Overview

Ouroboros: a cutting-edge microbial platform engineered to revolutionize the recycling of critical metals like Li, Mn, Co, and Ni from spent lithium-ion batteries. Our project harnesses the power of synthetic biology to create a sustainable, eco-friendly solution for metal recovery, meticulously designed around three innovative modules: bioleaching, biosorption, and biomineralization. Each module plays a pivotal role, transforming waste into valuable resources while minimizing environmental impact. Join us on a journey where nature meets technology in the quest for a circular, greener future.

Firstly, we use Aspergillus niger to produce citric acid and combine it with glucose oxidase (GOx) displayed on yeast cells to generate gluconic acid and hydrogen peroxide, facilitating efficient co-extraction of metal components from battery waste. Next, we use yeast surface display technology to precisely capture and separate metal ions from the leachate using specific metal-binding peptides and we also built a unique “fishing net” system to enhance the biosorption. Finally, we constructed an E. coli system with high expression of urease to induce biomineralization, precipitating captured metal ions as carbonates for recovery and reuse.

Fig. 1 Project Flow Diagram

In addition to the biological components, we have developed the necessary hardware to support and integrate these processes, ensuring the scalability and practicality of the system.

Fig. 2 Hardware Diagram

This recycling method not only significantly reduces pollutant emissions and energy consumption in the treatment of spent lithium batteries but also improves resource recovery rates through an optimized and efficient resource utilization strategies.

Here is detailed described the rationale behind the design of the parts of our system as well as an overview of our experimental design.

Module 1: Bioleaching

Bioleaching (also known as biohydrometallurgy) is an emerging green technology that uses microorganisms and their metabolic products to dissolve minerals and extract metals. It can also be used to recover and separate metal elements from waste materials. This project uses bioleaching technology to process the black powder obtained from spent lithium batteries that have been artificially disassembled and discharged, recovering four primary metals: Li, Mn, Co, and Ni.

Leachate Production

Aspergillus niger was chosen as our primary acid-producing microorganism. Under low pH and high oxygen concentration, it metabolizes glucose from the medium into pyruvate and then converts it into citric acid via the TCA cycle. The citric acid accumulates in the cells and is secreted into the extracellular space, serving as the leaching acid and complexing agent.

Fig. 3 Metabolism of Aspergillus niger

We utilized surface display techniques as a complement to co-extraction. We displayed glucose oxidase (GOx) on the surface of yeast cells. GOx catalyzes the oxidation of glucose to gluconic acid in a single step, which acts as the leaching acid. We were pleased to find that the byproduct, hydrogen peroxide, serves as a leaching reductant. By culturing the engineered yeast into glucose-rich media, we can obtain large quantities of gluconic acid and hydrogen peroxide (surface display technology will be discussed in the biosorption section).

Fig. 4 GOx Design Diagram

We chose “Spent medium bioleaching” as our leaching method. This “microbe-metal separation” leaching approach eliminates the need to consider microbial tolerance to heavy metal ions, allows for accelerated reactions by heating during leaching, and ensures that leached metal elements exist as free ions in the solution rather than being adsorbed onto the microbial membrane.

Fig. 5 Different methods of fungus bioleaching of refinery spent catalyst.

Module 2: Biosorption

Some trace metal ions in living organisms are coordinated with metal ion binding peptides (MIBPs), which inspired us to explore the possibility of achieving selective adsorption of metal ions from the leachate by displaying specific binding peptides on the surface of yeast cells. This method has been used in some studies for the bioremediation of heavy metal ion pollution in water. Since the representative and valuable metal ions in the black powder leachate are lithium, manganese, nickel, and cobalt, we chose binding peptides for these four metals to be displayed on Pichia pastoris to allow different engineered yeast strains to specifically adsorb each metal ion, facilitating subsequent recovery.

Metal-Binding Peptide Yeast Surface Display

Yeast surface display (YSD) is a “whole-cell” platform used for the heterologous expression of proteins immobilized on the yeast's cell surface. YSD combines the advantages of eukaryotic systems, such as post-translational modifications, correct folding, and glycosylation of proteins, with ease of cell culturing and genetic manipulation. It allows for protein immobilization and recovery. Additionally, proteins displayed on the yeast cell surface may exhibit enhanced stability against changes in temperature, pH, organic solvents, and proteases.

