Part1 Bioleaching

Result1: Design and Optimization of Leaching Solution

Bioleaching is an emerging green technology that utilizes microorganisms and their metabolic products to separate and recover metal elements from waste materials or ores. The principle of this technology mainly includes two categories: one primarily utilizes microbial metabolic products to interact with the leachate, extracting metal elements through proton action, complexation reactions, and redox reactions; the other mainly relies on the electrostatic forces and secondary bonds of mycelium or cell walls to extract metal elements.

Considering that the leaching solution generally requires acids, complexing agents, and reducing agents, we selected citric acid as the main leaching agent. As a tricarboxylic organic acid, it not only possesses acidic properties but also serves as an excellent complexing agent and reducing agent. Given that a single leaching acid may struggle to adapt to the complex metal ion environment of black powder leaching, we referenced catalyst recovery industries and preliminarily selected gluconic acid and hydrogen peroxide or ascorbic acid as supplementary acids and reducing agents in the leaching solution.

Fig. 1 Schematic Diagram of Leaching Solution Design

During the leaching process, temperature, solid-liquid ratio, acid concentration, and reducing agent concentration all affect the leaching effectiveness. To explore the optimal leaching conditions for black powder, we set up a series of gradient leaching experiments as shown in the table below.

Table 1 Leaching Experiment Gradient Conditions

The middle column of the table contains the theoretical optimal values collected from multiple literature sources. To reduce the experimental burden, we modified only one variable in each experiment while keeping the other parameters at their theoretical optimal values, resulting in a total of 15 experimental groups.

We conducted the experiments in a round-bottom flask equipped with a spherical condenser, and at 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h after the start of leaching, we extracted 1ml of leachate for subsequent analysis. Significant changes were observed during the experiment, with the leachate gradually transitioning from turbid to transparent, indicating that the designed leachate was effective for the leaching of black powder.

Fig. 2 Display of Leaching Effects at Different Time Points

In order to qualitatively analyze the leaching effect on the black powder samples, we employed graphite furnace atomic absorption spectrometry (AAS) to detect the concentrations of four metal ions in each sample group.

Fig. 3 AAS Detection Standard Curve Chart

As shown in the Fig.3, it can be observed that within the first half hour of the leaching process, the samples had already reached their maximum leaching rate, and the leaching rates of each element were able to reach approximately 70%-90%, indicating that the leaching effect met expectations.

Fig. 4 Leaching Rate under Theoretical Optimal Conditions from 0.5 to 6 hours

Based on a large amount of experimental data results,we derived the optimal leaching parameters for the black powder: 70°C, 100 rpm, with a solid-liquid ratio of 15 g/L, 1M citric acid, 0.4M gluconic acid, 0.6M hydrogen peroxide, or 0.25M ascorbic acid (the leaching agent concentration can be scaled according to the solid-liquid ratio).

Fig. 5 a) Rotation Speed Adjustment;b) Solid-Liquid Ratio Adjustment; c) Temperature Adjustment;d) Vitamin C Content Adjustment;e) Hydrogen Peroxide Content Adjustment; f) Citric Acid Content Adjustment;g) Gluconic Acid Content Adjustment

Result2: Leaching Solution Production and Constructio

After selecting the leaching agents, we need to choose a suitable microorganism to produce the substances we require. Initially, we selected Aspergillus niger as the producer of citric acid and gluconic acid, as it is a commonly used strain in the industrial production of citric acid. However, considering that there is a certain competitive relationship between these two pathways, to avoid placing an excessive metabolic burden on Aspergillus niger, we decided to use Aspergillus niger as the engineered strain for citric acid production and purchased the strain CICC2315. Furthermore, through literature review, we noted that glucose is oxidized to gluconic acid in a one-step reaction catalyzed by glucose oxidase (GOx), and the byproduct of this reaction is the reducing agent hydrogen peroxide that we require.

Therefore, we decided to utilize yeast surface display technology, employing Pichia pastoris to display GOx, and then co-cultivating the cultivated yeast with a glucose-containing medium to obtain a large amount of gluconic acid.

2.1 Cultivation and Optimization of Aspergillus niger

In the cultivation of Aspergillus niger, based on Professor JIA's experience and preliminary literature research, we chose the classic ME medium as the first-generation citric acid production medium, with the formulation of ME medium being 30 g/L malt extract and 5 g/L peptone. We observed clear images of Aspergillus niger growth under a confocal microscope.

Fig. 6 Actual Morphology of Aspergillus niger under Confocal Microscopy

In order to verify the acid production capability of Aspergillus niger CICC2315, we employed high-performance liquid chromatography (HPLC) to analyze the citric acid concentration in the culture medium of Aspergillus niger after 5 days of cultivation.

Fig. 7 HPLC Detection Chart

In order to verify the acid production capability of Aspergillus niger CICC2315, we employed high-performance liquid chromatography (HPLC) to analyze the citric acid concentration in the culture medium of Aspergillus niger after 5 days of cultivation.

In the analysis, we found that Aspergillus niger could indeed produce citric acid in the medium we formulated, but the yield was only 0.38 mg/mL, whereas the literature indicates that Aspergillus niger can produce citric acid at levels reaching 50 mg/mL. This indicates that the acid production capability of Aspergillus niger in the ME medium we used is significantly weaker than that reported in the literature, and it cannot meet the requirements for citric acid concentration in the leaching process.

