Microbial recovery of rare earth elements from various waste sources: a mini review with emphasis on microalgae.

Rare Earth Elements (REEs) are indispensable in contemporary technologies, influencing various aspects of our daily lives and environmental solutions. The escalating demand for REEs has led to increased exploitation, resulting in the generation of diverse REE-bearing solid and liquid wastes. Recognizing the potential of these wastes as secondary sources of REEs, researchers are exploring microbial solutions for their recovery. This mini review provides insights into the utilization of microorganisms, with a particular focus on microalgae, for recovering REEs from sources such as ores, electronic waste, and industrial effluents. The review outlines the principles and distinctions of bioleaching, biosorption, and bioaccumulation, offering a comparative analysis of their potential and limitations. Specific examples of microorganisms demonstrating efficacy in REE recovery are highlighted, accompanied by successful methods, including advanced techniques for enhancing microbial strains to achieve higher REE recovery. Moreover, the review explores the environmental implications of bio-recovery, discussing the potential of these methods to mitigate REE pollution. By emphasizing microalgae as promising biotechnological candidates for REE recovery, this mini review not only presents current advances but also illuminates prospects in sustainable REE resource management and environmental remediation.

Rare earth elements applied to phytoremediation: Enhanced endocytosis promotes remediation of antimony contamination with different valence levels in Solanum nigrum L.

Antimony (Sb) pollution poses a noteworthy risk to human health and ecosystem sustainability, therefore effective, eco-friendly, and widely accepted restoration methods are urgently needed. This study introduces a new approach of using La(III) foliar application on Solanum nigrum L. (S. nigrum), a cadmium hyperaccumulator, to improve its photosynthetic and root systems under Sb stress, resulting in a higher biomass. Notably, La(III) also enhances endocytosis in root cells, facilitating efficient and non-selective remediation of both Sb(III) and Sb(V) forms. The absorption of Sb by root cell endocytosis was observed visually with a confocal laser scanning microscope. The subcellular distribution of Sb in the cell wall of S. nigrum is reduced. And the antioxidant enzyme activity system is improved, resulting in an enhanced Sb tolerance in S. nigrum. Based on the existing bibliometric analysis, this paper identified optimal conditions for S. nigrum to achieve maximum translocation and bioconcentration factor values for Sb. The foliar application of La(III) on plants treated with Sb(III), Sb(V), and a combination of both resulted in translocation factor values of 0.89, 1.2, 1.13 and bioconcentration factor values of 11.3, 12.81, 14.54, respectively. Our work suggests that La(III)-enhanced endocytosis of S. nigrum root cells is a promising remediation strategy for Sb-contaminated environments.

Roles of pH and phosphate in rare earth element biosorption with living acidophilic microalgae.

The increasing demand for rare earth elements (REEs) has spurred interest in the development of recovery methods from aqueous waste streams. Acidophilic microalgae have gained attention for REE biosorption as they can withstand high concentrations of transition metals and do not require added organic carbon to grow, potentially allowing simultaneous sorption and self-replication of the sorbent. Here, we assessed the potential of Galdieria sulphuraria for REE biosorption under acidic, nutrient-replete conditions from solutions containing ≤ 15 ppm REEs. Sorption at pH 1.5-2.5 (the growth optimum of G. sulphuraria) was poor but improved up to 24-fold at pH 5.0 in phosphate-free conditions. Metabolic activity had a negative impact on REE sorption, additionally challenging the feasibility of REE biosorption under ideal growth conditions for acidophiles. We further examined the possibility of REE biosorption in the presence of phosphate for biomass growth at elevated pH (pH ≥ 2.5) by assessing aqueous La concentrations in various culture media. Three days after adding La into the media, dissolved La concentrations were up to three orders of magnitude higher than solubility predictions due to supersaturation, though LaPO4 precipitation occurred under all conditions when seed was added. We concluded that biosorption should occur separately from biomass growth to avoid REE phosphate precipitation. Furthermore, we demonstrated the importance of proper control experiments in biosorption studies to assess potential interactions between REEs and matrix ions such as phosphates. KEY POINTS: • REE biosorption with G. sulphuraria increases significantly when raising pH to 5 • Phosphate for biosorbent growth has to be supplied separately from biosorption • Biosorption studies have to assess potential matrix effects on REE behavior.

