WETLAB

In this sense, we describe the production of lanthanide-binding peptides in E. coli and the subsequent testing of said sequences under bioleaching conditions. One of the most important parts of our lab work has been world’s first cell surface expression of the lanthanide-binding protein Lanmodulin on Saccharomyces cerevisiae, followed by the evaluation of the adsorption capabilities of our novel recombinant yeast colony. We also tested phage display as an effective and reproducible method for the identification of new lanthanide binding-peptide sequences. These sequences could in turn be used to continue the previously mentioned process of protein evaluation.

For efficient peptide purification and protein attachment, we chose a Strep-tag rather than a His-tag due to its diminished propensity for protonation within the lower pH range, as would be in a possibly envisioned bioleaching process 4.

Furthermore, to employ the peptides on a matrix besides a fungal surface – for example through immobilization on a hydrophobic bead – we decided to undertake experimentation on a polystyrene microtiterplate using an LCI tag to immobilize the peptides.

The pET28a-Strep-17x-LCI vector, containing an expression cassette, a kanamycin resistance and both Strep-tag and an LCI-anchor, fused with a 17 amino acid long linker to ensure flexibility and freedom of the fused peptides, was obtained from our supervisor.

Central to the evaluation is the binding capability and affinity of the designated peptides to lanthanoids. To measure this, we chose neodymium (in solution as Nd³⁺) as a stand in for lanthanoids in general for multiple reasons. Testing for multiple (or all) lanthanoids would have been very extensive, proof of concept is sufficient for us, and we also have reason to believe affinity within the periodic group is similar for all lanthanoids, since all enter solution as trivalent cations 5 and have similar atomic radii and affinity of the same peptide for different lanthanoids has already been shown. 6 Also, neodymium has more real-world use and higher demand than some other representatives of lanthanoids 7. We measured neodymium concentration through the already previously used Arsenazo III assay 8,9 which we derived for use on microtiterplates.

Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against original concentration. The neodymium solution applied to the peptides had a starting neodymium concentration of c0(Nd³⁺) = 25 µM and c0(Nd³⁺) = 50 µM. 94 µL of this was treated with immobilized peptides (applied to each well were 25 µM *100 µL respectively) Nd01, Nd02, MC, EF1 and EF4. For the calibration curve neodymium solutions of 6,25 µM, 12,5 µM, 25 µM and 50 µM were used.

All absorbance data plotted has been deducted the absorbance of the respective blank. The measurements for the calibration curve were done in duplicates while the measurements for the peptide supernatants were done in triplicates. Affinity of peptides was underestimated so that standard curve was extended by the 0 µM absorbance measurement for the calculation of neodymium concentration of the peptide treated supernatant.

Before this, we had to answer the question what neodymium concentration to expect from using an industry e-waste sample and if acidic leaching would even be feasible for mobilizing components of interest. For this we wanted to bioleach a sample using acids directly produced by Aspergillus niger or Acidithiobacillus ferrooxidans, yet, due to safety concerns we abandoned this idea. Instead, we conducted acidic leaching on an e-waste sample from industry provided from STENA Recycling and a NdFeB-Magnet at different pHs. Our results show a significant amount and rising tendency of neodymium being mobilized at lower pH.

Having successfully immobilized the peptides, validated selectivity and produced an authentic e-waste leachate, for our proof of concept, we wanted to recover neodymium from a real world, heterogeneous industry e-waste leachate. For this we chose two reliably performing peptides, again immobilized them and treated them with our previously quantified leachate. Our results show that we were able to recover over 68 % of neodymium from the industry leachate using our experimental setup.

To realistically test the functionality of our envisioned cell surface display system for the isolation of rare earth elements from leaching solutions, we chose to express Lanmodulin (LanM) on Saccharomyces cerevisiae EBY100. LanM is a native lanthanide-binding protein containing four EF-domains capable of specifically binding to lanthanides 10 for the expression on a fungal surface.

The decision to use Saccharomyces cerevisiae as a proof-of-concept organism was made because filamentous fungi are less accessible and require specific selection and testing for particular applications. S. cerevisiae was chosen due to its widespread availability of genetic modification tools, the abundance of relevant strains, and the ability to induce the formation of pseudohyphae 11, which could mimic the functionality of fungal filaments in our project.

To differentiate between surface-binding through LanM and natural biosorption, a negative control only containing pYD1 was used. A defined number of cells were incubated in neodymium solutions of different concentrations for two hours. Afterwards, the cells were separated via centrifugation and the neodymium concentration in the supernatant was determined using a calibration curve.

The minimal variance in absorbance between the supernatant of the negative control and the calibration curve suggests that, at these low cell concentrations, the negative control is incapable of adsorbing substantial quantities of Nd³⁺.

In contrast, the supernatant of the positive control containing pYD1-LanM exhibited noticeably lower absorbance for each initial concentration. Therefore, it appears that the Lanmodulin surface construct has allowed the mutant cells to bind neodymium ions in significantly larger quantities compared to natural biosorption of cells not expressing LanM despite the low number of cells being present during the assay.

The percentage of bound neodymium ions varied from 24% up to 60 %, the mean value amounted to 40%. For the highest initial concentration of 50 µM, 64 mg neodymium was bound per 1 g of cell dry weight during incubation. It is expected that higher concentrations of neodymium would lead to the mass of bound neodymium to be elevated even further as saturation did not seem to occur yet. However, the calibration curve stops showing linearity for higher concentrations.

In conclusion, the mass of bound neodymium ions exhibits a direct correlation with the initial neodymium concentration and notably varies between cells that express LanM on their surface and those that do not. Mutant cells expressing LanM on their surface are capable of binding higher amounts of Nd than cells only expressing the pYD1 construct.

We were able to identify one sequence potentially able to bind neodymium. The relatively low number of new sequences is only due to our limited capacity of sequencing new colonies. We have isolated a great number of additional positive colonies which could be used to gain additional lanthanide-binding sequences, e.g., via next generation sequencing methods.

These new sequences could prove to be a great source of novel lanthanide-binding peptides potentially applicable for rare earth elements recycling. To confirm and characterize their ability to bind to neodymium and other lanthanides effectively, additional experiments that specifically assess their binding capabilities, similar to those showcased in our evaluation of peptides for bioleaching applications, are necessary to be carried out.

Simultaneously, we would need to continue our protein evaluation process, to identify new lanthanide binding sequences. The first logical step would be the testing of the novel sequences, we were able to identify with our phage display. To develop a fully functional fungal filtration system, it would be crucial to find suitable peptide candidates capable of withstanding prolonged exposure to acidic environments.

In summary, we have achieved all major goals we set for our laboratory work, laying the necessary groundwork for the creation of a unique approach to the recycling of rare earth elements from electronic wastes. Besides this major achievement, we were also able to create an experimental framework for other groups to expand and conduct their own experiments upon.