EXPERIMENTS

Construct Design

Goal

Designing a Plasmid that has all the desired parts that are needed to be able to produce and purify the peptides.

Methods

A backbone plasmid provided by Schwaneberg group contained following features:

Fig 1. | Initial plasmid pET28a_ Strep-17x-LCI (5534 bp) for gene expression of peptides in E. coli.

T7 Promotor:
The T7 promoter used to regulate gene expression of recombinant proteins. The T7 promoter is a DNA sequence 18 base pairs in length to the transcription start site at +1 (5' - TAATACGACTCACTATAG - 3') that is recognized by T7 RNA polymerase.

T7 Terminator:
A Strep-tag is a peptide that consist of 8 amino acids which originates from the bacterium Streptomyces avidinii. The Strep-tag has a strong affinity for Strep-Tactin, which is used for purification of the fusion proteins or in our case peptides via affinity chromatography. Also, the Strep-tag has a diminished propensity to be protonated at low pH rather than a His-Tag, which would be useful for recovery of peptides from acidic leaching solution or streptavidin-mediated peptide coupling at low pH. This could for example be useful to test immobilization density at low pH using HRP-ELISA.

LCI:
LCI is a 47 amino acid long sequence that allows binding of the fusion peptide to polystyrene; thus, the peptide can be immobilized on microtiter plates. This is particularly important for determining the affinity with the AS III assay. 1

Linker:
The peptide sequence of interest was employed on a 17x linker to guarantee flexibility and accessibility of the binding sequence. This was to ensure unhindered peptide-ion interaction.

Gene of interest:
Five different peptides known by literature were investigated. 2,3,4 Our criteria for selecting the different peptides were largely based on the selectivity for lanthanoids, especially Nd, and the ability of the peptides to bind these compounds of interest at very low pH conditions. These properties were quantified by our subsequent experiments.
Amino acids sequences of the chosen peptides:

EF1: DPDKDGTIDLKE 2
EF4: NPDNDGTIDARE 2
Nd01: GLHTSATNLYLH 3
Nd02: DSARSSGNIYGL 4
Most common: YIDTNNDGWYEGDELLA 4

For the designed constructs, we implemented codon optimized base sequences analogous to the above shown amino acid sequences.

Building Construct

Goal

To generate the plasmid for the peptide production via PCR reaction and to confirm the favored insertion of the peptide through gel electrophoresis and sequencing.

Methods

The PCR method was used to modify a plasmid into containing the needed peptide sequence. The used plasmid already contained the needed parts for the production (promotor, terminator), the purification (Strep-Tag) and the immobilization on polystyrene (LCI). After the PCR was conducted the insertion of the sequence into the plasmid was checked through agarose gel electrophoresis which allows the assessment of whether the PCR produced the intended DNA fragments and was then verified through DNA sequencing. Afterwards a two-step PCR was conducted, and its success was also checked through agarose gel electrophoresis and verified through DNA sequencing. Subsequently the rest of the PCR product was purified through a purification set and the concentrations were measured using a Nanodrop spectrophotometer. Followed by the execution of a restriction digestion using the restriction enzyme DpnI.

Comment PCR (three-step)
We noticed that the amount of primer needed adjusting as well as the number of cycles for step 1 of the PCR and the temperature for the attachment of the primer in step 2.

Comment PCR (two-step)
Since the three-step PCR was not yielding the hoped results, we utilized the two-step PCR which grants the respective primers used, specific annealing conditions while also carrying out the overlap extension to implement the insert in the product. For this the temperature is lowered facilitating primer binding to ss DNA and the DNA polymerase produces a new DNA strand from the primer.

Protocols

Results

PCR (three-step):
By observing the band sizes in the gel image and not being able to locate bands at 6000 kb we conclude that the PCR was ineffective thus the process of the PCR was repeated with a different polymerase called Q5 DNA polymerase. This polymerase makes fewer errors during DNA replication and because of its low error rate causes minimal mutations in the replicated DNA.

PCR (two-step):
The location of bands in the gel at 6000 kB suggests that the PCR was successful, but the actual conformation is done by DNA sequencing carried out by the company eurofins. After validation through sequencing of the PCR product, we had successfully produced our vectors containing insert. These we could now use for amplification and the following expression tests.

Transformation

Goal

Producing specific lanthanide-binding peptides (Nd01, Nd02, MC, EF1 and EF4) by expressing them in E. coli cells using our vectors produced from pET28a-Strep-17-LCI. Inserting plasmids containing the DNA-sequences of the peptides into cells of E. coli (transformation) and subsequently confirming the presence of the correct plasmid-sequence in the cells.

Methods

To achieve a successful transformation, two steps are executed: At first, the transformation of the cells with the plasmid is performed and in a second step the insertion of the correct and complete plasmid is verified through plasmid preparation.

In a first step, chemically competent cells need to be produced. For that, cultivated bacteria cells of E. coli strain BL21 were prepared for the transformation by treatment with reagents TFB I and TFB II and frozen until usage. To perform the transformation the competent cells were thawed, incorporated within the pET28a-Strep-17-LCI plasmid (containing one of the lanthanide-binding peptides Nd01, Nd02, MC, EF1 and EF4) and exposed to a heat shock, which results in the insertion of the plasmid into the cells. Afterwards, the cells were plated on a selection medium containing ampicillin. The plates are incubated over night at 37°C.

Colonies with the ability to grow on the plates represent candidates for successfully transformed clones. In a second step, the insertion of the complete and undamaged pET28a-Strep-17-LCI construct was confirmed. To achieve this, a plasmid preparation was conducted, utilizing a NucleoSpin™ kit. This includes extracting all plasmid-DNA the clones possess via alkaline lysis and isolating it using a DNA-microcolumn. Subsequently, the success of the plasmid extraction was tested through gel electrophoresis. At last, the plasmids were sequenced and for permanent storage of the transformed cells, glycerol-cultures are produced and frozen at -80°C.

Protocols
Every step is executed on a clean bench.

Production of chemically competent cells

Used buffers and solutions:
Prepare all solutions with cell culture grade water and chemicals of highest purity. Rinse bottle and culture flasks with sterile water before use. Prepare buffers fresh or one evening before and cool down at 4°C!

TFB I:
The quantities given are for a volume of 100 mL.

Top up with water to 95 mL, control final pH and adjust to pH 5.8 with 0.2 M acetic acid. Add cell culture grade water up to 100ml.

TBF II:
The quantities given are for a volume of 100 mL.

Add water to 95 ml, control final pH and adjust to pH 7 with 1 M NaOH.

Results

After several negative attempts, transformed colonies growing on ampicillin medium could be detected for all peptides (figure 2).

Fig 2. | Colonies of transformed E. coli cells growing on selective medium. 450 µl of the transformed cells, each containing one of the peptides, were plated on LB medium agar plates containing ampicillin. The plates were incubated at 37°C over night. The top left plate contains clones producing Nd01, the top right plate the cells expressing Nd02, the bottom right plate clones expressing EF 1 and the bottom left plate cells producing EF 4.

As illustrated in figure 2, different numbers of colonies as summarized in figure 3 grew on the plates.

Fig 3. | Number of colonies growing on selective medium and expressing one of the five peptides.

The gel electrophoresis of the elutes from the plasmid preparation extracted from the clones showed the following results (figure 4).

Fig 4. | Gel electrophoresis of the elutes, 3 samples per peptide were given into the pockets. Slot 1-3: Nd01; Slot 4-6: Nd02; Slot 7-9: EF1; Slot 10-12: EF4; Slot 13-15: MC; Slot 17: 1kb ruler.

As seen in figure 4, every single one of the five plasmids shows at least one slot with a band around the expected length of 500bp.

Samples of the plasmid-DNA isolated from the clones (Nd01, Nd02, EF1, EF4 and MC) were sent to sequencing, the results were positive for all peptides (100% alignement with the expected sequence).

For later usage glycerol stocks of the clones were produced and conserved at -80°C.

Production of Peptides and their Purification (Exp. 1: Overnight Cultures)

Goal

For our expression, we need to produce cultures in which we can induce expression on a large scale, to inoculate these larger cultures, smaller ONCs are required.

Methods

Production of ONC in test tubes with inoculation cultures being picked from plates.