Boosting plant resilience: The promise of rare earth nanomaterials in growth, physiology, and stress mitigation.

Rare earth elements (REE) have been extensively used in a variety of applications such as cell phones, electric vehicles, and lasers. REEs are also used as nanomaterials (NMs), which have distinctive features that make them suitable candidates for biomedical applications. In this review, we have highlighted the role of rare earth element nanomaterials (REE-NMs) in the growth of plants and physiology, including seed sprouting rate, shoot biomass, root biomass, and photosynthetic parameters. In addition, we discuss the role of REE-NMs in the biochemical and molecular responses of plants. Crucially, REE-NMs influence the primary metabolites of plants, namely sugars, amino acids, lipids, vitamins, enzymes, polyols, sorbitol, and mannitol, and secondary metabolites, like terpenoids, alkaloids, phenolics, and sulfur-containing compounds. Despite their protective effects, elevated concentrations of NMs are reported to induce toxicity and affect plant growth when compared with lower concentrations, and they not only induce toxicity in plants but also affect soil microbes, aquatic organisms, and humans via the food chain. Overall, we are still at an early stage of understanding the role of REE in plant physiology and growth, and it is essential to examine the interaction of nanoparticles with plant metabolites and their impact on the expression of plant genes and signaling networks.

Prospecting for rare earth element (hyper)accumulators in the Paris Herbarium using X-ray fluorescence spectroscopy reveals new distributional and taxon discoveries.

BACKGROUND: Rare earth elements (REEs) are increasingly crucial for modern technologies. Plants could be used as a biogeochemical pathfinder and a tool to extract REEs from deposits. However, a paucity of information on suitable plants for these tasks exists. METHODS: We aimed to discover new REE-(hyper)accumulating plant species by performing an X-ray fluorescence (XRF) survey at the Herbarium of the Muséum national d'Histoire naturelle (MNHN, Paris, France). We selected specific families based on the likelihood of containing REE-hyperaccumulating species, using known taxa that accumulate REEs. A total of 4425 specimens, taken in the two main evolutionary lineages of extant vascular plants, were analysed, including the two fern families Blechnaceae (n = 561) and Gleicheniaceae (n = 1310), and the two flowering plant families Phytolaccaceae (n = 1137) and Juglandaceae (n = 1417). KEY RESULTS: Yttrium (Y) was used as a proxy for REEs for methodological reasons, and a total of 268 specimens belonging to the genera Blechnopsis (n = 149), Dicranopteris (n = 75), Gleichenella (n = 32), Phytolacca (n = 6), Carya (n = 4), Juglans (n = 1) and Sticherus (n = 1) were identified with Y concentrations ranging from the limit of detection (LOD) >49 µg g-1 up to 1424 µg g-1. Subsequently, analysis of fragments of selected specimens by inductively coupled plasma atomic emission spectroscopy (ICP-AES) revealed that this translated to up to 6423 µg total REEs g-1 in Dicranopteris linearis and up to 4278 µg total REEs g-1 in Blechnopsis orientalis which are among the highest values ever recorded for REE hyperaccumulation in plants. It also proved the validity of Y as an indicator for REEs in XRF analysis of herbarium specimens. The presence of manganese (Mn) and zinc (Zn) was also studied by XRF in the selected specimens. Mn was detected in 1440 specimens ranging from the detection limit at 116 µg g-1 up to 3807 µg g-1 whilst Zn was detected in 345 specimens ranging from the detection limit at 77 µg g-1 up to 938 µg g-1. CONCLUSIONS AND IMPLICATIONS: This study led to the discovery of REE accumulation in a range of plant species, substantially higher concentrations in species known to be REE hyperaccumulators, and records of REE hyperaccumulators outside of the well-studied populations in China.

Role of indigenous microbial communities in the mobilization of potentially toxic elements and rare-earth elements from alkaline mine waste.