1) Carbon source: Aspergillus niger produces citric acid in the TCA cycle. The carbon source in the culture medium should be considered in terms of both concentration and type. High concentrations of carbon sources can induce Aspergillus niger to continuously carry out the tricarboxylic acid cycle, resulting in a large production of citric acid; whereas different types of carbon sources will affect the metabolic pathways of glycolysis. The ideal carbon source should be a monosaccharide that can be directly absorbed and utilized—glucose. The carbon source in the first-generation ME medium is malt extract, which is a complex mixture containing carbohydrates such as glucose and maltose. This not only has a low concentration but also makes substances like maltose less favorable for direct absorption and utilization.

2) pH:The pH of the culture environment will influence the citric acid production of Aspergillus niger from two aspects: in a low pH culture environment, citric acid will exist in its molecular rather than ionic form, and the low pH environment will better activate proton pumps, promoting the export of citric acid by Aspergillus niger; at the same time, the low pH environment will affect the activity of relevant metabolic enzymes, promoting citric acid production while inhibiting the production of other byproducts. The specific pH values are shown in the Fig. 8.

The first-generation culture medium did not consider the adjustment of pH, and we decided to adjust the initial pH in the subsequent culture medium to promote citric acid production by Aspergillus niger.

Fig. 8 Relationship between Culture pH and Citric Acid Production by Aspergillus niger

3) Nitrogen source: The utilization of carbon sources by Aspergillus nigerr consists of two competing pathways: metabolism and growth reproduction. Reducing the supply of nitrogen sources in the culture medium will limit protein formation, thereby inhibiting the growth reproduction of Aspergillus niger, allowing it to convert more of the absorbed carbon sources into citric acid.

In the first-generation culture medium, the nitrogen source was provided by peptone, with a calculated C/N ratio of approximately 7.99. This culture medium is more suitable for the growth of Aspergillus niger rather than citric acid production. We decided to use 2 g/L of sodium nitrate as the nitrogen source in the subsequent culture medium, and after calculation, the adjusted C/N ratio can reach 151.5, which greatly promotes citric acid production.

In order to improve the citric acid yield of CICC2315, we redesigned the culture medium. After considering the effects of carbon source, nitrogen source, and pH on the type and yield of acids produced by Aspergillus niger, we chose to use the CD medium as a template, changing the carbon source to glucose and the nitrogen source to sodium nitrate, and adjusting the glucose concentration, medium pH, and carbon-to-nitrogen ratio to 125 g/L, 5, and 151.5, respectively. The final culture medium formulation is 125 g/L glucose ,2 g/L NaNO₃ ,1 g/L K₂HPO₄ ,0.5 g/L MgSO₄·7H₂O ,0.5 g/L KCL ,0.01 g/L FeSO₄.

In order to compare the differences in growth and acid production capabilities of two strains of Aspergillus niger in ME and CD media, we inoculated the same quantity of Aspergillus niger spores in both media and cultured them under the same conditions (30°C, 220 rpm).

By comparing Fig. 9a and Fig. 9b, it can be observed that in the ME medium, the mycelial pellets of Aspergillus niger are smaller and more numerous; whereas in the CD medium, the diameter of the mycelial pellets of Aspergillus niger is slightly larger than that in the ME medium, while their quantity is lower, indicating that our regulation of the nitrogen source has been very successful.

Fig. 9 Comparison of Culture Medium Effects

Fig. 10 shows the changes in pH of the ME and CD media at different cultivation times. Overall, the pH of both media exhibits a downward trend; however, it can be observed that the pH of the CD medium decreases at a faster rate, while the pH of the ME medium begins to rise after 66 hours. In conjunction with the literature, we analyze that in the CD medium, glucose, as a simpler monosaccharide, is more easily absorbed and utilized by Aspergillus niger, thereby increasing the acid production rate of Aspergillus niger and accelerating the decrease in pH of the CD medium. In the ME medium, the larger mycelial pellets of Aspergillus niger accelerate the consumption of carbon sources in the medium, and after 66 hours, they may begin to utilize citric acid in the medium as a carbon source, leading to an increase in pH.

Fig. 10 pH Variation of Culture. a) represents ME medium; b) represents CD medium.

In order to determine the actual acid production of Aspergillus niger in the two media, we measured the citric acid content in the media using HPLC. As shown in Fig. 11, it can be observed that through the repeated iterations of our medium formulation, the citric acid content increased from 0.32 g/L to 39.87 g/L, demonstrating a several hundred-fold increase in yield, which indicates that our medium regulation has been highly effective.

Fig. 11 Variation of Citric Acid Yield in Different Culture Media.

Thus, we have basically determined that the Aspergillus niger cultures used subsequently will all be prepared using the CD medium: 125 g/L glucose ,2 g/L NaNO₃ ,1 g/L K₂HPO₄ ,0.5 g/L MgSO₄·7H₂O , 0.5 g/L 0.5 g/L KCL ,0.01 g/L FeSO₄ .The culture was incubated for 5 days under conditions of an initial pH of 5, 220 rpm, and 30°C, resulting in a culture liquid with a pH of around 2.5.

2.2 Yeast Surface Display of GOx

We utilized the surface display plasmid constructed in the bioadsorption section, employing the Gibson assembly method to insert the GOx gene into it, and subsequently introduced it into Pichia pastoris, thereby obtaining a recombinant strain capable of surface displaying glucose oxidase.

Fig. 12 a) GOx Plasmid Design Diagram;b) GOx Protein Gel Image.

For the constructed recombinant yeast, we incubated it in a glucose-containing medium. Compared to the wild-type control group, the yeast displaying GOx on its surface was able to lower the pH of the medium more significantly. This preliminary indicates that a reaction converting glucose to gluconic acid occurred in the medium containing the recombinant strain. However, when we were preparing to accurately detect the gluconic acid content using HPLC, we unfortunately discovered contamination in the recombinant strain, and due to time constraints, we were unable to proceed with the next verification step.