Protocols
Every step is executed on a clean bench.

Results

All ONC grew over night and could be used to inoculate shake flasks for expression test.
Had to be redone for Nd01 and Nd02 because expression test was negative.

Production of Peptides and their Purification (Exp. 2: Expression Test with SDS-PAGE as Detection Method)

Goal

Verifying that our transformed cells can produce the used peptides.

Methods

Start following the SOP for inoculating shake flasks. Measure OD of all cultures with TB-Medium as a blank until it is < 0.5. Induct the shake flasks with IPTG in a 1:1000 dilution. Shake it at 25°C at 220 rpm for 24 h. Transfer the cells into falcon and centrifugate for 30 min at 4°C at 4000 rpm and remove supernatant. Follow SOP for production of cell lysate with sonificator. Centrifugate cell lysate for 30 min at 4°C at 4000 rpm. Filter supernatant and use it for following steps. Follow SOP for SDS-PAGE.

Protocols

Results

SDS-PAGE in first round of expression test: Expression test was successful for EF1, EF4 and MC

Fig 5. | Measured OD in first round of expression test.

The first round of the expression test was negative for Nd01 and Nd02. As the OD for these two was much lower than the OD of the other peptides there probably were not enough cells which were able to produce the desired peptide. This resulted in low yields which could not be detected by the SDS-PAGE.

For the second round we decided to use the Dot-Plot as detection method as it is much faster.

Fig 6. | Detection of peptides for EF1, EF4 and MC. Perfect Protein™ Marker was used as ladder.

Production of Peptides and their Purification (Exp. 3: Expression Test with Dot Plot as Detection Method)

Goal

Verifying that our transformed cells can produce the used peptides.

Methods

Start following the SOP for inoculating shake flasks. Measure OD of all cultures with TB-Medium as a blank until it is < 0.5. Induct the shake flasks with IPTG in a 1:1000 dilution. Shake it at 25°C at 220 rpm for 24 h. Transfer the cells into falcon and centrifugate for 30 min at 4°C with 4000 rpm and remove supernatant. Follow SOP for production of cell lysate with sonificator. Centrifugate cell lysate for 30 min at 4°C at 4000 rpm. Filter supernatant and put 2 ul of it onto a nitrocellulose membrane. Block membrane with PBS and 3% BSA for 1 h. Remove liquid and moisten membrane with Anti-Strep HRP (Horse Raddish Peroxidase) antibody in PBS and 3% BSA (dilution 1:5000). Wash 3 times with PBS und 0.05% Tween20. Dry membrane and incubate with 2 ml Blotting Solution TMP. Stop reaction with ddH2O.

Protocols

Results

SDS-PAGE in first round of expression test: Expression was test successful.
Since we used cell cultures with a higher OD in the second round of expression tests the amount of cell material was high enough to detect the desired peptides.

Fig 7. | Measured OD in second round of expression test.

Fig 8. | Detection of peptides Nd01 and Nd02.

Production of Peptides and their Purification (Exp. 4: Protein Purification)

Goal

Producing peptides for the peptide application evaluation and aliquotation of produced peptides for further use.

Methods

Start following the SOP for overnight cultures and glycerol stocks and then for inoculating shake flasks. Measure OD of all cultures with TB-Medium as a blank until it is >0.5. Induct with IPTG in a 1:1000 dilution. Shake it at 25°C at 220 rpm for 24 h. Transfer into falcon and centrifugate for 30 min at 4°C with 4000 rpm and remove supernatant. Follow SOP for production of cell lysate with sonificator. Centrifugate cell lysate for 30 min at 4°C at 4000 rpm. Filter the supernatant. The Äkta with an Affinity chromatography column, StrepTrap™ XT by Cytiva is used as described in the instruction manual. With some of the collected fractions an SDS-PAGE is run to proof the success of expression. Follow SOP for BCA.

Protocols

Results

There was no cell growth for Nd02 in the first round of protein expression, so the experiment was redone for that peptide.

The SDS-PAGEs detected the expression of all peptides.

Fig 9. | Track 1 to 4 show Aktä fractions 6,9,12 and 15 of Nd01. Track 5 shows EF1. Track 6 shows EF4. Track 7 shows MC. Blue Prestained Protein Ladder of Gel was used as ladder.

Fig 10. | Track 1 shows Äkta fraction 3, track 2 Äkta fraction 4, track 3 Äkta fraction 5, track 4 Äkta fraction 6 and track 5 Äkta fraction 7 of Nd02. Blue Prestained Protein Ladder of Gel was used as ladder.

The highest protein yield for Nd01 is detected in Äkta fraction 9. The highest protein yield for Nd02 is detected in Äkta fraction 5.

The BCA shows that the highest protein yield is received with MC.

Fig 11. | Concentration for all peptides determined with BCA.

All peptides can be produced by our transformed cells in significant amounts. As the highest concentration occurs for MC, we choose these two for further experiments with fungi. The concentration of Nd02 is much lower than the concentration of the other peptides. There were growth issues for this peptide several times which leads us to the conclusion that there is proper expression of this peptide in our cells.

Arsenazo III Assay Adaptation

Goal

To assess peptides and leaching of e-waste, we need to measure lanthanoid concentrations. For this, it is necessary to have a measurement method which is reliable and can be used for our experiments. Adapt commercially available AS III assay for our experiments.

Methods

Adapting the AS III assay for our experiment was done following the procedure in Hogendoorn et al.. The purpose of this paper was the quantification of lanthanoid concentrations of cultivation media. However, in this research the goal is the adaptation of quantification of the concentration of Neodymium (Nd) in different solutions. To achieve this goal, binding experiments of Nd with different peptides in an aqueous environment were performed. Furthermore, different bioleaching solutions were analyzed. The scale of the experiments was set to samples in microtiter plates (MTPs) with a volume of 200 µl. Instead of a cultivation medium by Hogendoorn et al. a certain sample solution was analyzed.

To scale up the sample size from 1 µL to 200 µL, the number of components were adjusted keeping the ratio the same. The absorbance was measured at 650 nm for different concentrations of Nd to determine a linear regression. For colored solutions the characteristic wavelength must be taken into consideration, which may interfere with the assay.

Protocols

Results

The assay can easily be adapted to be used in microtiter plates by multiplying the amount of the used components in the original paper by the factor of the scale down – 1/5 – and then performing the assay in the wells of MTPs. For the first trial, Nd was dissolved in the prepared buffer. Yet the resulting linear range for the assay was only down to about 100 µM and the prepared stock solution of 1 mM was cloudy. Through solving the Neodymium trichloride in Millipore water a clear stock solution was obtained and a linear range down to 1 µM. Two resulting calibration curves were plotted in the peptide affinity experiment.

For the quantification of industry samples/bioleaching experiments, the characteristic peak at 650 nm which is used by the assay may be overlayed when other photoactive components are in solution which shows the missing peak at a to high concentration of the sample obtained from the industry solution leached at pH 0.

Examining Immobilised Peptides on MTP using HRP-ELISA

Goal

Estimating the immobilisation density of the peptides in order to generate fundamental data to use for further affinity and selectivity tests

Methods

To quantify the immobilisation density of the peptides, they are in a first experiment immobilized on a microtiter plate (5 µM, 100 µL per well) and analysed using HRP-ELISA. In a second trial, different concentrations of the peptides were used following the same method (10 µM, 5 µM, 2,5 µM, 1,25 µM, 0,625 µM) to assess at which concentration the wells are saturated with immobilised peptides.

Protocols

Results

The resulting absorbance data of the first experiment is presented in figure 12. The data shows that the peptides could all be immobilized in similar amounts, with Nd02 exhibiting higher absorbance and Nd01 showing lower absorbance compared to the other peptides.

Fig 12. | Adsorbance at 405 nm after application of HRP-ELISA on five peptides. The peptides were immobilised on a MTP using using 100 μL of 5 μM peptide solution. Afterwards, HRP-ELISA was applied and the absorbance at 405 nm was measured using a ClarioStar plate reader. The measurements were performed in triplicates.

The absorbance data generated during the saturation experiment is visualized in figure 13. This demonstrates that saturation was not achieved, and the absorbance increases up to a peptide concentration of 10 μM. An exception to this is peptide EF 4, which exhibits the highest absorption at 5 μM.