This study aims to evaluate the role of indigenous microorganisms in the mobilization of potentially toxic elements (PTE) and rare-earth elements (REE), the influence of the bioavailability of carbon sources that might boost microbial leaching, and the generation of neutral/alkaline mine drainage from alkaline tailings. These tailings, with significant concentrations of total organic carbon (TOC), were mainly colonized by bacteria belonging to the genera Sphingomonas, Novosphingobium and Solirubrobacter, and fungi of the genera Alternaria, Sarocladium and Aspergillus. Functionality analysis suggests the capability of these microorganisms to leach PTE and REE. Bio-/leaching tests confirmed the generation of neutral mine drainage, the influence of organic substrate, and the leaching of higher concentrations of PTE and REE due to the production of organic acids and siderophores by indigenous microorganisms. In addition, this study offers some insights into a sustainable alternative for reprocessing PMC alkaline tailings to recover REE.

The stimulatory effect and mechanism of low-dose lanthanum on soybean leaf cells.

Light rare earth elements (LREEs) have been long used in agriculture (i.e., mainly via aerially applied LREE fertilizers) based on the fact that low-dose LREEs promote plant growth. Meanwhile, the toxic effects of low-dose LREEs on organisms have also been found. However, the cellular and molecular mechanism of low-dose LREEs acting on organisms remain unclear. Plants are at the beginning of food chains, so it is critical to uncover the cellular and molecular mechanism of low-dose LREEs on plants. Here, lanthanum (La) and soybean were the representatives of LREEs and plants, respectively. The effects of low-dose La on soybean leaves were investigated, and the stimulatory effect and mechanism of low-dose LREEs on leaf cells were revealed. Specifically, clathrin-mediated endocytosis (CME) activated by low-dose La is an influx channel for La in soybean leaf cells. The intracellular La and La-activated CME jointly disturbed multiple forms of intracellular homeostasis, including metallic element homeostasis, redox homeostasis, gene expression homeostasis. The disturbed homeostasis either stimulated cell growth or caused damage to the plasma membrane of soybean leaf cells. These results provide new insights for clarifying the cellular and molecular mechanisms of low-dose LREEs as a class of stimulators instead of nutrients to stimulate plants.

A perspective on the role of lanthanides in biology: Discovery, open questions and possible applications.

Because of their use in high technologies like computers, smartphones and renewable energy applications, lanthanides (belonging to the group of rare earth elements) are essential for our daily lives. A range of applications in medicine and biochemical research made use of their photo-physical properties. The discovery of a biological role for lanthanides has boosted research in this new field. Several methanotrophs and methylotrophs are strictly dependent on the presence of lanthanides in the growth medium while others show a regulatory response. After the first demonstration of a lanthanide in the active site of the XoxF-type pyrroloquinoline quinone methanol dehydrogenases, follow-up studies showed the same for other pyrroloquinoline quinone-containing enzymes. In addition, research focused on the effect of lanthanides on regulation of gene expression and uptake mechanism into bacterial cells. This review briefly describes the discovery of the role of lanthanides in biology and focuses on open questions in biological lanthanide research and possible application of lanthanide-containing bacteria and enzymes in recovery of these special elements.

Broad-spectrum and effective rare earth enriching via Lanmodulin-displayed Yarrowia lipolytica.

The traditional mining processes of rare earth elements (REEs) are accompanied by the production of a large number of acid mine drainage rich in REEs. A wide-adaptive, low-cost and environmentally friendly biosorbent is an attractive technology to enrich and recycle REEs from the liquid wastes. To construct a broad-spectrum and efficient biosorbent, a novel REEs-binding protein Lanmodulin (LanM) is successfully displayed on the cell surface of a fungus, Yarrowia lipolytica, for the first time, and the adsorption capacities for various REEs are studied. The LanM-displayed Y. lipolytica shows significantly enhanced adsorption capacities for multiple REEs, achieving the highest reported values of 49.83 ± 2.87 mg Yb /g DCW, 50.38 ± 1.46 mg Tm /g DCW, 49.94 ± 3.61 mg Er /g DCW and 48.72 ± 3.09 mg Tb/g DCW, respectively. Moreover, the LanM-displayed Y. lipolytica possesses a high selectivity for REEs over other common metal cations and excellent suitability under acidic conditions. The kinetics and equilibrium analysis of biosorption processes agree well with the pseudo-first kinetic and Langmuir isotherm model. Based on the FTIR and SEM-EDS analysis, the chelation with phosphate/carboxylate groups dominates the Yb binding in LanM-displayed cells, and LanM enhances the adsorption performances by introducing more binding sites with high selectivity towards a wide range of REEs. Thus, the LanM-displayed Y. lipolytica investigated in this study exhibits prosperous potential for the enriching/removal of REEs from acid mine drainage.