Result3: Selection of Leaching Method

In bioleaching, there are primarily three existing leaching methods, namely "One-step bioleaching", "Two-step bioleaching" and "Spent medium bioleaching". The first two methods involve directly mixing the unseparated microbial culture with the material to be leached, with the distinction that One-step bioleaching adds the leachate during the microbial growth phase, while Two-step bioleaching allows the microbial strain to proliferate to the stationary phase before adding the leachate. The latter method separates the microorganisms from the culture medium and uses pure culture medium to leach metal ions.

Fig. 13 Schematic Diagram of Three Different Leaching Methods

Considering the toxic effects of heavy metal ions contained in black powder on cells, the leaching rate of One-step bioleaching is usually lower, although it shortens the cultivation time. We are considering selecting a more suitable method for the project through experiments between Two-step bioleaching and Spent medium bioleaching.

We believe that each of these two methods has its own advantages and issues as follows:

1. Two-step bioleaching, due to its mixing of microorganisms with black powder, provides leaching effects that include the adsorption of metal ions by the microbial biofilm, in contrast to Spent medium bioleaching, which only uses pure culture medium. Additionally, this method allows the microbial strain to continuously utilize the culture medium to produce acid. However, this approach requires consideration of the survival conditions of the microorganisms, such as the tolerance of the microbial strain to metal ions, as well as the inability to heat the leaching process, and the need to consider the interactions between the two types of microorganisms when mixing the microbial cultures.

2. Spent medium bioleaching, as it only uses pure culture medium to leach black powder, does not require consideration of the survival conditions of the microorganisms, and it can be heated to improve leaching efficiency while also allowing for flexible mixing ratios of microbial cultures. However, this leaching method will inevitably result in a loss of leaching rate.

Experiment 1: Searching for Optimal Leaching Conditions.

After understanding this basic information, we had an in-depth discussion with Professor Zhang Guimin from Beijing University of Chemical Technology. Considering the driving effect of biofilm adsorption on metal leaching, Professor Zhang suggested that the leaching effect of the Two-step bioleaching method might surpass that of the spent medium bioleaching method. In order to compare and validate the differences in leaching rates between Two-step bioleaching and Spent medium bioleaching, we constructed a set of control experiments to select the leaching method with the optimal combination of leaching rate and leaching time.

Fig. 14 a) Two-step bioleaching Gradient Time Physical Diagram; b) Spent Medium Bioleaching Gradient Time Physical Diagram;c) Leaching Rate Diagram.

Fig. 14a and 14b show the changes in pH of the microbial liquid during the two leaching methods over a 2-hour period, as well as photographs taken during the leaching process. From Fig a and b, it can be seen that the Spent medium bioleaching method, due to its introduction of heating conditions, nearly completed the leaching in just about 30 minutes, compared to the two-step bioleaching method which has a leaching time lasting several days. The final leaching rates of the two methods are quite similar, with the leaching rate of the Spent medium bioleaching method even slightly higher than that of the two-step bioleaching method, which contradicts our analytical hypothesis.

We observed that during the leaching process of the Two-step bioleaching method, the leaching solution experienced a transition from turbidity to clarity. We believe this is due to the adsorption of metal ions by the mycelium, resulting in a significant amount of leached metal ions and black powder being adsorbed onto the surface of the microbial membrane. At the same time, the changes in pH during the cultivation process led to the black Aspergillus producing a byproduct acid—oxalic acid—beyond citric acid, which may cause the precipitation of Ni or Co ions. Considering all factors, the Two-step bioleaching method may release more metal ions from the black powder, but it does not allow them to exist in the solution in a free ionic form. This means that if the metal ion content in the solution is measured using AAS to characterize the leaching rate, the leaching rate of the Two-step bioleaching method will appear lower.

Experiment 2: Verifying the Adsorption of Metal Ions by Aspergillus niger.

To verify whether the black Aspergillus has an adsorption effect on metal ions, we conducted scanning electron microscopy—energy dispersive spectroscopy (SEM-EDS) analysis on samples of black Aspergillus before and after leaching. From the images, it is clear that a large number of metal ions are adsorbed on the surface of the black Aspergillus, with local elemental concentrations even exceeding 50%, which represents an unacceptable loss of such a large amount of element adsorption. (Due to the limitations of the instrument, the Li content could not be detected, and the detection limit for Co is relatively high.)

Fig. 15 a) Pure Aspergillus niger SEM; b) Pure Aspergillus niger EDS;c) "Two-step method" Aspergillus niger SEM;d,e) "Two-step method" Aspergillus niger EDS

Therefore, considering the aspects of leaching time and leaching rate, we ultimately decided to select the Medium bioleaching method as the final leaching approach.

In summary, in the bioleaching section, we successfully constructed a dual-acid combined leaching system based on the cultivation of black Aspergillus and yeast surface display of GOx, and determined the Medium bioleaching method as the final leaching approach.

Result4: Future Directions and Plans for Experiments

4.1 GOx Surface Display in Yeast

In the future, we will continue to develop the GOx surface display technology. After successfully introducing the gene into Pichia pastoris, we will explore the effects of yeast culture medium and cultivation methods on the yields of gluconic acid and hydrogen peroxide. Once we obtain reliable GOx surface display yeast and cultivation methods, we will mix the corresponding black Aspergillus culture liquid with the yeast co-culture liquid in a certain ratio and conduct leaching experiments to verify whether our hypothesis regarding the combined leaching process of the two microbial liquids can achieve a high leaching rate of black powder.