Fig. 6 Schematic of Metal-Binding Peptides Displayed on Yeast Surface and the genetic construct

Through extensive literature research, we selected 10 metal-binding peptides for surface display. These peptides are linked to the Pir1 anchor motif via linkers and displayed on the surface of Pichia pastoris. Due to issues with some peptides being too long or too short, we optimized their sequences for ease of surface display.

Fig. 7 Structure Diagram of Some Metal-Binding Peptides

Polyester Nonwoven Fabric and Yeast Binding

Engineered yeast needs to be fixed in the adsorption tank to achieve effective adsorption. Polyester nonwoven fabric can bind with Pichia pastoris through extracellular polymeric substances (EPS), forming an active biofilm on the surface and allowing effective yeast fixation. As the biofilm matures, some dead cells within the biofilm can lead to biofilm detachment, a phenomenon that can be applied to regulate yeast detachment in the engineering process.

Fig. 8 Polyester Nonwoven Fabric Used for Yeast Fixation

Considering the limited metal-binding capacity of peptides in their monomeric state, we further enhanced adsorption efficiency by employing a self-assembly strategy based on the surface display of metal ion-binding peptides. A cellular system with a mechanism analogous to "fishing" was designed, wherein metal ion-binding peptides are simultaneously secreted in high throughput into the solution while being displayed on the surface. The peptides will self-assemble in situ, using the surface-displayed peptides as anchor points, encapsulating a large quantity of metal ions during the assembly process. Furthermore, the resulting assembled structures will provide additional binding sites.

Fig. 9 “fishing net” system design diagram

Module 3: Biomineralization

This module utilizes biomineralization to convert metal ions (Li, Mn, Co, Ni) separated from the black powder in the biosorption module into corresponding carbonate precipitates. These carbonates are then used in various ratios to form new high-performance electrode materials, achieving green recycling of lithium-ion batteries.

Surface Display of SazCA in E. coli BL21 (DE3)

Inspired by iGEM23_MSP-Maastricht, we designed a strategy for surface display of SazCA (carbonic anhydrase from Sulfurihydrogenibium azorense) in E. coli. SazCA, one of the fastest known carbonic anhydrases, is expressed fused with the outer membrane protein INPN of E. coli through genetic engineering, exposed to the extracellular environment. This allows efficient catalysis of CO₂ hydration to form bicarbonate, promoting the formation of carbonate minerals. This design focuses on improving enzyme-substrate contact efficiency and is expected to enhance mineralization efficiency.

Fig. 10 Visual Representation of the SIMD Fusion Protein

Urease from S. pasteurii Recombinant Expression in E. coli

In our experiments with E. coli BL21 (DE3) displaying SazCA for biomineralization, we faced challenges with suboptimal mineralization results (details are in the engineering section). The carbonate mineralization process is usually more effective under alkaline conditions, where carbonate ions (CO₃^2-) are more stable and contribute to mineral phase formation. However, the formation of carbonic acid (H₂CO₃) from the reaction between SazCA and CO₂ in water lowers the pH, creating an acidic environment that reduces carbonate ion concentration and impedes mineral phase stability, affecting mineralization efficiency.

We transitioned to expressing the highly efficient urease gene from S. pasteurii in E. coli, constructing a dual-plasmid system to enhance expression efficiency. Urease catalyzes the hydrolysis of urea to generate ammonia and carbonate ions, raising the pH and carbonate ion concentration. Additionally, carbonate ions generated intracellularly diffuse to the extracellular space along the concentration gradient, with the highest concentration at the cell membrane and nucleation sites provided by the microbial membrane. Urease effectively induces mineralization of Li, Mn, Co, and Ni ions to form carbonate precipitates. This conversion allows for microbial-induced carbonate precipitation under alkaline conditions, improving mineralization efficiency.

Fig. 11 Recombinant Urease Mineralization Model Diagram

Reference