A novel green strategy for biorecovery of valuable elements along with enrichment of rare earth elements from activated spent automotive catalysts using fungal metabolites.

Metals recovery from spent automotive catalytic converters (SACCs) has gained great attention due to high metal content of SACCs and their potential to pollute the environment. This study presented a novel green strategy for treating SACCs using oxalic acid-enriched spent culture medium from Aspergillus niger cultivations. To enhance oxalic acid production, the Central Composite Design (CCD) was applied, which demonstrated that glucose (27.06 g/L), NaNO3 (0.9 g/L), disodium oxalate (7.7 g/L), MnSO4·H2O (0.28 g/L), and ethanol (0.65%(v/v)) were the optimum values leading to production of 15.3 g/L oxalic acid. The results of metals biorecovery with the fungal metabolites showed that pulp density of 15 g/L, temperature of 60 °C, and leaching time of 6 h resulted in the highest extraction of 99.1% Al, 99.3% Si, 82.2% Mn, 91.9% Zn, 17.6% Ba, 99.5% Fe, 92.2% Sr, 35.7% Ti, 60.9% Pt, and 73.7% Pd, as well as maximum enrichment of rare earth elements (REEs) in the residual powder. The EDX-mapping analysis indicated that the concentration of ∑REEs was nearly 8% in the initial waste powder, while it reached around 81% in the residual powder after bioleaching. The bioleaching mechanism was further analyzed by characterizing the bioleaching residues through XRD, FTIR, and FESEM analyses.

Microorganisms Accelerate REE Mineralization in Supergene Environments.

Exogenic deposits are an important source of rare earth elements (REEs), especially heavy REEs (HREEs). It is generally accepted that microorganisms are able to dissolve minerals and mobilize elements in supergene environments. However, little is known about the roles of microorganisms in the formation of exogenic deposits such as regolith-hosted REE deposits that are of HREE enrichment and provide over 90% of global HREE demand. In this study, we characterized the microbial community composition and diversity along a complete weathering profile drilled from a regolith-hosted REE deposit in Southeastern China and report the striking contributions of microorganisms to the enrichment of REEs and fractionation between HREEs and light REEs (LREEs). Our results provide evidence that the variations in REE contents are correlated with microbial community along the profile. Both fungi and bacteria contributed to the accumulation of REEs, whereas bacteria played a key role in the fractionation between HREEs and LREEs. Taking advantage of bacteria strains isolated from the profile, Gram-positive bacteria affiliated with Bacillus and Micrococcus preferentially adsorbed HREEs, and teichoic acids in the cell wall served as the main sites for HREE adsorption, leading to an enrichment of HREEs in the deposit. The present study provides the first database of microbial community in regolith-hosted REE deposits. These findings not only elucidate the crucial contribution of fungi and bacteria in the supergene REE mineralization but also provide insights into efficient utilization of mineral resources via a biological pathway. IMPORTANCE Understanding the role of microorganisms in the formation of regolith-hosted rare earth element (REE) deposits is beneficial for improving the metallogenic theory and deposit exploitation, given that such deposits absolutely exist in subtropical regions with strong microbial activities. Little is known of the microbial community composition and its contribution to REE mineralization in this kind of deposit. Using a combination of high-throughput sequencing, batch adsorption experiments, and spectroscopic characterization, the functional microorganisms contributing to REE enrichment and fractionation are disclosed. For bacteria, the surface carboxyl and phosphate groups are active sites for REE adsorption, while teichoic acids in the cell walls of G+ bacteria lead to REE fractionation. The above-mentioned findings not only unravel the importance of microorganisms in the formation of supergene REE deposits but also provide experimental evidence for the bioutilization of REE resources.

Inflammation and accompanied disrupted hematopoiesis in adult mouse induced by rare earth element nanoparticles.