4.2 Exploring New Application Scenarios

We will review more literature and attempt to expand the application scenarios to mining, soil heavy metal pollution remediation, and other areas by adjusting the types and proportions of organic acids produced by microorganisms, thereby enhancing the value of the project. Additionally, we will explore the synergistic effects of different organic acids in the bioleaching process, aiming to optimize the leaching efficiency and recovery rates of valuable metals. This comprehensive approach will not only contribute to the development of sustainable biotechnological solutions but also address environmental challenges associated with heavy metal contamination.

Part2 biosorption

Result1: Detection of the Binding Ability of MBPs to Metal Ions by ITC

Metal-binding peptides (MBPs) can bind metal ions through the interaction between specific amino acid residues and metal ions. The side chain functional groups of certain amino acids in MBP can form coordination bonds with metal ions. Like "claws", they grab metal ions to form stable metal-peptide complexes and reduce the free concentration of metal ions.

Here, peptides were characterized by isothermal titration calorimetry (ITC) with respect to their binding capacity for the respective target ion and binding experiments were performed on other ions.We used a Nano-ITC for our experiments using a standard protocol. The measuring cell contained 350 µL buffer with 0.2 mM peptide. The syringe was filled with 2 mM LiCl, NiCl₂, MnCl₂ or CoCl₂ solution in the same buffer. The injections were performed in 20 single steps of 2.5 µL each. The stirring speed was 350 rpm. Using Launch NanoAnalyze, perform data evaluation.The isothermal titration experiments revealed the cyclic nickel-binding peptide with the motif CNAKHHPRCGGG showed complexation with Ni²⁺ ions with an affinity of KD= 5.53x10^-5 M. However, no interaction was detected between the nickel-binding peptide and Li⁺, Mn²⁺, and Co²⁺. The nickel-specific peptide binds only selectively to nickel.

Fig. 16 Isothermal titration calorimetry results of NBP4 with different mental ions.

Result2:Immobilization of Pichia pastoris

We placed the sterilized polyester nonwoven fabric and activated Pichia pastoris in an incubator for co-culture. Then, we counted the yeast attached to the fabric by shaking and the dilution plating method to evaluate its fixation ability. According to the experimental results, at 4 hours after inoculation, compared with other time points, the number of viable bacteria on the membrane was relatively small, and its fixation density was 8.39×10³ CFU/cm². This indicates that only a small amount of bacteria adhered to the fabric. The main reason may be that the yeast inoculated in the liquid medium at this time is in the lag phase and has not yet undergone rapid growth and reproduction. At 12 hours, the number of viable bacteria increased significantly to reach 1.50×10⁶ CFU/cm². On the one hand, it is because the yeast is in the logarithmic growth phase and grows and reproduces in large numbers. On the other hand, it is because a large number of proliferated yeast adhere to the fabric.At 20 hours, the number of viable bacteria reached the highest value of 1.42×10⁷ CFU/cm². At 24 hours, the number of viable bacteria was 5.71×10⁶ CFU/cm², which was slightly lower than that at 20 hours. It is speculated that a small amount of yeast may have fallen off. Judging from the comprehensive experimental results, polyester nonwoven fabric shows good yeast fixation ability in a short time. This provides a basis for subsequent engineering, enabling us to consider adsorption by attaching yeast with surface display to polyester nonwoven fabric.

Fig. 17 Polyester nonwoven fabric and Pichia pastoris are co-cultured in a 24-well plate.

Fig. 18 Viable cells of P.pastoris on polyester nonwoven. a) Unit area; b) Unit weight.

Result3: Surface Display of Metal-Binding Peptides

3.1 Construction of Plasmid

In order to express metal-binding peptides on the yeast surface, it is necessary to construct an efficient surface display plasmid first. Using Pichia pastoris as the expression vector, we designed the plasmid sequence.

After investigation, we found that the anchoring motif pir1 performs well in the surface display proteins of Pichia pastoris. Therefore, we connected pir1 to the target protein through a linker and used the α-factor secretion signal to initiate secretion. In addition, in order to facilitate the subsequent detection of the content of surface display proteins, we added a Myc tag at the end of Pir1, which can recognize the corresponding antibody. We selected the GAP promoter and the AOX terminator to enable the effective expression of these sequences. The Bleomycin resistance gene is applied to the plasmid by us to facilitate the screening of strains into which the plasmid is introduced.

Fig. 19 Agarose Gel Electrophoresis diagram of surface display plasmid containing MBPs gene.

3.2 Construction of Engineered Yeast

After the plasmid construction is completed, we introduce it into E.coli for amplification. After the amplification is completed, we extract the plasmid and transform it into Pichia pastoris by electrical stimulation. Finally, through colony PCR, we determined that the plasmid was successfully introduced into yeast.

Fig. 20 Agarose Gel Electrophoresis diagram of engineered yeast obtained by electrotransformation after colony PCR.

3.3 Verification of Surface Display Effect

Since the constructed surface plasmid contains a Myc tag, an immunofluorescence detection can be performed on it by ELISA method. If it is successfully displayed on the cell surface, obvious red fluorescence can be observed on its surface. Conversely, if the re is no obvious red fluorescence on the surface, it means that the display is unsuccessful. According to the indirect yeast immunofluorescence method for sample preparation, we performed fluorescence microscope detection. As shown in Fig. 21 below, the control group GS115 has no fluorescence, while the CBP1-pir cells have strong fluorescence, and there is a halo around the cell edge. Since Pichia pastoris GS115 is a sphere, the surface display protein is evenly distributed on the cell surface and is relatively concentrated at the cell edge. Therefore, the brightness at the cell edge is the highest, indicating successful displa.

