Simultaneous silver recovery and bactericidal bionanocomposite formation via engineered biomolecules

Abstract

Biomolecules natively possess specific interactions with metals and thus can be promising tools for precious metal recovery from solution phase. In this study, we genetically engineered a biomolecule consisting of a silver-binding peptide and a cellulose-binding domain for simultaneous silver recovery and bactericidal bionanocomposite formation. This is the first research demonstrating this rapid, green and eco-friendly concept for one-pot silver recovery and antimicrobial material synthesis.

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Precious metals, such as gold, silver and platinum, have been widely used in today's industries due to their unique catalytic activity, conductivity and biocompatibility.¹ As the quantity of precious metals and their mining are very limited, their recovery from different sources has been attracting more attention. To this end, several efforts, including chemical precipitation, chemical coagulation, ion-exchange, electrochemical technology, membrane process and ultrafiltration, have been extensively investigated.² However, these methods are either too expensive, lacking of specificity, or not environmentally-friendly. In contrast, biomolecules natively possess specific interactions with metals and have been considered as promising tools to recover precious metals from aqueous solutions or wastewaters with low cost and high efficiency.³ Nevertheless, most of these studies were using complex biomolecules, such as bacteria, fungi, algae, or agricultural and industrial byproducts, which may result in the lack of specificity and the overproduction of biomass.⁴ Therefore, an alternative way to overcome these issue is using protein-based biomolecules as their interactions with metals are specific⁵ and their sizes are much smaller than cells.⁶ However, protein-based systems have so far failed to attract much attention due to the tedious protocol and the high cost associated with purification.

With recent advances in genomics, proteomics and metabolomics, designer biomolecules for specific applications can be easily created though gene manipulation.⁷ Apart from the specific and rapid interaction with target metals, a good candidate for precious metal recovery should also be able to bring the chelated metal out of the aqueous phase with high efficiency but low cost. Furthermore, it would be even better if the designer biomolecule can also assist the formation of functional product for certain purpose. Silver is one of the most commonly used precious metals in medical applications due to its antimicrobial properties to prevent infections⁸ and plasmonic properties for diagnostics and therapeutics.⁹ However, aside from its high cost, the raising concerns regarding the potential adverse effect of leached silver on environment and human health are also reasons for the urgent need of silver recovery. To this end, in this study, a designer biomolecule was created for direct precious metal recovery and bionanocomposite formation from artificial wastewater stream. This designer biomolecule contained a silver-binding peptide (AgBP) at its N-terminus for the capture of silver ions from wastewater stream and a cellulose-binding domain (CBD) at the C-terminus for the subsequent removal of captured silver from the aqueous phase via cellulose binding and precipitation (Fig. 1).

Fig. 1 The designer biomolecule contained a silver-binding peptide (AgBP) at its N-terminus for the capture of silver ions from wastewater stream and a cellulose-binding domain (CBD) at the C-terminus for the subsequent removal of captured silver from the aqueous phase via cellulose binding and precipitation.

To construct this biomolecule, a gene fragment coding for the CBD from Clostridium thermocellum was amplified from pScaf-ctf¹⁰ with the forward primer 5′-GCCGCATATGGATCCGACCAAGGGAGC-3′ and the reverse primer 5′-CCGCCTCGAGTACTACACTGC CACCGGG-3′. The PCR product was then digested and ligated into BamHI and XhoI double digested pET24a(+) plasmid (Merck KGaA, Germany) to form pETCBD. Thereafter, a gene encoding the AgBP (NPSSLFRYLPSD) identified from a combinatorial phage display peptide library by Naik et al.,¹¹ was constructed via overlapping oligonucleotide method and inserted into the Nhe1 and BamH1 digested pETCBD vector to generate pETAgCBD. Sequences of the resulting plasmids were confirmed by DNA sequencing. The AgBP–CBD fusion protein was expressed in Escherichia coli BL21 (DE3) [fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI:PlacUV5:T7 gene1) i21 Δnin5] cultured in LB medium supplemented with 50 mg mL⁻¹ kanamycin at 37 °C. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM for induction. Cells were harvested by centrifugation, resuspended in Tris buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl), and lysed by ultrasonic disruption using a sonicator.

Due to its high affinity toward cellulose, CBD has been exploited as an affinity tag for the purification and immobilization of heterologous fusion proteins onto cellulose supports.¹² In particular, the CBD/cellulose affinity system is attractive as it does not require a derivatized matrix, and cellulose is abundant and available in a variety of inexpensive forms.¹³ To check the functionality of the CBD in the designed biomolecule, cellulose binding test was performed (Fig. 2). 200 μL of cell lysate was mixed with 20 mg of Avicel, a microcrystalline cellulose from Sigma, at room temperature for 30 min. After centrifugation, the supernatant was removed and collected as unbound portion. The pellet was then washed three times with pure water and incubated with 200 μL of Tris–NaOH (pH 11) for protein elution. A specific band corresponding to the AgBP–CBD fusion at 31 kDa was detected in whole-cell lysates, demonstrating the successful expression of the designed biomolecule. Upon addition of Avicel, only this band disappeared from the supernatant and was recovered after elution, indicating the preservation of its cellulose-binding functionality. A slightly smaller band was also detected after elution, suggesting the AgBP–CBD fusion was partially degraded while elution.