Rare earth element nanoparticles (REE NPs) or agents have been used extensively in various fields. Human exposure to REE NPs is an increasing concern. To date, REE NP-mediated comprehensive immune responses after incorporation into the body remain unclear. In our study, using gadolinium oxide NPs (Gd2O3) as a typical REE NP, we systematically investigated immune responses in vivo. The liver and spleen were the main sites where Gd2O3 retained and accumulated, while Gd2O3 content per unit tissue mass in the spleen was 4.4 times higher than that in the liver. Gd2O3 increased the number of monocyte-derived macrophages and myeloid-derived dendritic cells (M-DCs) in the liver. In the spleen, Gd2O3 caused infiltration of neutrophils, M-DCs, and B cells. The accumulation of Gd2O3 in the liver or spleen also contributed to an increased concentration of cytokines in peripheral blood. In both the bone marrow and spleen, Gd2O3 led to increased populations of hematopoietic stem cells (HSCs), multipotent progenitors, and common lymphoid progenitors. Compared to the decreased monocytes in peripheral blood on day 2, a significant decrease of circulating lymphocytes on day 7 was still observed, suggesting the exposure duration led to variable effects. This might be explained by the sustained accumulation of Gd2O3 in the liver and spleen. Together, our study systemically depicted the alterations in mature immune alterations together with hematopoiesis in both myeloid and lymphoid lineages induced by Gd2O3 exposure. Our findings will facilitate a comprehensive understanding of the interactions of immune system with REE NPs in vivo.

Effects of rare earth elements on bacteria in rhizosphere, root, phyllosphere and leaf of soil-rice ecosystem.

The effects of rare earth mining on rice biomass, rare earth element (REE) content and bacterial community structure was studied through pot experiment. The research shows that the REE content in rice roots, shoots and grains was significantly positive correlated with that in soil, and the dry weight of rice roots, shoots and grains was highly correlated with soil physical and chemical properties, nutrient elements and REE contents; The exploitation of rare earth minerals inhibited a-diversity of endophytic bacteria in rhizosphere, root, phyllosphere and leaf of rice, significantly reduced the abundance index, OTU number, Chao, Ace index and also significantly reduced the diversity index-Shannon index, and also reduced uniformity index: Pielou's evenness index, which caused ß-diversity of bacteria to be quite different. The exploitation of rare earth minerals reduces the diversity of bacteria, but forms dominant bacteria, such as Burkholderia, Bacillus, Buttiauxella, Acinetobacter, Bradyrhizobium, Candida koribacter, which can degrade the pollutants formed by exploitation of rare earth minerals, alleviate the compound pollution of rare earth and ammonia nitrogen, and also has the function of fixing nitrogen and resisting rare earth stress; The content of soil available phosphorus in no-mining area is lower, and the dominant bacteria of Pantoea formed in such soil, which has the function of improving soil phosphorus availability. Rare earth elements and physical and chemical properties of soil affect the community structure of bacteria in rhizosphere and phyllosphere of rice, promote the parallel movement of some bacteria in rhizosphere, root, phyllosphere and leaf of rice, promote the construction of community structure of bacteria in rhizosphere and phyllosphere of rice, give full play to the growth promoting function of Endophytes, and promote the growth of rice. The results showed that the exploitation of rare earth minerals has formed the dominant endophytic bacteria of rice and ensured the yield of rice in the mining area, however, the mining of mineral resources causes the compound pollution of rare earth and ammonia nitrogen, which makes REE content of rice in mining area significantly higher than that in non-mining area, and the excessive rare earth element may enter the human body through the food chain and affect human health, so the food security in the REE mining area deserves more attention.

Precision Engineering of 2D Protein Layers as Chelating Biogenic Scaffolds for Selective Recovery of Rare-Earth Elements.

Rare-earth elements, which include the lanthanide series, are key components of many clean energy technologies, including wind turbines and photovoltaics. Because most of these 4f metals are at high risk of supply chain disruption, the development of new recovery technologies is necessary to avoid future shortages, which may impact renewable energy production. This paper reports the synthesis of a non-natural biogenic material as a potential platform for bioinspired lanthanide extraction. The biogenic material takes advantage of the atomically precise structure of a 2D crystalline protein lattice with the high lanthanide binding affinity of hydroxypyridinonate chelators. Luminescence titration data demonstrated that the engineered protein layers have affinities for all tested lanthanides in the micromolar-range (dissociation constants) and a higher binding affinity for the lanthanide ions with a smaller ionic radius. Furthermore, competitive titrations confirmed the higher selectivity (up to several orders of magnitude) of the biogenic material for lanthanides compared to other cations commonly found in f-element sources. Lastly, the functionalized protein layers could be reused in several cycles by desorbing the bound metal with citrate solutions. Taken together, these results highlight biogenic materials as promising bioadsorption platforms for the selective binding of lanthanides, with potential applications in the recovery of these critical elements from waste.