Fig. 21 Bright field and immunofluorescence detection image of surface displayed CBP1 strains with emission wavelength of 488 nm. a) Immunofluorescence detection diagram of the control group; b) Immunofluorescence detection diagram of the experimental group.

We have learned and practiced how to construct an effective plasmid that can be used for surface display, and successfully obtained multiple strains that can display different metal-binding peptides.

Result4: Detection of Adsorption Effect of Engineered Yeast

After obtaining the engineered yeast, we designed some experimental schemes to quantitatively detect their adsorption effect on target metal ions, and further observed the surface display of metal-binding peptides by scanning electron microscopy.

To detect the adsorption effect, we mixed the culture medium of engineered yeast and the prepared single metal solution in a certain proportion. After 2 hours, we took it out and centrifuged it, and stored the supernatant and precipitate respectively. Drawing on the experience of two other experimental groups, we chose to use a graphite furnace to detect the concentration of metal ions in the supernatant and scanning electron microscopy (SEM) to observe the surface display effect of yeast in the precipitate.

Fig. 22 Flow chart for detecting the adsorption effect of engineered yeast.

4.1 Quantitative detection of adsorption effect by graphite furnace

We carried out the operation of collecting the adsorption system at 2 hours. After centrifuging the collected mixture, we took the supernatant and filtered out the remaining bacteria with a filter membrane. Since the metal ion concentration in the supernatant is high after adsorption is completed, deionized water and dilute nitric acid need to be usedfor dilution. After preparing a sample with an appropriate concentration, it is detected by a graphite furnace. By calculating the ratio of the reduced metal concentration in the supernatant to the originally added metal concentration, we obtained the adsorption rate of the engineered strain to the target metal ion. The engineered strains with cobaltion-binding peptides displayed on the surface, including CBP1-pir and CBP2-pir, have an adsorption rate of Co²⁺ in the system of more than 70%, showing good adsorption effects. Among them, the strain displaying CBP2 has a slightly higher adsorption rate than the strain displaying CBP1, and it is also the strain with the highest adsorption rate among the detected strains, which is 76.46%. The engineered strains used for adsorbing Ni²⁺ also show a certain adsorption effect, and the adsorption rate is about 15% - 30%. Through comparison, it can be found that the adsorption efficiency of Ni²⁺ is NBP4-pir > NBP2-pir > NBP1-pir. Relatively speaking, the efficiency of the engineered strain adsorbing Mn²⁺ is lower, but the adsorption efficiency of the best-performing strain also reaches 11%. Among them, the adsorption efficiency is mntR-pir > TssS-pir > BH2807-pir.

It is also worth noting that in the adsorption experiment of metal-binding peptides on non-target ions, we found that CBP2-pir showed good adsorption specificity for Ni²⁺ and Co²⁺. Also within the adsorption time of 2 hours, its adsorption rate for Co²⁺ is 76.46%, while the adsorption rate for Ni²⁺ is only 3.63%. Considering that Ni²⁺ and Co²⁺ are difficult to be separated by other adsorption methods, CBP2 may be a great way to solve this problem.

Fig. 23 The adsorption rate of engineered strains adsorbing target metal ions within 2 hours. a) The adsorption rate of CBP1-pir and CBP2-pir for Co²⁺; b) The adsorption rate of NBP1-pir, NBP2-pir and NBP4-pir for Ni²⁺; c) The adsorption rate of mntR-pir, TssS-pir and BH2807-pir for Mn²⁺.

4.2 Observation of yeast surface by scanning electron microscope

The yeast cell precipitates after adsorption completion are used for electron microscope sample preparation. We use a scanning electron microscope to observe yeast cells with metal-binding peptides displayed on the surface. In Figure 9, (a) and (b) are the control group GS115; (c) and (d) are the strains with surface-displayed BH2807; (e) and (f) are the strains with surface-displayed mntR; (g) and (h) are the strains with surface-displayed TssS. By comparing the photos, it can be found that the roughness of the cell surface from large to small is: mntR-pir > TssS-pir > BH2807-pir > GS115. According to the performance of GS115 as the control group, we initially infer that the roughness of the cell surface is related to the displayed protein. According to the detection results of the graphite furnace, the adsorption efficiency is mntR-pir > TssS-pir > BH2807-pir. Combining the order of the roughness of the cell surface, we further speculate that the more displayed proteins, the rougher the cell surface may be.

Fig. 24 Scanning electron microscope images of yeast strains. a) GS115 (×25000 times); b) GS115 (×40000 times); c) BH2807-pir(×25000 times); d) BH2807-pir (×40000 times); e) mntR-pir (×25000 times); f) mntR-pir(×40000 times); g) TssS-pir(×25000 times); h) TssS-pir(×40000 times).

We used a graphite furnace and scanning electron microscopy (SEM) to qualitatively measure the ability of surface-displayed strains to adsorb target metal ions, verified their effectiveness, and initially selected strains with better adsorption effects. Although the adsorption effect of individual strains is acceptable, on the whole, the adsorption efficiency still needs to be improved. We will continue to work hard with this as our goal.

Result5:Self-assembling of NBP4 (Nickel-Binding Peptide 4)

The binding sites of monomeric metal ion-binding peptides are limited, while self-assembled peptides can form different binding sites. Therefore, we attempt to improve the binding efficiency of metal ion-binding peptides through a self-assembly strategy. Through model prediction, NBP4 is an excellent peptide that can self-assemble. Therefore, we have carried out a series of verification experiments for NBP4.

