen in the soil structure, and low microbial communities are factors that negatively affect plant growth. This project provides practical field-scale applications for the use of phytostabilization, which uses plants to create a vegetation cap that stabilizes metals in the root zone while preventing wind and water erosion in mine tailings. The project is divided into three main studies: (1) the assessment of the translation of successful greenhouse results to the field of phytostabilization using compost-assisted direct planting. This includes the use of different rates of compost as an amendment and different desert native plant species in addition to some potential parameters that could be used as indicators of a successful modification of biochemical and physical environment from a disturbed soil towards a more healthy soil when compost assisted direct planting phytostabilization is used; (2) the second study aims to evaluate the effect of the phytostabilization strategy on reducing windborne transport of particle and metal(loids) following the establishment of the vegetation cap. The results indicate that the vegetation resulted from direct planting decreases dust emissions from IKMHSS mine tailings; and (3) the third study focuses on one of the most important requirements for phytostabilization application in the field, the performance of the different plant species selected from the greenhouse studies. This performance was evaluated as the metal accumulation in aerial plant tissue based on metal concentration guidelines from the National Research Council as well as changes in the composition of plant species and canopy cover with time. The results derived from the translation of compost–assisted direct plating based on successful greenhouse results are showing the capacity of this technology on a field scale by maintaining a canopy cover over time that decreases mobilization by not hyper-accumulating metals in the aerial tissue and by preventing windborne particle dispersion with the potential of disrupting contamination pathways.

Particulate and dissolved forms of arsenic and heavy metals are released from legacy mine tailings, particularly in (semi-) arid environments where tailings remain barren of vegetation and therefore highly susceptible to erosion. This leads to contamination of adjacent ecosystems and increased risk to public health. Establishment of a vegetative cap using amendments, such as composted organic matter to enhance plant growth, may be employed to reduce both physical erosion and leaching, but the impacts of such practices on molecular-scale mechanisms controlling metal(loid) speciation and lability remain poorly understood. Here we report on subsurface biogeochemical transformations of metal(loid)s in a phytostabilization field study at a Superfund site in Arizona, USA, where a legacy pyritic tailings (4,000 mg kg^-1 As, 2,438 mg kg^-1 Pb, 6,142 mg kg^-1 Zn, 13.25% Fe, and 11.71% S, averages for the top 0.5 m) has undergone oxidation in the top 1 m. Tailings were amended in the top 20 cm with 10%, 15%, and 20% composted organic matter by mass and seeded with native halotolerant plant species. All field treatments and the uncomposted control received irrigation of 0.36 ± 0.03 mm y^-1 in addition to 0.25 ± 0.16 mm y^-1 of precipitation, resulting in water input of 144% the annual precipitation rate. The field trial incorporated four annual samplings from 2010 – 2013. Sampling consisted of a single core of 90 cm in length and 2.54 cm in diameter collected from each field plot that was subsequently sectioned into 20 cm depth increments for analysis by synchrotron Fe and As X-ray absorption spectroscopy (XAS) coupled with quantitative chemical extraction methods. Subsurface stabilization of arsenic by Prosopis juliflora (mesquite) was investigated by bulk and micro synchrotron XAS and multiple-energy microscale fluorescence mapping combined with chemical digestion of plant samples following 1, 2, and 3 months of growth in greenhouse microcosms as well as 14 and 36 months of growth at the field site. Results indicate persistence of oxidizing conditions following compost amendment in surface tailings despite addition of organic matter, development of heterotrophic microbial communities and irrigation of a poorly draining medium. Compost amendment of 20% corresponded with evidence of higher oxidative pyrite weathering activity at 40-60 cm depth during phytostabilization compared to treatments of 0% or 10% compost for which the highest oxidative pyrite weathering activity was observed closer to the surface at 20-40 cm depth.. Despite observed downward transport of As, Fe, Zn, Mn, Pb, Ni, Cu, Cr, V, and Co during phytostabilization, ≥ 75% of total As was found to be attenuated by ferrihydrite in surface depths. Attenuation of Mn, Co, and Ni was observed below 40 depth by tailings receiving compost amendment relative to the irrigated control. Root associated As(V) was immobilized on the root epidermis bound to ferric sulfate precipitates and within root vacuoles as trivalent As(III)-thiol complexes. Rhizoplane associated ferric sulfate phases were dissimilar from the bulk tailings mineralogy shown by XAS and exhibited a high capacity to scavenge As(V) with As:Fe ratios 2x higher than the compost amended growth medium, indicating a root surface mechanism for their formation or accumulation. Results indicate that arsenate attenuation in semi-arid mine tailings during phytostabilization greatly depends on the presence of high concentrations of Fe(III) (oxyhydr)oxide minerals with a high capacity for arsenic adsorption.