Generation of a Gluconobacter oxydans knockout collection for improved extraction of rare earth elements.

Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency.

Biodegradable freestanding rare-earth nanosheets promote multimodal imaging and delivers CRISPR-Cas9 plasmid against tumor.

Designing nanomaterials for bio-imaging and drug delivery for advanced cancer therapy with biodegradability and biocompatibility is a promising but challenging frontier. Herein, we assembled biodegradable and biocompatible ultrathin rare-earth erbium/dysprosium nanosheets that improve contrast in multimodal bio-imaging settings (MRI and X-ray CT) and deliver CRISPR-Cas9 plasmid to treat tumors.

Low-dose lanthanum activates endocytosis, aggravating accumulation of lanthanum or/and lead and disrupting homeostasis of essential elements in the leaf cells of four edible plants.

Rare earth elements (REEs) are emerging as a serious threat to ecological safety due to their increasing accumulation in environments. The accumulation of REEs in environments has significantly increased its accumulation in the leaves of edible plants. However, the accumulation pathway of REEs in the leaves of edible plants are still unknown. In this study, lanthanum [La(III), a widely used and accumulated REE] and four edible plants (soybean, lettuce, pakchoi, and celery) with short growth cycles were selected as research objects. By using interdisciplinary research techniques, we found that low-dose La(III) activated endocytosis (mainly the clathrin-mediated endocytosis) in the leaf cells of four edible plants, which provided an accumulation pathway for low-dose La in the leaf cells of these edible plants. The accumulation of La in the leaf cells was positively correlated with the intensity of endocytosis, while the intensity of endocytosis was negatively correlated with the density of leaf trichomes. In addition to the accumulation of La, low-dose La(III) also brought other risks. For example, the harmful element (Pb) can also be accumulated in the leaf cells via La(III)-activated endocytosis; the homeostasis of the essential elements (K, Ca, Fe, Mg) was disrupted, although the chlorophyll synthesis and the growth of these leaf cells were accelerated; and the expression of stress response genes (GmNAC20, GmNAC11) in soybean leaves was increased. These results provided an insight to further analyze the toxicity and mechanism of REEs in plants, and sounded the alarm for the application of REEs in agriculture.

Toxicity of representative mixture of five rare earth elements in juvenile rainbow trout (Oncorhynchus mykiss) juveniles.

Rare earth elements (REEs) are contaminants of increasing interest due to intense mining activities for commercial purposes and ultimately released in the environment. We exposed juvenile rainbow trout (Oncorhynchus mykiss) to a representative mixture of the five most abundant REEs for 96 h at concentrations similar found in lakes contaminated by mining activities at 0.1, 1, 10, and 100X whereas the 1x mixture contained cerium (Ce, 280 µg/L), lanthanum (La, 140 µg/L), neodymium (Nd, 120 µg/L), praseodymium (Pr, 28 µg/L), and samarium (Sm, 23 µg/L). We investigated the expression of 14 genes involved in oxidative stress, DNA repair, tissue growth/proliferation, protein chaperoning, xenobiotic biotransformation, and ammonia metabolism in the liver. In addition, DNA damage, oxidative stress (lipid peroxidation or LPO), inflammation (cyclooxygenase or COX activity), detoxification mechanisms (glutathione-S-transferase activity or GST), and labile zinc were determined in gills. The data revealed that genes involved in oxidative stress-catalase (cat), heat shock proteins 70 (hsp70), and glutamate dehydrogenase (glud) were upregulated while glutathione S-transferase (gst) and metallothionein (mt) gene expressions were downregulated. The mixture was genotoxic and increased labile Zn in gills of exposed trout. These changes occurred at concentrations 600 times lower than the LC50 for this mixture indicating effects below the 1X concentration. Based on principal component analysis and concentration-dependent reponses, the following sublethal effects were considered the most important/significant: DNA strand breaks (genotoxicity), labile Zn, cat, gst, hsp70, sparc, mt, and glud. These effects of fish juveniles are likely to occur in environments under the influence of mining activities.