Fig 13. | Plotting absorbance at 405 nm in dependency of peptide concentrations. The peptides were immobilized on a 96 well microtiter plate using 100 μL using in ascending concentrations of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. The absorbance after the application of HRP-ELISA was measured using a ClarioStar plate reader. The measurements were performed in triplicates.

Comment: Nanodrop. Prior to the experiments described above, an attempt was made to determine the success of immobilization using Nanodrop. The peptides were immobilized using the same procedure (100 μL, 10 μM), and the concentration of peptides in solution was determined using Nanodrop before and after the incubation period. Since no significant difference in peptide concentration was observed, HRP-ELISA was applied.

Discussion: Our data depicted in figures 12 and 13 demonstrate successful immobilization of the peptides on the MTP at pH 8. However, within a concentration range of 10 μM, three peptides did not exhibit saturation of absorbance and consequently, immobilization density. To provide a more precise assessment of saturation, higher peptide concentrations would need to be employed. Nevertheless, it is anticipated that due to their small size, the peptides bind very well to the surface of the MTP and at a higher concentration immobilisation saturation can be achieved.

Raw data

Fig 14. | Mean absorbance at 405 nm of the five peptides after applying HRP-ELISA. The peptides were immobilized on a 96-well microtiter plate (100 μL, 5 μM), and the absorbance was measured using a ClarioStar plate reader. The measurements were performed in triplicates.

Fig 15. | Mean absorbance at 405 nm of four peptides after applying HRP-ELISA. The peptides were immobilized on a 96-well microtiter plate using 100 μL using in ascending concentrations of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. The absorbance was measured using a ClarioStar plate reader. The measurements were performed in triplicates.

Peptide Absorbance

Goal

Assessing the peptides absorbance for their use in bioleaching process. Determining the peptide absorbance under the conditions of the Arsenazo III assay in the presence of various peptide constructs.

Methods

To detect possible interactions of the tested peptides with the Arsenazo III assay, citrate/phosphate buffer, Millipore water, Tris-HCl (50 mM, pH 8, 200 μL) or protein construct were employed with the Arsenazo III assay. 1:4 and 4:1 ratios of peptides to water were tested to assess the significance of the proteins effect on the absorbance. Tris-HCl buffer was used as a blank sample, since the proteins were solved in a TrisHCl buffer. Under these conditions five peptides were tested (Nd01: 25.79 µM, Nd02: 47.0 µM, EF1: 33.903 µM, EF4: 33.487 µM, MC: 50.013 µM). Each absorbance was measured using the modified Arsenazo III assay at 650 nm. The mean values and standard deviation were then determined.

Protocols

Results

Several absorbance tests were performed on different peptides, whereby the dependency between the absorbance with the concentration of peptides was analyzed. Therefore, the absorbance between a high and low peptide concentrated solution was compared. In all performed experiments the concentration was increased by the factor of four for the higher concentrated peptide solution. In the following part, the measured data were summarized in graphs. The first experiment was performed with Nd01 as peptide (Fig. 16).

Fig 16. | Absorbance data of MTP of two water to peptide ratios with peptide Nd01 following the Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against volumetric ratios. The ratios are in reference to the water/peptide ratio. Blank values were subtracted from the measured absorbance values.

In a solution with a low concentration of peptide Nd01, an absorbance up to 0.043 can be observed. With an increase of the Nd01 concentration (four times) the absorbance value increases by 39% as well to value of 0.066. This was expected, since with a higher peptide concentration more molecules are in solution leading to a higher absorbance. To validate this trend, the experiment was repeated, whereby, the peptide was changed to Nd02 (Fig. 17).

Fig 17. | Absorbance data of MTP of two water to peptide ratios with peptide Nd02 following the Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against volumetric ratios. The 80% to 20% ratio consisted of 78.4 µL of ddH2O and 19.6 µL of the peptide Nd02, the 20% to 80% ratio is the inverse of these volumes. Blank values were subtracted from the measured absorbance values.

As in Fig. 16, the solution with the higher Nd02 concentration leads to a higher absorbance value (increased by 23%) than the solution with the higher amount of water. However, with an absorbance value of 0.051 for the low concentrated Nd02 solution a higher absorbance was obtained (Fig. 17) than with the low concentrated Nd01 solution (Fig. 16). Whereby, the absorbance for the higher concentrated Nd02 solution is similar to the value achieved with Nd01 (Fig. 16). Next on, the experiment was repeated with EF1 as peptide (Fig. 18).

Fig 18. | Absorbance data of MTP of two water to peptide ratios with peptide EF1 following the Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against volumetric ratios. The 80% to 20% ratio consisted of 78.4 µL of ddH2O and 19.6 µL of the peptide EF1, the 20% to 80% ratio is the inverse of these volumes. Blank values were subtracted from the measured absorbance values.

The absorbance is higher by 7% in the higher concentrated EF1 solution (0.042) than in the lower concentrated one (0.038). In comparison, the absorbance here obtained is significantly lower than in the test performed with Nd01 (Fig. 16) and Nd02 (Fig. 17). However, the trend of a higher absorbance in the higher concentrated peptide solution than in the lower concentrated solution was observed as well. The next experiment was performed with EF4 (Fig. 19).

Fig 19. | Absorbance data of MTP of two water to peptide ratios with peptide EF4 following the Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against volumetric ratios. The 80% to 20% ratio consisted of 78.4 µL of ddH2O and 19.6 µL of the peptide EF4, the 20% to 80% ratio is the inverse of these volumes. Blank values were subtracted from the measured absorbance values.

Similar values for the absorbance as in the experiment with EF1 were measured with EF4. The absorbance for the lower concentrated EF4 solutions was 0.037 and for the higher concentrated EF4 solution a value of 0.043 was reached. Therefore, the trend was confirmed here as well. Next on, the experiment was performed with MC as the peptide (Fig. 20).

Fig 20. | Absorbance data of MTP of two water to peptide ratios with peptide MC following the Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against volumetric ratios. The 80% to 20% ratio consisted of 78.4 µL of ddH2O and 19.6 µL of the peptide MC. Blank values were subtracted from the measured absorbance values.

With the lower MC concentrated solution an absorbance of 0.047 was obtained, which is similar to the values measured with Nd01 (Fig. 16), EF1 (Fig. 18) and EF4 (Fig. 19). However, with an absorbance of 0.078 a significantly increase by 39% was achieved with the higher MC concentrated solution than in the lower concentrated one. Compared to the previous measurements, the highest absorbance was reached with MC as the peptide.

Significances of the discrepancy between a solution containing peptides in contrast to a solution containing no peptide were calculated as follows: $$ Median absorbance * (3 * Std. Deviation).

These significances were than compared within a range comprised of the median absorbances of the blank and the respective ratio, to determine the peptides level of impact on the absorbance in the Arsenazo III assay.

Fig 21. | Significance of discrepancy between solutions containing the peptides against solutions with no peptides. Method of calculation can be found above. Green colour-coding indicating significant impact on the AS III assay. Red colour-coding indicating no significant impact on AS III assay.

Comment: To only determine the absorbances of the peptides other factors were eliminated by measuring a blank sample, consisting of citrate/phosphate buffer, millipore water and TrisHCl and the AS III assay.

Discussion: Overall, interactions between the Arsenazo III assay with each tested peptide was observed. Furthermore, increasing the concentration of peptide in solution a higher absorbance was achieved in all experiments. This effect can be elucidated by increasing the peptide to water ratio by four times the original amount (see figures 16-20), since more molecules are in solution. Depending on the peptide, this increase in absorbance can vary. Observing increases of 20% to 40% with peptides Nd01, Nd02 and MC and rather low increases in absorbance with peptides EF1 and EF4. To determine the impact of the absorbance of the peptides within the AS III assay conditions, significances had to be calculated (see figure 21). The median value of the blank samples, concerning the respective ratio, and the median absorbance of the corresponding water-to-peptide ratio, were established as the lower and upper limits of a defined range (see figure 22). Once the calculated significance value surpasses these thresholds of the range, it is indicative of a significant impact of peptide absorbance on the assay. Significant effect of the peptides on the absorbance were observed in the 80% to 20% water-to-peptide ratio sample for Nd01 and in the 20% to 80% water-to-peptide ratio sample for Nd02. No significant impact of the peptides on the absorbance were observed in the other samples. Subsequently, it becomes evident that certain peptides exhibit a minimal impact on the measured absorbances in the context of the AS III assay, while other peptides lead to a higher impact on the absorbance. For the subsequent experiments, we have opted to continue recording control samples of the protein in order to facilitate the identification of any deviations in the AS III assay test data that may arise during our experiments.