Ce travail a pour objectif la mise en place d'un procédé de phytostabilisation sur un ancien site industriel, la Vieille Usine d'Auzon (43, France). La caractérisation pédochimique du site atelier a révélé une pollution polymétallique par l'As, Pb, Sb, Cd et Cu. L'As, de par sa concentration totale et sa biodisponibilité dans le sol, est considéré comme le polluant le plus présent et le plus toxique. L'étude floristique réalisée sur le site a relevé une flore métallicole dominée par des pseudométallophytes électives telles que Agrostis capillaris, Equisetum arvense, ou les Euphorbiacées. De manière générale, la majorité des espèces présentes sur la friche industrielle a accumulé de très faibles quantités de polluants (As, Pb) dans ces parties aériennes. Dans un deuxième temps, les travaux ont porté sur les effets induits par les ETM au niveau physiologique et biochimique chez des modèles végétaux, Agrostis capillaris, Solanum nigrum, Vicia faba et Cannabis sativa. Ces données ont mis en évidence la sensibilité de V. faba et la tolérance de S. nigrum à la pollution aux ETM, un maintien de l'activité physiologique mais un ralentissement de la croissance de C. sativa sur sol pollué et une tolérance adaptative d'A. capillaris à la pollution du sol. Par ailleurs, une culture en hydroponie de S. nigrum et V. faba en présence d'As inorganique, arsénite et arséniate, à différentes concentrations a permis de mettre en évidence deux effets de l'As. Celui-ci a provoqué une altération de la structure et du fonctionnement des PSII, plus sensibles à l'As(III), et, une inhibition de l'activité oxygénase et carboxylase de la Rubisco, cible privilégiée de l'As(V). Enfin, ces résultats ont été utilisés dans le cadre de l'expérimentation in situ pour la validation d'un procédé de phytostabilisation sur la friche industrielle d'Auzon. Les effets de la combinaison d'une association d'espèces prairiales et d'un amendement de grenaille de fer zérovalent ou/et de compost ont pu être caractérisés. L'ajout de grenaille de fer au sol complété par un apport de compost a accentué la réduction de la disponibilité de l'As et dans une moindre mesure de Sb. De plus, il a permis d'enrichir le sol en élément minéraux et organiques et a ainsi facilité l'implantation d'un couvert végétal constitué d'espèces végétales à phénotype d'exclusion. Cette expérience in-situ a donc permis de mettre en avant la faisabilité d'un procédé de phytostabilisation dans la dépollution et la revalorisation de sites industriels. / This work aims to the establishment of a phytostabilization process on an old industrial site, "La Vieille Usine" in Auzon (43, France). The pedochemical characterization revealed a polymetallic pollution by As, Pb, Sb, Cd, Cu. The As, by its total content and its bioavailability, is considered as the most concentrated and toxic. A floristic study noted a metallicolous flora dominated by elective pseudometallophyte species such as Agrostis capillaris, the dominant specie, Equisetum arvense, or Euphorbiaceae. Broadly speaking, the majority of species on the industrial site has accumulated very low amounts of pollutants (As, Pb) in these shoots. To better understand the trace element effects on different plants, Agrostis capillaris, Solanum nigrum, Cannabis sativa and Vicia faba were grown on the Auzon’s soil under controlled conditions. These data highlighted (i) the sensitivity of V. faba and the tolerance of S. nigrum to trace element pollution; (ii) for C. sativa, the maintenance of its physiological activity but a slowdown of its growth; (iii) the adaptative tolerance of A. capillaris to soil pollution. In addition, a hydroponics culture of S. nigrum and V. faba in the presence of different concentrations and different oxidation degrees (arsenite and arsenate) of inorganic As allowed to identify two main effects of As at physiological and biochemical level. First, it alters the structure and function of PSII, which seems more sensitive to As(III), and, secondly, it inhibits the carboxylase and oxygenase activities of Rubisco, the main target of As(V). These data were then used to conduct an in-situ experiment based on a phytostabilization process on the industrial wasteland of Auzon. The effects of the combination of an grassland species association with a zerovalent iron shot and/or compost amendment were characterized. The addition of iron shot on the soil completed with compost decreased the available fraction of As and to a lesser extent Sb. Moreover, the amendments allowed to enrich the soil with mineral and organic elements and to facilitate the establishment of vegetal cover composed by tolerant plants with an exclusion phenotype. This in-situ experience enabled to highlight the feasibility of a phytostabilization method in the depollution of industrial sites as in their enhancement.

Challenges to the reclamation of pyritic mine tailings arise from in-situ acid generation that severely constrains natural revegetation. While microbial communities that participate in acid generation through iron and sulfur (FeS) oxidation in acidic aquatic environments are well studied, relatively little information is available concerning the initial dynamics of in-situ soil acidification due to microbial FeS oxidation that occur in moderately acidic conditions. This research characterizes the taxonomic composition and behavior of microbial FeS oxidizing communities across a pH gradient from moderately acidic to highly acidic environmental conditions. We combine results from a 7-year compost-assisted phytostabilization field study with a controlled microcosm enrichment experiment that was conducted in an artificial soil matrix to follow the influence of pH on development of the soil microbiome. Microcosm results show that biological activity significantly increases the acidification rate in moderately acidic pH conditions in comparison to abiotic controls. Taxonomic profiles of the microbial communities in the microcosms and from the field study reveal that populations associated with both heterotrophic and lithotrophic activity (Alicyclobacillaceae, Acetobacteraceae and Xanthomonadaceae) dominate during acidification in moderately acidic conditions. These results suggest that chemoheterotrophs are an important element of the microbial community that help enable, directly and indirectly, lithotrophic FeS oxidation across moderately acidic conditions. Taken together, this research suggests that shifts of microbial populations associated with pH transitions have the potential to be used as bioindicators of the present and future status of the phytostabilization process.