5.1 In Vitro Self-Assembly of Chemically Synthesized Polypeptides

Considering that the self-assembly of metal-binding peptides may be different under different conditions, in order to make the self-assembled N4 have a regular structure and good performance, based on our analysis of the structure of the N4 binding peptide and research on self-assembly-related literature, we control the concentration of N4 at 2 mg/ml. At the same time, we designed a series of experiments to explore the optimal conditions for N4 self-assembly from aspects such as time, temperature and catalyst.

At first, since the 32-mer of N4 in the simulation result has ductility, we speculated that the self-assembly of N4 may show a fibrous state. After literature research, we chose to self-assemble at room temperature (25℃) for 24 hours. However, it is not effective and shows a "pseudo-fiber" state in AFM. When we observe it again after a few hours, the "pseudo-fiber" state turns into an irregular spherical object. We guess that the self-assembled polypeptide we want may be this spherical aggregate rather than fibrous. After theoretical analysis, we hypothesized that there are two possible pathways for N4 self-assembly: low-temperature-induced hydrogen bond formation, in which hydrogen bonds are the main driving force for self-assembly; and high-temperature-promoted hydrophobic interaction, in which hydrophobic interaction is the main driving force. For this reason, we self-assembled at 4℃ and 37℃ and observed the microstructure at 24 h and 48 h to determine the optimal self-assembly temperature and time.

For each group of samples, we observed the morphology of self-assembly at 24 h and 48 h respectively. Obviously, we can see that at 48 h, the self-assembly is spherical and microspheres appear. At 48 h, spherical self-assembled polypeptides appeared in all three groups. Among them, the 37℃ group showed a higher polypeptide density and a considerable size. However, the assembly formed by N4 is not regular and does not present a spherical shape but a "tadpole-shaped" with a small tail.

In addition, considering the specific binding affinity of the imidazole group to the histidine R group for metal ions, we considered using a catalytic amount of metal ions to induce N4 to self-assemble into regular spheres. We selected four common metal ions Ni²⁺, Mn²⁺, Co²⁺, and Ca²⁺, all of which are divalent metal ions with coordination ability.

We added four types of metal ions of 0.1 mM Ni²⁺, Mn²⁺, Co²⁺, and Ca²⁺ respectively, and observed their morphologies after self-assembly at 37℃ for 24 hours and 48 hours. At the same time, we measured the particle size and distribution of the self-assembled polypeptide s pheres at 48 hours. Through the AFM characterization experiment, we can clearly observe from the image that under the condition of 37℃, under the catalytic amount of Ni, N4 forms a large number of uniform spherical assemblies after 48 hours of assembly. Among various met al ions tested by induction, Ni shows the most effective induction effect, independently verifying the specific binding ability of N4 and Ni.

Fig. 25 a) AFM topography; b) Characterization of the height of nanoparticle.

From the experimental results, we successfully made N4 complete self-assembly, and found the best time, temperature and catalyst, and obtained N4 that can be assembled well.

5.2 Co-Incubation of Self-Assembled Polypeptides with Cells

After obtaining the self-assembled N4, we need to design experiments to confirm that it can be well adsorbed on the specific binding site of yeast and confirm that it can increase the adsorption amount of nickel ions. On this basis, we also explored the influence of adsorption time on the adsorption amount.

We added the self-assembled N4 to the yeast liquid cultured at 30℃ and 200 rpm for 2 days, and co-incubated it at 30℃ and 200 rpm for 24 hours so that the self-assembled N4 can be well bound to the yeast. Then, it was cultured with 40 mM nickel ions by shaking to judge the adsorption effect. We found that it is difficult to clearly see the morphology of the yeast surface by SEM. So we used EDS to analyze the content of nickel on the surface of yeast cells and indirectly confirm whether the polypeptide is adsorbed on the yeast by comparing with the blank control. At the same time, we also designed a cell-free experiment to observe the reaction of the polypeptide solution without yeast adsorption with nickel ions and scan its nickel content in EDS.

Due to the particle size issue, it is difficult to directly observe the morphology of the small spheres under SEM. However, through elemental analysis of the electron energy spectrum, it can be found that in the co-incubation experiment of assembled cells, we can detect a very obvious difference in Ni element content on the yeast surface and the substrate surface. We have reasons to believe that the yeast surface is indeed loaded with polypeptide assemblies enriched in Ni elements. In the monomer and cell co-incubation group, we can clearly find that almost no high concentration of Ni is detected on the cell surface. Since a single peptide contains two histidines, assuming that every four histidines can complex one Ni, then each mole of polypeptide can bind half a mole of Ni. Based on such a rough theoretical calculation, we estimate that the theoretical value of Ni element content on the polypeptide self-assembly is about 2.5%, and the actually measured Ni content is about 2.3%. This is very in line with our calculation results. At the same time, we also tested the nickel element analysis results of the co-incubation of polypeptide solution and nickel ions, and found that it is slightly higher than that of assembled cells and almost reaches the theoretical maximum value of nickel adsorption.

Fig. 26 a) Scheme of the combination of the assemblies; b) EDS analysis of electronic images; c) EDS spot scanning carbon element content distribution image; d) EDS point scanning images of oxygen content distribution; e) EDS spot scanning image of nickel content distribution; f) EDS surface scanning nickel content layered image; g) Total spectrum of element distribution map of self-assembled N4 yeast incubated with Ni²⁺ for 3h in blank control group; h) Total spectrum of element distribution map of self-assembled N4 yeast incubated with Ni²⁺ for 3h; i) Analysis of 3h EDS of N4-added yeast incubated with Ni²⁺ (experimental group); j) 3h EDS analysis of yeast that has been added with N4 incubated with Ni²⁺ (taking no liquid at the edge as control group); k) Analysis of 6h EDS of N4-added yeast incubated with Ni²⁺ (experimental group); l) 3h EDS analysis of yeast that has been added with N4 incubated with Ni²⁺ (no liquid at the edge was taken as control group).