Raw data

Fig 22. | MTP absorbances for Nd01, Nd02, EF1, EF4 and MC.

Fig 23. | Calculated median absorbances of 80% to 20% and 20% to 80% water to peptide ratio.

Peptide Affinity Tests

Goal

To assess the peptides for their use in a bioleaching process, it is crucial to determine their binding capacity to lanthanoids (in acidic environment). Quantification of the affinity of the previously produced peptides (Nd01, Nd02, MC, EF1 and EF4) to Neodymium (Nd).

Methods

To quantify the binding capacity, peptides were immobilized on a microtiter plate (MTP) (25 µmol ∙ 100 µL per well). To each plate Neodymium solutions with different known concentrations (pH 2.8) were added. After 30 min incubation with gentle shaking, the solutions were removed from each well containing the immobilized peptides and transferred to another microtiter plate. The resulting concentration was determined by using the previously adapted Arsenazo III assay. Simultaneously, a calibration curve was generated to classify the results. For the first trial an equimolar concentration (25 µM ∙ 94 µL) of Nd was applied to the immobilized peptides and a calibration curve was taken in parallel for 25 µM, 12.5 µM, 6.25 µM and 3.125 µM all in 94 µL. In the following trial the immobilized peptides were treated with 25 µM ∙ 94 µL and 50 µM ∙ 94 µM and the difference to the applied solutions was measured. In a follow-up experiment, the calibration curve was extended for the concentrations 50 µM, 25 µM, 12.5 µM and 6.25 µM.

Protocols

Results

To evaluate the binding of Nd ions with different peptides, the absorbance of peptide-treated Nd solutions and Nd solutions without peptides at different concentrations of Nd were measured. The collected data was summarized in a graph (Fig. 24).

Fig 24. | Absorbance data of MTP with transferred calibration curve and peptide treated neodymium solution supernatant after Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against original concentration. 94 µL Nd solution of c0(Nd3+) = 25 µM was treated with immobilized peptides Nd01, Nd02, MC, EF1 and EF4 (applied to each well 25 µM ∙ 100 µL respectively). For the calibration 6.25 µM, 12.5 µM and 25 µM Nd solutions were used. The absorbance of the respective blank was subtracted from the measured data. The measurements for the calibration curve were performed two times, while the measurements for the peptide supernatants were performed three times and each median value was determined and used in this graph.

The untreated supernatant with c0(Nd3+) = 25 µM has an absorbance value of 0.167. The lowest absorbance of 0.104 was obtained with EF1. Whereby, with peptides MC and Nd02 the highest absorbance value of 0.127 was reached. Therefore, a decrease of the absorbance of the peptide treated Nd solutions was observed. The absorbance value was reduced by 24 – 38 % by adding peptides. Since the absorbance is correlated to the concentration of the substance, a decrease of the concentration of Nd by adding peptide was suggested. However, since more molecules are in solution, a certain decrease of the absorbance in the peptide containing solutions was expected. To further determine the affinity of the peptides Nd01, Nd02, MC, EF1 and EF4 to Nd ions, the molar affinity at equimolar exposure of immobilized peptides with neodymium solution was determined (tab. 1).

Fig 25. | Molar affinity at equimolar exposure of the immobilized peptides to Nd solutions (Fig. 24) were determined. ∆c(Nd³⁺) is the difference in neodymium concentration from the supernatant treated with peptide to the untreated supernatant. K_a is the calculated binding constant as quotient of ∆c(Nd³⁺)) divided by c(Peptide).

Fig 26. | Absorbance data of MTP peptide treated neodymium solution and untreated peptide neodymium solution following Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of supernatant is plotted against original concentration. The neodymium solution had an original neodymium concentration of c0(Nd3+) = 25 µM and 50 µM respectively. 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. The measured absorbance data were subtracted from the absorbance of the respective blank. The untreated peptides were measured two times and the peptide-treated supernatants were measured three times, each median value was determined and used in this graph.

A reduction in absorbance – and thus neodymium concentration – was observed for all peptides except Nd01. However, due to poor regressive fit the neodymium concentration of the peptide treated supernatant cannot be quantified. The experiment is presented for the sake of completeness and to show the varying binding tendencies/behavior of the peptides.

Fig 27. | Absorbance data of MTP with calibration curve and peptide treated neodymium solution supernatant following Arsenazo III-assay. 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. The measurements for the calibration curve were performed two times, while the measurements for the peptide supernatants were performed three times and each median value was determined and used in this graph.

A reduction in neodymium concentration in the supernatants treated with peptides was observed. The resulting affinity of immobilized peptides treated with 25 µM and 50 µM neodymium solution is given in the following figure 28. Based on the experiments conducted before, the affinity of the peptides and concentration of bonded Nd in solution was underestimated. Thus, the standard curve for very low neodymium concentrations was not plotted. The regression was extended for the values of the blank to allow quantification of the samples. Therefore, the standard curve for very low neodymium concentrations was not plotted. To provide quantification of the samples, the regression was extended for the blank sample values.

Fig 28. | Bonded Nd in the solution and molar affinity of (25 µM) immobilized peptides exposed to 25 µM and 50 µM neodymium solution each for experiment 3. ∆c(Nd³⁺) is the difference in neodymium concentration from the supernatant treated with peptide to the untreated supernatant. K_a is the calculated binding constant as quotient of ∆c(Nd^(3+) ) divided by c(Peptide).

Regarding the different performances of peptides in the binding experiments in terms of binding capacity, a range of affinities based on the collected data are summarized in the following table.

Fig 29. | Affinity range of the binding of Nd to the different peptides. Calculated affinity for peptides Nd01, Nd02, MC, EF1 and EF4. K_a is the calculated affinity constant. The interval limits are taken from figure 28 and 29.

Comments:
We experimented with different setups for the experiment, we settled on this setup because it allowed us to do the AS III assay in a solution without any interference of the buffer or the Arsenazo chemical in the Peptide-Ion binding process.

We noticed that it is necessary to include blanks of the individual immobilized peptides in the calculation for their absorbance data, since treatment of a blank with an immobilized peptide.

Also, we conducted this experiment multiple times to find a different varying binding behavior of the peptides for the applied 25 µM and 50 µM neodymium solution.

As the paper regarding the AS III assay suggests, for evaluating lanthanoid concentrations above 10 µM an amount of has been found to give a more reliable calibration curve.

We also experimented with two calibration curves, one being added into a well and then transferred to another well by using a pipette and one being directly added without it being transferred to another well by using a pipette. We found that these curves differed significantly as such that the curve of the directly added solution had overall lower absorbance rates than the transferred curve.

Discussion:
Our data shown in figures 24, 26 and 27 suggests that the peptides are capable of binding dissolved Nd in significant amounts but with different affinities. The resulting range of affinity is displayed in figure 29. Due to various factors, the range of affinities is significantly large. For the immobilization density, the quantification of immobilized peptides on the MTP after treatment with the sample was difficult to obtain. Since the best immobilization density was achieved at pH 8, it was suspected that the MTP is coated with less peptide than we estimated, as incubation with a sample (pH 2.8) may decrease the immobilization density by destabilizing the LCI-polystyrene interaction. Thus, the actual binding capacity would be even higher than the one we calculated based on the initial peptide concentration for the immobilization. The varying performance observed may be due to many different factors and is not always plausible to us. For example, it is unclear to us how a lower concentration in the 50 µM supernatants rather than the 25 µM supernatant results for peptides EF1 and EF4 in experiment 2. We suspect, since the neodymium salt was solved in Millipore water and the dilution was done with Millipore water whose pH was increased to 2.8 by titration with 1 M hydrochloric acid, the actual pH of the sample for the 50 µM solution would have had a higher pH than the 25 µM sample. This causes a different equilibrium for binding and changes parameters for the colorimetric assay as the paper we derived the AS III assay from shows. It is also necessary to take into account that the affinity of the peptides for other lanthanoids may differ. However, we have reason to believe that the affinity for lanthanides is generally in similar ranges, since all lanthanides enter solution as trivalent cations with very similar atomic radii. While our data shows, that the peptides are suitable for use in an acidic bioleaching process – immobilized as in our setup or in solution and later recovered – other affinities (and immobilization densities) must be assumed for leaching at lower pH, basic leaching or leaching with other lanthanoid-complexing components. This is due to the peptides either forming a coordination bond as a complex or just electrostatic interaction at different degrees of protonation of the peptides depending on the pH of the applied leaching.

