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Systems-level identification and comparison of Acidithiobacillus ferrooxidans tolerance mechanisms to cobalt, lithium, and nickel

EMSL Project ID


The United States’ clean energy transition is reliant on sustainable and secure mineral supply chains as clean energy technologies require 4-6X the critical minerals of the conventional analogs (e.g., electric vs. gasoline vehicles). Considering the domestic supply of critical minerals such as lithium, cobalt, and nickel are not expected to meet the project demand, efficient recycling of e-wastes such as lithium-ion batteries will become increasingly critical to the “security and resilience of the Nation’s critical infrastructure” (BER mission, Biohydrometallurgical approaches to e-waste recycling (e.g., the use of microorganisms to leach metals from e-wastes such as batteries) are a promising alterative to pyro- or hydro-metallurgiacal appraoches owing to the predicted lower energy inputs, lower GHG emissions, and reduced toxic waste generation, but improved understanding of fundamental underlying biological processes is necessary towards technology development and deployment.

Chemoautolithotropic bacteria, such as Acidithiobacillus ferrooxidans, utilize CO2 as a carbon source and oxidize iron and/or sulfur for energy, and further have been shown to be viable biohydrometallurgical platforms. The role of these microorganisms in natural environments has been the subject of study for many decades motivated in-part by their role in acid mine drainage and commercial mining operations. However, metal tolerance mechanisms beyond iron and copper have been sparsely studied and almost never in combination, yet the natural environments in which A. ferrooxidans and other chemoautolithotrophs are found contain a multitude of minerals. Understanding the mechanisms by which chemoautolithotrophic bacteria interact with and/or tolerate high concentrations of metals will directly advance our ability to “control the function of biological and ecological systems” (EMSL 2021 Strategic Plan, p. 3).

This project aims to understand the molecular basis for A. ferrooxidans tolerance and adaption to high concentrations of cobalt, nickel, and lithium. These metals were chosen as (a) they are present in the natural environment and thus A. ferrooxidans has likely developed specific tolerance mechanisms, and (b) they are the three primary metal components of lithium-ion battery cathode materials that need to be extracted in biohydrometallurgical processes. We hypothesize that tolerance mechanisms to mixed metals will be different than individual metals, and thus by studying the intersection (and divergence) of Co/Ni/Li biological tolerance processes we will identify both specific and general biological processes. A systems-level multi-omics approach is warranted as metal tolerance mechanisms are known to involve both proteins (e.g., efflux pumps) and metabolites (e.g., siderophores/metallophores). Our approach is to study wild-type A. ferrooxidans as the baseline and evolve A. ferrooxidans to increasing concentrations of individual or mixed metals via established tolerance adaptive laboratory evolution methodology. Whole-genome resequencing of control and evolved isolates with increased tolerance and/or growth rate under these conditions will be conducted to identify causal genetic mutations. EMSL’s molecular science instruments – specifically mass spectrometers for proteomics and metabolomics, and RNA-Seq equipment – will be utilized to characterize the transcriptomic, proteomic, and metabolic basis for metal tolerance/adaptation. This data will directly correspond to the DNA resequencing data such the result of this work will be a genotype-to-phenotype understanding of A. ferrooxidans tolerance to mixed Co, Ni, and Li.

Project Details

Project type
Exploratory Research
Start Date
End Date


Principal Investigator

Allison Werner
National Renewable Energy Laboratory

Team Members

Kalen Rasmussen
National Renewable Energy Laboratory

Young-Saeng Avina
National Renewable Energy Laboratory

Gregg Beckham
National Renewable Energy Laboratory