In this part of the experiment, we successfully verified that the assembly can bind to cells, and the assembly bound to cells can adsorb a large amount of nickel ions. At the same time, we also verified that compared with monomers, the assembly does have an indispensable advantage. We think this comes from the fact that the assembly provides additional binding sites. Not only are there more adsorption sites for metal ions, but also more peptide segments can bind to cells.

PART 3 Biomineralization

Result 1: Plasmid Construction and Transformation

Due to the urease gene of S. pasteurii, which encodes a large protein complex composed of seven subunits, constructing this gene on a single plasmid presents significant challenges. Therefore, we followed the methodology of the iGEM23_BUCT team and divided the urease gene into two segments, which were subsequently cloned into separate plasmids.The genes UreA, UreB, and UreC were inserted into the pET28a vector (T7 promoter), while UreE, UreF, UreG, and UreD were inserted into the pET21a vector (T7 promoter). The constructed plasmids are illustrated in the figure below. The ribosome binding site (RBS) sequences preceding each subunit gene reference the article "Rational Control of Calcium Carbonate Precipitation by Engineered E.coli," where the natural RBS translation initiation rates of the efficient urease from S. pasteurii were assessed. They found that the natural RBS translation initiation rates for the genes ureA, ureB, ureC, ureE, ureF, ureG, and ureD were 3792 au, 15,425 au, 4358 au, 12,201 au, 2.1 au, 4006 au, and 49 au, respectively. Subsequently, they reconstructed the urease biosynthetic pathway using synthetic RBS, resulting in translation initiation rates for the genes ureA, ureB, ureC, ureE, ureF, ureG, and ureD of 5000 au, 10,000 au, 5000 au, 10,000 au, 1000 au, 1000 au, 1000 au, 5000 au, and 1000 au, respectively, in order to create new strains.

Fig. 27 Plasmid Maps of pET28a (Containing Genes UreA, UreB, UreC) (Left); Plasmid Map of pET21a (Containing Genes UreE, UreF, UreG, UreD) (Right)

Initially, we designed UreE, UreF, UreG, and UreD for cloning into the pET21b vector (ProD promoter); therefore, we recombined the target genes UreE, UreF, UreG, and UreD into the pET21a plasmid, which contains the T7 promoter. We designed the primers vector-for, vector-rev, fragment-for, and fragment-rev. Following the receipt of the primers, we performed PCR and reverse PCR to linearize the target genes and the vector, respectively. The BB fragment represents our final vector, pET21a, with a band length of 5310 bp after reverse PCR, while the INSERT corresponds to the target gene UreE-UreF-UreG-UreD, with a band length of 2698 bp after PCR. Running a nucleic acid gel confirmed that the observed band lengths matched the expected values.

Fig. 28 PCR Analysis of pET21a Gibson Assembly Reconstructed Plasmid on Nucleic Acid Gel (where BB: 5310 bp; INSERT: 2698 bp)

After gel extraction and purification of genomic DNA, we performed Gibson assembly to reconstruct the plasmid. The assembled DNA was then transformed into host cells for amplification and colony PCR screening. Ultimately, we retained the colonies with favorable sequencing results for plasmid extraction and dual plasmid transformation.

Fig. 29 pET21a transformation

Result 2: Urease Expression Validation

2.1 SDS-PAGE

After extensive literature research and consultations with the supervising professor, we determined that a low-temperature induction at 20 °C for 16 hours using 0.5 mM IPTG and 5 µM NiCl2 is more favorable for urease expression. Subsequently, we conducted SDS-PAGE to observe the expression of various urease subunits. The control group consisted of pJUMP, which does not express urease, while the experimental group included pET21a, pET28a, and dual plasmids. The samples were labeled as "pre" for uninduced and "post" for induced. We obtained whole-cell lysates via sonication (SNP) and supernatants through centrifugation (SN). The red boxes in the figure below indicate the bands corresponding to the sizes of the urease subunits. The darker intensity of these bands suggests a good translation and expression of the urease subunits.

Fig. 30 SDS-PAGE of pET21a and pET28a Dual Plasmids(UreC: 61.4 kD; UreD: 29.3 kD; UreG: 23.1 kD; UreF: 23.0 kD; UreE: 17.4 kD; UreB: 14.0 kD; UreA: 11.1 kD)

2.2 Quantitative Detection of Urease Activity

The Urease (UE) Activity Assay Kit was purchased from Boxbio for the quantitative detection of urease activity. The principle of the reaction is as follows: urease hydrolyzes urea to produce NH3-N, which, in a strongly alkaline medium, reacts with hypochlorite and phenol to generate a water-soluble blue dye, indophenol blue. The product exhibits a characteristic absorption peak at 630 nm, and the change in absorbance is used to characterize the urease activity. The dual plasmid experimental group is designated as A measurement, while the BL21 control group is designated as A control.

Fig. 31 Quantitative Detection of Urease Activity. a) Schematic diagram of the reaction principle for quantitative detection of urease activity; b) Standard curve for quantitative detection of urease activity;c) Bar chart of absorbance valuesΔA for quantitative detection of urease activity

Using the standard curve for urease activity detection from panel b), a series of calculations were performed to determine that the urease activity is 0.153 U/g DCW (U: the amount of enzyme that catalyzes the formation of 1.0 µmole of ammonia per minute at pH 7.0). Although this value is somewhat lower than the 4-6 U/g DCW reported in the literature, it still indicates a significant urease activity compared to the control group. This preliminarily demonstrates that our recombinant urease exhibits activity, although there is room for improvement. (Note: The unit definition for urease activity in this kit has been converted to align with the definition used in the paper "Rational Control of Calcium Carbonate Precipitation by Engineered E.coli," facilitating comparison with reported recombinant urease activities).