We conclude that the peptides have a significant binding capacity (table 28) in such an acidic environment and can bind neodymium even when immobilized which makes them suitable for use in a bioleaching application. The quantification given in table 29 may be not completely accurate due to uncertainty regarding the immobilization density and the affinity for other lanthanoids other than neodymium may also vary.

Raw data

Fig 30. | Supernatant neodymium concentration.

Fig 31. | Regression.

Fig 32. | Raw data experiment 2.

Fig 33. | Raw data experiment 2.

Fig 34. | Raw data experiment 3.

Fig 35. | Raw data experiment 3.

Peptide Selectivity Tests

Goal

Examining the peptide selectivity in solutions containing different metal ions (iron, neodymium, mixed solution with iron and neodymium, leaching solution) with peptide construct MC.

Methods

To evaluate the selectivity of the peptide, it is immobilized on a microtiter plate (25µM*100µL per well), following the application of the neodymium, iron, mix and leaching solution with different concentrations (neodymium, iron, mix: 10 µM, 5 µM, 2.5 µM, 1.25 µM, 0.625 µM; leaching: 1:10.000) (pH 2.8). After five minutes of incubation with gentle shaking these solutions were removed from the peptide containing wells and transferred to a new MTP. The absorbances were measured using the adapted Arsenazo III assay at 650 nm. Calibration curves were generated for all examined solutions. The use of the 1:10.000 dilution of the leaching solution was chosen based on the calibration curve generated for this solution.

Protocols

Results

To determine the binding selectivity of MC to iron (Fe), neodymium (Nd), different solutions were prepared. First, a solution containing only Nd as metal was tested (Fig. 36).

Fig 36. | Absorbance data of MTP with neodymium solution, without (blue) and with peptide treatment (orange) following Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against original concentration c0(Nd3+). The neodymium solutions applied to MC had concentrations of 0.625 µM, 1.25 µM, 2.5 µM, 5 µM and 10 µM. 94 µL of each solution was treated with immobilized peptide MC (applied to each well 25 µM*100 µL respectively). The measurements were performed three times to determine the median values, which are summarized in this graph.

A decrease in the concentration of Nd with peptide treatment was observed. This reduction occurred in every neodymium solution with varying molarity. The measured data without and with peptide treatment are summarized in the following figure 37.

Fig 37. | Calculated median absorbance and percentile reduction without and with peptide treatment at different neodymium concentrations.

Next on, the experiment was repeated with a solution containing only iron instead of Nd. The obtained data was graphically summarized (Fig. 38).

Fig 38. | Absorbance data of MTP with iron solution, without and with peptide treatment following Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against original concentration. The iron solution applied to the peptides had an ascending concentration from 0.625 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM. The absorbance at 10 µM concentration was left out due to it being recognized as an outlier. 94 µL of each solution was treated with immobilized peptide MC (applied to each well were 25 µM*100 µL respectively). The measurements were performed in triplicates.

A slightly decrease in concentration was observed with peptide treatment than in the one without. The obtained data without and with peptide treatment is provided in the subsequent figure 39.

Fig 39. | Calculated median absorbance and percentile reduction without and with peptide treatment at different iron concentrations.

The next experiment was performed with solution containing Nd and Fe. The measurements were summarized in a graph (Fig. 40).

Fig 40. | Absorbance data of MTP with mixed solution, containing neodymium and iron ions, without and with peptide treatment following Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against original concentration. The mixed solution applied to the peptides had an ascending concentration from 0.625 µM, 1.25 µM, 2.5 µM, 5 µM. 10 µM, 94 µL of each solution was treated with immobilized peptide MC (applied to each well were 25 µM*100 µL respectively). The measurements were performed in triplicates.

Comparing the measured data of the solution with and without peptide treatment, a significant decrease of the absorbance was observed in all peptide treated solutions. Whereby, the highest gap was observed at c0(Nd3+, Fe3+) = 5 µM. Here the absorbance increased by 50% using the peptide treated solution. The measured data is summarized in table 41.

Fig 41. | Calculated median absorbance and percentile reduction without and with peptide treatment at different mixed solution (neodymium and iron) concentrations.

Fig 42. | Absorbance data of MTP for calibration curve following Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) for the detection of neodymium via the AS III Assay. For the calibration curve neodymium solutions of descending concentrations 100 µM, 10 µM, 1 µM and 0.1 µM were used. To 94 µL of neodymium solution, 100µL of pH 2.8 H2O were added. The absorbance data plotted has been deducted with the absorbance of the respective blank. The measurements for the calibration curve were done in quadruplets.

Fig 43. | Resulting regression for the calibration of leaching solution derived from magnets solved in citric acid at equimolar exposure of immobilized peptides.

Fig 44. | Absorbance data of MTP with leaching solution (derived from magnet leaching) without and with peptide treatment following Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of peptide supernatant is plotted against original concentration. The leaching solution applied to the peptides had a concentration of 0.1 µM (1:10.000 dilution). This concentration was deduced based on the calibration curve for the neodymium and leaching solution. 94 µL of each solution was treated with immobilized peptide MC (applied to each well were 25 µM*100 µL respectively). The measurements were performed in triplicates.

A decrease of the absorbance was observed following peptide treatment. Lower dilutions were not employed due to the interference of the leaching solution with the Arsenazo III assay stemming from its color.

Fig 45. | Calculated median absorbance and percentile reduction without and with peptide treatment at different leaching solution concentrations.

Comment: After exploring various experimental setups, we selected this configuration because it allowed us to perform the AS III assay in a solution without interference from the buffer or Arsenazo chemical during the Peptide-Ion binding process.

We observed that including blanks with immobilized peptides in absorbance data calculations is necessary for constructing an accurate calibration curve. In this experiment, we omitted blank measurements since only relative reduction values were evaluated, rendering blank differences unnecessary. We conducted multiple repetitions of the experiment to investigate how the binding behavior of peptides varies with ascending and descending molarity of solutions.

Discussion: Overall, applying MC as peptide led to a decrease in absorbance as shown in figures 36, 38, 40, and 42. In a solution with varying molarities of pure neodymium, a relative reduction in neodymium concentration of up to 25% was observed (see figure 37).

In contrast to the pure neodymium solution, a pure iron solution was tested, which exhibited a slight reduction in absorbance, although, at a minimal level below 1% (see figure 39). An outlier value was detected, characterized by an unusually high absorbance. This test, rather than aiming for quantitative results, was performed to confirm the functionality of the Arsenazo III assay. Specifically, the Arsenazo III assay was utilized to solely detect changes in neodymium concentrations. This test confirmed that interference with the assay by other ions, such as iron, did not occur.

To assess the ability of neodymium binding by our peptide construct MC in the presence of other ions, solutions with equal amounts of iron and neodymium molarities were employed. A reduction in absorbance was observed, suggesting that neodymium was bound by the peptide even in the presence of iron (see figure 41). Notably, the reduction in absorbance was even more pronounced compared to the pure neodymium solution, reaching up to 50%. In conjunction with the iron solution test, it can be assumed that iron ions did not bind under these conditions and the reduction in absorbance was solely due to neodymium ion binding.

In practical testing, our peptides were subjected to a leaching solution derived from magnets, dissolved in citric acid (pH 2.8). A calibration curve was determined to establish the appropriate molarity for the effectiveness of the Arsenazo III assay (figure 42 and 43). Comparable absorbances were recorded at a 1:10.000 dilution (0.1 µM). Peptide selectivity testing revealed a reduction in absorbance after peptide treatment of the leaching solution, suggesting that ions were effectively bound by the peptide construct. When comparing the reduction values at similar ranges of molarity in the mixed solution (0.625 µM) and the leaching solution (0.1 µM), it can be inferred that similar binding affinity for neodymium was exhibited by the peptide in both cases (figure 41 and 45).