Fig. 2 SDS-PAGE analysis of samples taken over the course of the cellulose binding experiment. Lane 1: total cell lysates of the E. coli expressing AgBP–CBD; Lane 2: supernatant (unbound fraction) after binding and precipitation of the AgBP–CBD/cellulose complex; Lane 3: eluted AgBP–CBD from the complex (bound fraction).

To illustrate the silver-binding functionality of the designer biomolecule, Avicel-bound AgBP–CBD (0.1 mg-protein per mg-Avicel) was prepared and silver recovery was performed. Briefly, soluble fraction from crude extract was exposed to Avicel prewashed with pure water. After 30 min of gentle agitation at room temperature, Avicel/AgBP–CBD complexes were recovered by centrifugation at 3500g for 3 min. The slurry was then washed three times with pure water and incubated in an aqueous solution of 330 ppm silver nitrate at room temperature. Avicel incubated with E. coli lysates not expressing the fusion protein was used as a control. As shown in Fig. 3a, with 50 mg of Avicel, around 180 ppm of silver ions was removed from the supernatant with the Avicel/AgBP–CBD complexes whereas only around 30 ppm removal was observed with the Avicel control, indicating that the AgBP–CBD fusion was responsible for silver binding and removal. To gain more information regarding the specific relation between the concentration of silver in solution and the amount of silver adsorbed on the Avicel/AgBP–CBD complexes when both phases are in equilibrium, the isotherm data were fitted to Langmuir model as eqn (1).

where C_e and q_e are the equilibrium concentration of silver in the solution (ppm) and the amount of silver adsorbed per unit weight of adsorbent (mg g⁻¹), respectively. K_L is the Langmuir coefficient related to the strength of adsorption (ppm) and q_m is the maximum adsorption capacity (mg g⁻¹). It was found that the Langmuir model gave a satisfactory fitting to the adsorption isotherm with a maximum adsorption capacity of 332 mg g⁻¹ (Fig. 3b), implying the adsorption of silver onto Avicel/AgBP–CBD complexes followed the Langmuir isotherm.

Fig. 3 (a) Time course for silver adsorption by bare cellulose and biomolecule-functionalized cellulose. (b) Isotherm for silver adsorption by the biomolecule-functionalized cellulose.

To check the selectivity of the designer biomolecule, competitive adsorption experiments were carried out in a multi-metallic solution containing three different monovalent metals, namely Cu⁺, K⁺ and Ag⁺, at different ratio (Fig. 4a). Briefly, 200 mg of Avicel/AgBP–CBD complexes were incubated with multi-metallic solutions for 1 hour with gentle agitation at room temperature. After centrifugation, the supernatants were collect and the concentrations of the three metals were analysed by an Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). The Ag⁺ adsorption capacity of the designer biomolecule in the mixed solution was appreciably high. It reached more than 90% of the total adsorption capacity of the designer biomolecule when the concentration of Ag⁺, Cu⁺ and K⁺ in the mixture was 200, 100 and 100 ppm, respectively. Even when the concentration of Cu⁺ and K⁺ was elevated to 200 ppm, the selectivity was still as high as 85%, demonstrating a great selectivity of the designer biomolecule toward silver recovery. In addition, since most of the waste solutions containing metal ions are acidic, the adsorption of silver ions to the Avicel/AgBP–CBD complexes under various pH was evaluated. With an initial silver concentration of 200 ppm, the amount of silver adsorbed by Avicel/AgBP–CBD complexes was not significantly changed when the pH was above 3 (Fig. 4b). However, the adsorption capacity of Avicel/AgBP–CBD complexes decreased to around 90 ppm when the pH was reduced to 2. This reduction was mainly due to the denaturation of AgBP–CBD biomolecules as notable amount of precipitates were observed.

Fig. 4 (a) Competitive adsorption of silver to the biomolecule-functionalized Avicel. (b) Effect of pH on silver adsorption by the biomolecule-functionalized Avicel.