Result 3: Validation of Recombinant Urease Biomineralization

After confirming that urease is indeed active, we proceeded to investigate the biomineralization of four metal ions: Li, Mn, Co, and Ni. Various methods were employed to validate our mineralization results.

3.1 Optical Microscopy Observation

Fig. 32 Results of Biomineralization Observed by Optical Microscopy. a) BL21(DE3) control group;b) Dual-plasmid experimental group for Li mineralization; c) Dual-plasmid experimental group for Mn mineralization;d) Dual-plasmid experimental group for Ni mineralization;e) Dual-plasmid experimental group for Co mineralization

It is evident that, compared to the BL21(DE3) control group, the dual-plasmid experimental group with recombinant urease exhibits significant differences in the mineralization of Li, Mn, Co, and Ni metal ions under optical microscopy: notable aggregation of the cells can be observed, with a distinct layer of material adhering around the bacteria.

3.2 Atomic Force Microscopy (AFM) Observation

Fig. 33 Results of Biomineralization Observed by AFM. a) Dual-plasmid experimental group for Li;b) Dual-plasmid experimental group for Mn;c) Dual-plasmid experimental group for Ni;d) Dual-plasmid experimental group for Co;e) BL21 control group

It can be observed that the bacteria in the control group are relatively dispersed, and the surrounding area appears flat. In contrast, the dual-plasmid experimental groups predominantly show aggregation of the bacteria, with noticeable elevation around them. AFM analysis confirms that a layer of material is indeed attached to the bacteria.

3.3 SEM and EDS Analysis

Fig. 34 Electron Microscopy Images of Lithium, Manganese, Nickel, and Cobalt Carbonate

Biomineralization(Note: The negative control uses wild-type BL21 bacteria co-cultured with metal ions without urease for biomineralization; the positive control involves the addition of urease to wild-type BL21 bacteria to simulate in vitro biomineralization; the experimental group uses BL21 bacteria expressing recombinant urease with dual plasmids for biomineralization.) a) Li negative control electron microscopy image;b) Mn negative control electron microscopy image;c) Ni negative control electron microscopy image;d) Co negative control electron microscopy image;e) Li experimental group electron microscopy image;f) Mn experimental group electron microscopy image;g) Ni experimental group electron microscopy image;h) Co experimental group electron microscopy image; i) Li positive control electron microscopy image;j) Mn positive control electron microscopy image;k) Ni positive control electron microscopy image;l) Co positive control electron microscopy image

As shown in the figure above, the surfaces of the Li, Mn, Ni, and Co negative control bacteria are relatively smooth. The Li experimental group and positive control group exhibit swelling at one or both ends of the bacteria. In the Mn experimental group, the bacteria show swelling at one end, with a ring-like swelling in the middle and additional swelling at both ends; in contrast, the positive control also forms two hemispherical carbonate precipitates around the bacteria. The Ni experimental group presents a swollen appearance at one end and a swelling in the middle of the bacteria, similar to the positive control. The Co experimental group primarily displays a swollen appearance that resembles a club shape at one end and a swelling in the middle of the bacterial surface, while the positive control group shows no significant mineralization on the surface of the bacteria. Next, we will perform EDS analysis on the Mn, Ni, and Co groups to further validate the metal adsorption and mineralization capabilities of our recombinant urease-expressing E.coli.

Fig. 35 EDS analysis of Mn. a)Schematic diagram of Mn carbonate biomineralization;b)EDS layered images of Mn;c)EDS analysis of Mn negative control;d)EDS analysis of the Mn experimental group;e)EDS analysis of Mn positive control

Fig. 36 EDS analysis of Ni. a)Schematic diagram of Ni carbonate biomineralization;b)EDS layered images of Ni; c)EDS analysis of Ni negative control;d)EDS analysis of the Ni experimental group; e)EDS analysis of Ni positive control

Fig. 37 EDS analysis of Co. a)Schematic diagram of Co carbonate biomineralization;b)EDS layered images of Co;c)EDS analysis of Co negative control;d)EDS analysis of the Co experimental group;e)EDS analysis of Co positive control

The EDS results indicate that the elemental content analysis of Mn, Ni, and Co follows the trend: positive control > experimental group > negative control. This result is normal and relatively ideal, as the positive control group involved the direct addition of urease, allowing metal ions to interact with urease and decompose urea, generating carbonate ions that precipitate as carbonates. Consequently, the metal element content is highest in this group. The recombinant urease E.coli adsorbs and mineralizes metal ions via its expressed urease. However, due to lower urease activity compared to that in the positive control group, the metal element content in the experimental group is slightly lower than in the positive control but significantly higher than in the negative control group. The negative control group, consisting of wild-type BL21 bacteria, does not express urease, rendering it incapable of biomineralization, resulting in the lowest metal element content.

In conjunction with the previously obtained SEM biomineralization images of Li, Mn, Ni, and Co, these findings effectively demonstrate that our recombinant urease-expressing E.coli can successfully facilitate mineralization.

Conclusion

This project developed a lithium battery waste resource recycling platform that efficiently recovers and reuses metals through three distinct modules: bioleaching, biosorption, and biomineralization. In the future, we aim to optimize the synergistic interactions among these modules, enhance mineralization efficiency, expand the application range of mineralization products, and explore technologies for large-scale production. These efforts will drive advancements in lithium battery metal recovery techniques and significantly contribute to the establishment of a sustainable lithium battery recycling system.