Based on the collected data, it can be inferred that the peptide MC exhibits a specific affinity for neodymium ions, displaying a pronounced selectivity towards this element. Conversely, the occurrence of iron ions binding was not detected within the experimental context. This leads to the conclusion that neodymium ions are selectively bound by the peptide construct MC, not only within the mixed solution but also within practical applications such as the leaching solution derived from magnets.

Raw data

Fig 46. | Absorbance of different ion solutions with and without peptide treatment.

Fig 47. | Microtiterplate (MTP) of selectivity experiment.

Fig 48. | Raw data of calibration curve for neodymium and leaching solution.

Fig 49. | Microtiterplate (MTP) of the calibration curve experiment.

Quantification of Bioleached Industry Sample

Goal

Leaching of industry samples at different pHs to quantify neodymium content of sample and concentrations resulting from different leaching. Our project envisages a process in which different sources of e-waste are bioleached and then the lanthanides are isolated from the solution by peptides. For this purpose, we want to test the bioleaching of e-waste on an actual waste sample from industry.

Methods

5 grams of industry sample obtained from STENA Recycling and 80 mL of hydrochloric acid with a pH of 0, 1, 2 and 7 were added into glass bottles while gentle, manual shaking. The solutions were incubated for 31 days. Samples were taken from the bottles and diluted millipore water to a pH of 2.8 (pH adjusted with hydrochloric acid) from undiluted to 1:100000. Following the AS III assay, the experiment was performed in a microtiter plate (MTP). A calibration curve was generated, and blank samples were prepared for each dilution of the sample solution.

Protocols

Results

The leaching approaches set up in the beginning are shown in the top of figure 50. On the bottom the leaching solutions after 30 days of incubation are shown.

Fig 50. | Leaching solutions of STENA Recycling industry sample. 5 g of sample treated with 80 mL of hydrochloric acid (pH 0, 1, 2 and 7) r at the start of leaching (top) and after 30 days of leaching (bottom).

The absorbance peak at 650 nm is significantly smaller when the yellow/green leaching solute of pH 0 leaching sample is added (Fig. 51).

Fig 51. | Different UV-Vis spectra taken up with a photometer. Each spectrum is taken up from a well in a MTP which holds 100 µL of citrate/phosphate buffer, 6 µL of 1 mM Arsenazo III and 94 µL of a varying sample. On the right of each spectrum the absorbance at 650 nm is noted. In a) this sample is Milipore water with an adjusted pH of 2.8. In b) undiluted bioleachate of the pH 0 bioleaching sample is used as the sample. In c) the 1 to 100 dilution of the pH 0 bioleaching sample is used as the sample.

Uptake of a UV-Vis spectrum with the photometer of AS III assay with the sample being made up of leaching solute of pH 0 revealed interference of a photoactive component in the solution. The absorbance peak at 650 nm in b) is significantly smaller than the peak in depiction a) or c) although the neodymium concentration should be higher than any dilution or water. Rather according to figure 51, the 1:100 dilution of the pH 0 leachate sample solution has a higher neodymium concentration than the undiluted solution, which simply does not make sense. This is because we assume that the absorbance at 650 nm correlates with the neodymium concentration. However, there is interference by which the peak at 650 nm is significantly reduced due to a photoactive component present in the leaching solution (Fig. 51). Thus, for the calculation of the concentration of leaching solutions, only values of such solutions are used, which are sufficiently diluted, so that the interfering effect is mitigated. We found the effect to be sufficiently mitigated at a dilution of 1:100.

Fig 52. | Absorbance data of MTP filled with samples of 1 to 100 leachate dilutions. Concentration of neodymium is plotted against absorbance at 650 nm. Neodymium concentrations of leaching solution samples diluted 1:100 with pH 2.8 milipore water obtained from 5 g of bioleached industry sample treated with hydrochloric acid (pH 0, 1, 2 and 7) incubated for 30 days.

Considering the previously noted peak interference of the photoactive components in the leachate at higher concentrations, the 1:100 dilution is used to quantify the sample. By plotting the standard curve of neodymium concentration against absorbance, the neodymium concentration of the leachate sample can be calculated based on the measured absorbance using the standard curve function. The results were summarized in the following table.

Fig 53. | Calculated neodymium concentration for leaching solutions of pH 0, 1, 2 and 7. Concentration of 1 to 100 dilution is result of calculation of concentration through regression function using measured absorbance for 650 nm. Concentration for undiluted solution is determined on calculated neodymium concentration for 1 to 100 dilution.

The highest concentration of neodymium was calculated for leaching solution of pH 0 with a concentration of c(Nd3+) = 993 µM. For the leachate at pH 1 the neodymium concentration c(Nd3+) = 300 µM for pH 2 c(Nd3+) = 142 µM and for the pH 7 c(Nd3+) = 71.9 µM were calculated. Taking this into account we conclude that our industry sample holds a significant amount of neodymium. This neodymium is brought into solution in greater amounts at a lower pH. The neodymium is brought into solution at significant enough amounts for it to be retrieved from peptides.

Comment: The e-waste sample obtained by STENA Recycling was very heterogenous, at the bottom of the container there was soil and smaller and denser particles. The samples we took for the leaching were taken from this soil-like material to achieve some equality in what is contained in the sample used and minimize bias of results due to different e-waste samples.

Discussion: It may have also been sufficient to use dilutions only for pH 0 and pH 1 leachates to mitigate peak interference while for others – pH 2 and 7 – the absorbance data of the undiluted or only 1 to 10 diluted sample could have been used to get a more precise calculation of the neodymium concentration. Also, the sample obtained from STENA Recycling was very heterogenous containing different solids of for example wood, different metals, soil, textiles and plastics. Higher neodymium concentrations could have been obtained from prepurification of the sample before bioleaching. This also means that the samples in the bottles were not exactly the same. The tendency in leaching behavior – more solubilization of neodymium at lower pH – can still be observed. Also leaching efficiency for different lanthanides may vary, which means lanthanides would possibly have higher or lower concentrations. Yet the tendency of better leaching at lower pH should persist. We conclude, acidic leaching leads to solubilization of neodymium which could potentially be bound out of solution by peptides. The lower the pH the more lanthanides go into solution. Which pH is the most efficient for a bioleaching process is still determined by many more factors. Especially the vitality conditions of the peptide producing/presenting and acid producing organism must be considered.

Raw data

Fig 54. | Raw data.

Fig 55. | Raw data.

Fig 56. | Raw data.

Proof of Concept for Extracting Lanthanoids from Bioleached E-Waste Solutions using Peptides

Goal

The previously produced peptides Nd02 and MC are immobilized and used to extract neodymium from a previously produced bioleaching solution of e-waste with pH 2.

Methods

The peptides Nd02 and MC are immobilized on an MTP (25 µM * 100 µL per well). The bioleaching solution produced beforehand (5 g of STENA Recycling's sample + 80 mL pH 2 hydrochloric acid, incubated 30 days) is diluted 1:10 with Milipore water with a final pH of 2.8. 94 µL of the solution were added into wells not coated with peptides and wells coated with peptides. The mixture was incubated for 30 min while gentle shaking. The solution was removed from the wells containing the immobilized peptides and transferred to another microtiter plate. The resulting concentration is determined using the previously adapted Arsenazo III assay. The difference in concentration from the solution which was not treated with peptides and the solution treated with peptides is calculated. In parallel, a calibration curve was generated to classify the results. The calibration curve was taken up for the concentrations 20 µM, 10 µM, 5 µM and 2.5 µM.

Protocols

Results

The measurement of the peptide untreated bioleaching solution is determined to have a concentration of c0(Nd3+) = 10.79 µM. Treating this on MTP immobilized peptides (25 µM * 100 µL respectively) results in a decrease in absorbance and thus neodymium concentration (Fig. 57).