An important feature of the silver-binding peptide used in this study is its ability to form silver nanoparticles (AgNPs).⁹ According to the mechanism proposed by Naik et al.,⁹ the silver-binding peptides interact with preformed silver nanoclusters or nuclei in the aqueous silver nitrate solution. The interaction of peptide with the clusters provides a chemically reducing environment around the cluster, thereby allowing further accelerated reduction of silver ions at the interface between peptide and metal. Thus, AgNPs were formed as a result of the Ag⁺ reduction and silver crystal formation. AgNP solution generally has a visible color depending on the nanoparticle size.¹⁴ When performing this study, we observed changes in the color of the composite, implying the formation of AgNPs on the Avicel/AgBP–CBD complexes. Compared to general antibiotics, one great benefit of nanosilver is its wide-spectrum antibacterial activities without the concern of developing drug tolerance.¹⁵ However, AgNPs have the propensity to aggregate in solution, causing loss of their bactericidal activity over time.¹⁶ Furthermore, direct use of AgNPs as disinfectants is highly risky as the residual nanosilver is too small to be eliminated from aqueous phase by filtration or centrifugation, making this potentially toxic to humans.¹⁷ Therefore, there is a need of stabilizing AgNPs to reduce their toxicity. Apparently, direct formation and immobilization of nanosilver on a supporting matrix via the mediation of AgBP–CBD biomolecules is a good way to address this issue.

To elucidate the formation of silver nanoparticles on the cellulose surface, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) image was performed. The SEM images (Fig. 5a) revealed that the Avicel surface was covered with AgBP–CBD biomolecule and nanosilver complexes. The TEM image (Fig. 5b) further confirmed that the adsorbed silver was in a nanoparticle structure with a diameter of around 20 nm. In addition, the elemental composition of the nanoparticles characterized by an energy dispersive X-ray spectroscopy (EDS) indicated that the element of the nanoparticles formed was silver (Fig. 5c). All of these confirmed the direct formation and immobilization of AgNPs on the cellulose surface by the designer biomolecule.

Fig. 5 (a) SEM image of the Avicel surface covered with AgBP–CBD biomolecule and nanosilver complexes. (b) TEM image of the AgNP formed by the biomolecule-functionalized Avicel. (c) EDS analysis for the AgNPs.

To demonstrate the antibacterial activity of this green nanobiocomposite, the bacteriological tests of the formed composites against a model gram negative bacterium E. coli and a model gram positive bacterium Staphylococcus aureus was performed. Bacterial suspension of around 5 × 10⁷ cells were inoculated to LB-medium for growth retardation test or plated onto LB-agar plates for disc diffusion test. Thereafter, 50 mg of dried nanobiocomposites collected after incubating with 100 ppm artificial wastewater for 1 hour were placed into the LB broth or onto the LB-agar plate as antimicrobial agents. On the other hand, 50 mg of dried Avicels collected after incubating with 100 ppm artificial wastewater was used as controls. Broth or plates without nanobiocomposites and Avicel were inoculated with the corresponding bacteria and used as blanks. Growth curves were obtained using spectrophotometer assisted absorption observations (Fig. 6a and d) whereas inhibition zones were obtained after 24 hours incubation (Fig. 6b, c, e and f). All the blanks and controls exhibited no inhibition effect on bacterial growth. In contrast, the bactericidal activity of the nanobiocomposites was confirmed as evidenced from the bacterial growth curves and the formed inhibition zones.

Fig. 6 Growth retardation test and disc diffusion test of the bionanocomposite against E. coli and S. aureus. (a) Growth curves of E. coli under different condition. (b) Control experiment of the disc diffusion test against E. coli. (c) Inhibition zone formed by the bionanocomposite against E. coli. (d) Growth curves of S. aureus under different condition. (e) Control experiment of the disc diffusion test against S. aureus. (f) Inhibition zone formed by the bionanocomposite against S. aureus. Blank: broth only; Control: broth with the Avicel that had been exposed to silver nitrile solution; BNC: broth with the Avicel/AgBP–CBD complexes that had been exposed to silver nitrile solution and formed AgNPs on it.

Conclusions

In summary, by combining the high affinity interactions between silver/silver-binding peptide and cellulose/cellulose-binding domain, the bactericidal bionanocomposite was successfully synthesized from wastewater streams containing soluble silver via the genetically engineered biomolecule. This bi-functional biomolecule was able to adsorb silver with high capacity, broad pH tolerance and good selectivity. In addition, the recovered silver formed nanoparticles and could be easily removed from wastewater streams by gravity precipitation. Furthermore, the fabricated bionanocomposites exhibited a desirable bactericidal activity against both E. coli and S. aureus. The capability of modulating the individual components of the system, such as the use of different capture peptides (metal-binding peptides), and/or pulling-down domains (material-binding domains), makes this method very flexible for the formation of a wide range of functional bionanocomposites.

The authors would like to thank the Ministry of Science and Technology of the Republic of China, Taiwan, for funding this research under the Contract no. NSC 101-2218-E−011-046-MY3. The authors are grateful to Prof. Wilfred Chen for the plasmid pScaf-ctf and to Prof. Jia-Yaw Chang for the TEM and EDS.

Notes and references

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