Fig 57. | Absorbance data of MTP with transferred calibration curve and untreated as well as peptide treated sample solution following Arsenazo III-assay. Absorbance at 650 nm as optical density (OD) of 1 to 10 diluted untreated pH 2 bioleaching solution and peptide supernatant is plotted against original concentration. The 1 to 10 diluted pH 2 bioleaching solution had an original neodymium concentration of c0(Nd3+) = 10.79 µM, 94 µL of this was treated with immobilized peptides (applied to each well were 25 µM * 100 µL respectively) Nd02 and MC. For the calibration curve neodymium solutions of 20 µM, 10 µM, 5 µM and 2.5 µM were used. The absorbance of the respective blank was subtracted from the measured data. The measurements for the calibration curve were performed two times, while the measurements for the peptide supernatants were performed three times and each median value was determined and used in this graph.

Fig 58. | Neodymium concentration of 1 to 10 diluted pH 2 bioleaching solution untreated and treated with immobilized peptides (figure 57) is given in the following table. c(Nd³⁺) is the calculated neodymium concentration of the measurements ∆c(Nd^(3+) ) is the difference in neodymium concentration from the supernatant treated with peptide to the untreated bioleaching solution. Percentage bound is the percentage of neodymium bound from solution by immobilized peptides.

The decrease in neodymium concentration of the sample solution is calculated to be 7.36 µM for Nd02 and 9.39 µM for MC. This means that 68.15 % and 86.94 % of neodymium respectively were bound from solution by Nd02 and MC.

Discussion: We were able to bind neodymium from a previously produced bioleaching solute of a sample obtained from industry (STENA Recycling) with our previously produced and immobilized peptides, and this at a significant amount (Fig. 57). As for the affinity test, we must still take into account factors of uncertainty, such as immobilization density.

Yet we conclude that the peptides we produced and immobilized can extract a large amount of neodymium from a produced bioleaching solution resulting from a very heterogenous industry sample. This demonstrates the applicability of the (immobilized) lanthanoid binding peptides Nd02 and MC for the extraction of neodymium (as a stand in for lanthanoids in general) from e-waste leaching solutions as they would be produced by STENA Recycling in a bioleaching process.

Raw data

Fig 59. | Raw data.

Fig 60. | Raw data.

Transformation of pet19b-LanM construct in E. coli

Goal

First, the pet19b vector containing the LanM gene was transformed into E. coli DH10𝛽 to create back up colonies for the vector and to further amplify it.

Methods

Chemically competent cells of E. Coli are treated with heat for successful transformation. The vector contains the gene for a β-lactamase, successfully transformed cells are selected by plating on agar containing carbenicillin (or other β-lactam antibiotics), an antibiotic which can be hydrolyzed by β-lactamase.

Protocols

Results

Multiple colonies grew on LB-Agar containing carbenicillin after transformation ensuring that the vector has been transformed successfully.

Amplification of LanM-gene

Goal

To perform effective restriction, the LanM-gene is being amplified with designed primers containing restriction sites for EcoRI and Xhol.

Methods

First, the plasmid was isolated from a transformed E. coli DH10β colony incubated in LB-medium containing carbenicillin overnight using a plasmid-miniprep kit. The LanM gene was amplified via PCR with designed primers introducing restriction sites. A 2.5%-agarose gel electrophoresis was performed to ensure the correct size of the amplicon.

Protocols

Initial PCR LanM

1. Mix ingredients for PCR samples.

2. Start the PCR program accordingly.

Results

Restriction of pYD1 and LanM

Goal

Restriction is used to create the pYD1-LanM expression vector for the surface display of LanM on the cell surface of S. cerivisiae.

Methods

The amplified LanM gene containing the restriction sites for EcoRI and Xhol was first purified using a PCR cleanup kit. The pYD1 vector was isolated from an obtained E. coli overnight culture containing pYD1. The purified amplicon, along with the purified vector obtained through a miniprep, underwent restriction enzyme treatment after ligation

Protocols

Restriction

Used restriction enzymes EcoRI, XhoI, DphI + other HF-enzymes (which cut in backbone)

0,5 μl per restriction enzyme, incubate for 1 hour at 37 °C

DNA Ligation

1. Mix the ingredients.

2. Incubate over night.

Results

See next experiment.

Transformation of pYD1-LanM in E. coli and sequencing

Goal

To ensure the insertion of LanM and to be able to amplify pYD1-LanM in back-up cultures, the restriction mix was transformed into E. coli.

Methods

Transformation of the restriction preparation in competent E. coli cells was achieved by heat shock, the transformants were selected with the help of carbenicillin containing LB-agar plates as the pYD1 vector contains a gene for a β-lactamase. To test the colonies for the existence of LanM, colony PCR using the primers pYD1-fw and –rev was performed following agarose gel electrophoresis. Lastly, the vector of positive colonies was sequenced.

Protocols

Colony PCR

1. Mix the compounds according to the table

2. Start the program accordingly.

Results

Three positive colonies containing pYD1-LanM were found showing a band of 476 bp proving the insertion of the 336 bp LanM-DNA between the 140 bp sequence between pYD1-fw and –rev. The other colonies only contained the pYD1-vector showing a band of 140 bp. Sequencing proved the successful LanM insertion.

Fig 62. | Colony PCR of transformed E.coli cells to test for the existence of pYD1-LanM.

Transformation of pYD1-lanM in S. cerivisiae EBY100

Goal

By transforming pYD1-LanM into S. cerivisiae, a recombinant strain for Lanmodulin surface display is created.

Methods

Purified pYD1-LanM was chemically transformed into cells of an overnight culture of S. cerivisiae EBY100 in YEP-medium. As the vector complements the yeast’s auxotrophy for tryptophane, transformed colonies were selected by plating them on minimal agar plates with leucine. To check whether the transformation worked, cells were also plated on minimal agar with tryptophane and leucine to ensure the cells did not die during the process. Grown colonies on plates without tryptophane were checked using colony PCR following agarose gel electrophoresis. Transformation also was repeated with pYD1 to have a negative control for further experiments. To rule out contaminations, the colons were microscoped.

Protocols

Colony PCR

1. Mix the compounds according to the table

2. Start the programm acoordingly.

Results

Multiple colonies grew after (repeated) transformation as the pYD1-vector complements the trp-auxotrophy. The microscoped cells ensured that these were yeast cells, ruling out contamination. The gel electrophoresis showed three positive colonies containing LanM (476 bp band) and one negative colony containing pYD1 without LanM insertion (140 bp bp band).

SDS-PAGE to detect LanM surface construct

Goal

By conducting an SDS-PAGE the existence of the surface-protein construct was tried to be verified.

Methods

After inducing expression in YNB-CAA containing Galactose for the colony containing pYD1-LanM as well as a negative control containing pYD1 and the EBY100 wildtype, samples were taken at 0, 6, 24 and 30 h. Before loading the samples into the gel, they were centrifuged and treated with DTT to detach surface construct.

Protocols

SDS-Page

Cast two SDS gels. They can be stored up to one week in the fridge.

It is possible to release the Ag2p protein fusion from the cell wall by treating with dithiothreitol (DTT). DTT reduces the disulfide bond holding Aga2p to Aga1p. It is possible that this could be exploited to purify the LanM. But displayed proteins tend to be glycosylated which can interfere with purification.

1. Spin down the cells that were removed and stored at 4°C at each time point. Remove supernatant carefully and resolve the cell pellets in 50 µl 100 µM DTT overnight.

2. Mix 1 mM DTT with 5 x SDS loading buffer 1:40.

3. Set up the SDS-PAGE gels into the chambers. Fill with SDS buffer.

4. Mix 16 µL of each sample with 4 µL DTT/SDS. Incubate for 10 min at 95°C.

5. Load SDS-gels according to scheme.

6. Let gel run: 80 V until the collection gel is passed, turn up to 120 V after and wait until the blue nearly fades out.

7. Stain for one hour in Coomassie Blue or for 5 min while heating in the microwave (stop heating shortly before its boiling, then incubate for 5 min).

8. Destain several times in dH2O until clear bands form.

Results

Despite multiple repetition, the surface construct did not show in any SDS-PAGE. Possible reasons were diverse but often the most probable error was the incorrect storage of DTT resulting in oxidation with air oxygen and therefore infunctionality of the reducing agent. In addition, the Coomassie available in the laboratory was flawed multiple times and other experiments conducted with it didn’t work as well.

Binding assays with recombinant strains

Goal

Binding assays with Arsenazo III were conducted aiming in proving increased binding of neodymium ions of S. cerivisiae cells containing pYD1-LanM in contrast to cells only containing pYD1.

Methods

First, the expression in the positive and negative control was induced. After 48 h hours, the OD was measured and a volume equivalent to 1 ml of an OD=1 was used for different neodymium concentrations. After centrifuging the cells, the supernatant was discarded, and the cells were resuspended in neodymium solutions of different concentrations for 2 hours. Lastly, the cells were centrifuged again, and the neodymium concentration of the supernatant was measured using Arsenazo III twice each to reduce errors. For the Blank absorbance value, the cells were incubated with water only. To create a calibration curve, pure neodymium solutions of the same concentrations were used.

Protocols

Adapted Arsenazo assay for cell binding assays

1. Adjust pH to 4 in a bottle of millipore water with HCl, this is the solution for NdCl3 solubilization.

2. Prepare a 100 µM NdCl3 solution in a 20 mL in a falcon and 0,1 mM Arsenazo solution in a 1,5 mL eppi.

3. Prepare phosphate-citrate buffer in a bottle with 84,15 ml 0,1 M citric acid and 15,85 ml 0,2 M Na2HPO4. Adjust pH to 2,7-2,8.

4. Prepare dilution series in 20 mL falcons. Take 10 ml of the 100 µM solution and add 10 ml of the acidic water to obtain a 50 µM solution. Repeat this process with the 50 µM solution. Continue diluting the solutions by the factor 2 until you reach a concentration of 1.50625 µM.

5. Take samples of your positive and negative colony after induction of expression for 48 h. You need a positive (pYD1-LanM) and negative (pYD1) sample for each NdCl3 concentration.

6. Take a volume equivalent to 1 mL of an OD of 1 or 0,02. Fill up with medium to 1 mL and remeasure OD to validate.

7. Centrifuge cells and discard the medium.

8. Resuspend the cells in neodymium solutions of defined concentrations. Use every concentration each for the positive and negative control.

9. Create one blank for each colony by performing step 8 with acidic water preparation without neodymium.

10. Incubate the cells for 2h. Centrifuge and use the supernatant for your assay.

11. Fill the wells of a microtiter plate with 100 µl phosphate-buffer, 98 µl incubated sample supernatant (or blank supernatant) or pure neodymium solution for your calibration and 2 µl 0,1 mM Arsenazo. Measurement is done in doublets to reduce error.

Fig 65. | Pipetting scheme for binding assays.

Results

For the first assay, the calibration curve showed linearity for up to 25 µM indicating a direct proportionality between neodymium concentration and absorbance at 650 nm. In the wells for the calibration there was no buffer present which however it did not affect the linearity nor the results. After incubation with cells using equivalently 1 ml of OD=1 there was no significant neodymium concentration present in the supernatant of both samples, as the absorbance of all solutions did not differ from the blank. This indicates all neodymium ions were adsorbed by the positive and the negative control. Neodymium ions therefore can be adsorbed naturally via biosorption.6

To be able to differentiate between natural adsorption and possibly increased binding due to Lanmodulin on the cell’s surface, the assay was repeated using 50 times less cells resulting in an OD of 0,02. In contrast to the calibration curve, there was almost no significant difference in the absorbance of the supernatant of the negative control only containing pYD1, indicating that the cells nearly adsorbed no neodymium due to the low cell concentration.

The supernatant of the positive control containing pYD1-LanM however showed significantly lower absorbance for every initial concentration. Therefore, it appears that the Lanmodulin surface construct has allowed the mutant cells to bind neodymium ions.

The binding efficiency was determined by calculating the concentration in the supernatant of the positive control with the help of the calibration curve and subtracting the quotient of concentrations from 1. The percentage of bound neodymium ions varied from 24% up to 60 %, for 25 µM and 50 µM the same efficiency of approximately 50% was calculated. Thus, Lanmodulin seems to have bound the neodymium ions in greater quantity than the natural biosorption and despite the low number of cells being used.

Additionally, the mass of bound neodymium ions in relation was calculated and related to the cell dry weight used for the binding assay. The amount and mass of bound neodymium ions by the positive control was determined by regarding the concentration of the calibration curve because the negative control did not bind neodymium in a significant order allowing simplification, 250 µL were used during incubation. As shown in figure 67, the mass of bound neodymium ions increased with initial neodymium concentration during incubation, indicating that the cells could have even bound more neodymium, however the calibration curve showed no linearity for higher concentrations. It is possible that the binding kinetic follows a Monot-kinetic as the mass of the bound neodymium ions for the lower concentrations are more error-prone due to the lower absorbance values meaning more neodymium could have possibly been bound.

Literature indicates around 0.7 g/L cell dry weight for an OD600 of 1 resulting in a dry weight of 14 µg present during the assay as equivalently 1 mL with an OD600 of 0,02 was used.7 However, this number should be regarded cautiously as it’s error-prone due to the low OD. The highest amount of bound neodymium ions was calculated for the highest initial concentration of 50 µM, where 64 mg neodymium was bound per 1 g of cell dry weight.

Fig 68. | Calculated values for the concentration of the supernatant after incubation with cells containing pYD1-LanM (c(pos)) for different initial concentrations equal to the concentrations of the calibration curve (c(cal)). With these concentrations, the binding efficiincy as well as the amount and mass of bound neodymium ions was calculated. Lastly, the mass of bound ions was set into relation with the cell dry weight presentduring the assay being approximately 14 µg.

Expand further results

Fig 69. | Calculated mass of bound neodymium ions by 14 µg of S. cerevisiae cells containing pYD1-LanM plotted against the initial concentration during incubation with cells containing pYD1-LanM (OD=0,02).

Fig 70. | Absorbance OD=1 Measured absorbance for the calibration (A(cal)) and for the supernatant of cells containing pYD1-LanM (A(pos)) and cells containing pYD1 (A(neg)) after 2 h incubation, an equivalent of 1 ml cells with an OD600 of 1 was used, each absorbance was measured in doublets for different initial concentrations.

Fig 71. | Absorbance OD=0,02 Measured absorbance for the calibration (A(cal)) and for the supernatant of cells containing pYD1-LanM (A(pos)) and cells containing pYD1 (A(neg)) after 2 h incubation, an equivalent of 1 ml cells with an OD600 of 0,02 was used, each absorbance was measured in doublets for different initial concentrations.

Biopanning

Goal

By using a phage library, phages binding specifically to immobilized Nd-ions are aimed to be isolated.

Methods

Biopanning experiments were conducted, which comprises multiple steps, including binding phages Neodymium-agarose and subsequently washing off excess phages. The strongest binders among the phages are then isolated. Prior to this, a negative biopanning step is carried out to eliminate phages that bind to NTA-Agarose, as outlined in the phage display protocol. Furthermore, phage amplification is performed using E. coli ER2738

Protocols

Results

Positively binding phages could be isolated.

Sanger-Sequencing of Isolated Clones

Goal

Identify the genetic sequence of isolated phages.

Methods

Isolated clones were picked and incubated in TBS buffer following a PCR with a high-fidelity polymerase. The PCR product was purified and sent to Sanger sequencing.

Protocols

Colony PCR for phage amplification:

1. Pick isolated clones from agar plate generously with pipette tip and dissolve in 50 µL TBS. Rub pipette tip against tube gently to dissolve everything.

2. Incubate for at least 2 h better ON at 4°C.

3. Centrifuge for 10 min at 4°C and 10,000 x g to pellet cell debris and agarose and transfer supernatant to new PCR tube.

4. Perform PCR under following conditions.

5. Isolate PCR product after gel electrophoresis for further use with Kit.

6. Use purified PCR product for Sanger sequencing (Eurofins).

Results

Results: After multiple repition, 8 bands could be identified with four being relativly weak. The last three amplicons showed a shorter size indicating a loss of the peptide insert sequence which was proven by sequencing. One DNA sequence showing an insertion could be identified via sequencing, defining a 12 amino acid peptide sequences which may bind to Nd ions:

VVGSSGSTIPFP

The other isolated phages have lost the insert sequence or the sequence could not be properly identified. More time and better sequencing methods like NGS would have been needed to identify more sequences.