CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application No. 63/263,648, filed November 5, 2021, and U.S. Provisional Patent Application No.63/351,754 filed June 13, 2022, the entire contents of each are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DE-AR.0001339 awarded by the Department of Energy. The government has certain rights in the invention.

FIELD OF THE INVENTION

The disclosed technology is generally directed to a bacterial hydrogel. More particularly the technology is directed to a bacterial hydrogel for metal recovery.

BACKGROUND OF THE INVENTION

Modem technological advances in the electronics industry raise new challenges in environmental sustainability, specifically, the recovery, recycling, and remediation of electronic waste (e-waste). Due to the increasing demand for sophisticated electronic devices, the production of e-waste globally per year is around 45 million tons and with a presumed annual growth rate of 3 to 4%, the volume of e-waste could reach 120 million tons by 2050. Without proper handling and recycling, e-waste can cause massive damage to both the environment and public health by continuously releasing hazardous heavy metals (e.g. Cd, Cr, As, Pb and Hg) and organic compounds such as polychlorinated biphenyls, polychlorinated diphenyl ethers, and dibenzofurans. E-waste is also considered a valuable “urban mine” as it contains precious materials such as gold (Au), platinum group metals (PGM, e.g., platinum (Pt), Palladium (Pd)) and Rare Earth Elements (e.g., Neodymium (Nd), Indium (In), Yttrium (Y), Gallium (Ga)) with purity that is orders of magnitude higher than the richest natural ores. The present challenge is the efficient recovery of valuable products from e-waste. BRIEF SUMMARY OF THE INVENTION

Disclosed herein are hydrogels comprising electrochemically active bacteria and reduced graphene oxide integrated with the outer membrane of the bacteria and methods of making and using the same for metal recovery.

Another aspect of the technology comprises hydrogels prepared by culturing a precursor solution comprising the bacteria and graphene oxide, wherein culturing the precursor solution reduces the graphene oxide and integrates the reduced graphene oxide with the outer membrane of the bacteria. The precursor solution may be cultured under anaerobic conditions for a time sufficient to reduce the graphene oxide and integrate the reduced graphene oxide with the outer membrane of the bacteria. The hydrogels described herein may be prepared without the use of an inactive cross-linked material such as alginate.

Another aspect of the technology comprises methods of metal recovery with the hydrogels disclosed herein. Suitably, the method may comprise contacting the hydrogel with a metal, such as a metal in a leachate.

Also disclosed are bioreactors utilizing the hydrogels disclosed herein. The bioreactor may comprise a leachate tank holding a leachate solution; a nutrition tank holding a nutritional solution; a mixing tank fluidly coupled to the leachate tank and the nutrition tank such that the leachate solution and the nutritional solution can be combined in the mixing tank to form a combined solution; any of the hydrogels described herein; at least one cartridge having an inlet and an outlet, wherein the cartridge holds the hydrogel, wherein the mixing tank is fluidly coupled to the inlet; and a pump, wherein the pump directs the combined solution to flow from the mixing tank to the inlet, through the cartridge, and exit through the outlet as an effluent.

Another aspect of the technology provides for a method of removing metal species from a leachate solution comprising flowing the leachate and a nutritional solution into the inlet of a bioreactor cartridge and over the hydrogel to form an effluent that exits the bioreactor cartridge through the outlet.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

Figure 1. Electrochemically active bacteria reduce and self-assemble around GO to form hydrogels (a) Scheme of the bio-initiated self-assembly. During this process, the metabolic electron of PV-4 drives the reduction of GO to form B-rGO intimately integrated with the outer membrane of PV-4. The intermolecular forces such as hydrophobic interaction and DLVO forces drive the self-assembling of PV-4 and B-rGO to construct a free-standing hydrogel.

Figure 2. Self assembly conditions Plot showing self-assembly results for different concentrations of GO and bacteria.

Figure 3. Mechanical stability and morphology of hydrogels A tweezer manipulation demonstrating the structural integrity of this hydrogel. The hydrogels can be formed in (a) various shapes and (b) free-standing arrays according to the container in which the precursor solution is placed.

Figure 4A. A two probe electrical measurement of the hydrogel shows a conductivity at 0.4 mS/cm.

Figure 4B. Methyl orange degradation was used to test the viability of the hydrogel encapsulated PV-4 which demonstrates biocatalytic activity superior to native biofilms. UV-vis spectroscopy of MO solutions before (squares) and after (circles) treatment by hydrogels. The decolonization of MO was verified by the vanishing of its absorption peak centered at 464 nm, which further confirms the viability of encapsulated PV-4 in the hydrogel and indicates that metabolic rate of encapsulated PV-4 can be advanced by the sufficient mass- and charge- transport of this hydrogel.

Figure 5. Pd recovery efficiency of hydrogels (a) Hydrogels were initially cultured in media containing 100 ppm ofPd ions to perform Pd recovery, which gave a maximum recovery efficiency at 70%; (b) Following a 48-hour period, another 100 ppm of Pd ions was introduced into the culture media to enhance the concentration driven Pd mass transport. Results indicate that the hydrogel can recover a total of 140 ppm of Pd ions after 96 hours; the mass transport is a factor that limited recovery rate. (n=3) Plots show the Pd concentration in the solution at time points.

Figure 6. Morphological control of hydrogels and its influence in Pd recovery efficiency (a) molding methods to construct hydrogels with different size and 3-D profiles (Scale bar: 1 cm); and (b) efficiency of hydrogels with total surface areas of 1225 mm ² (cylindrical) and 2156 mm ² (square prism). Results indicate that increasing the surface area can promote the Pd recovery efficiency (60 % vs. 35 %) while reducing the start-up times (immediately vs. 3 hours) of the hydrogels. (n=3)

Figure 7. Pd recovery rate of microcables in simulated e-waste streams. EAB/GO/ Alginate microcables (grey bars) and EAB/GO microcables (black bars) were cultured in media containing 100 ppm of Pd ions for 7 days to perform continuous Pd recovery. Results indicate that both microcables lost their biocatalytic activities after the 4th day of recovery. EAB/GO/ Alginate microcables achieve 60% Pd recovery rate while EAB/GO microcables achieve 80% Pd recovery.

Figure 8. SEM characterization of hydrogel after Pd recovery (a) low magnification image; (b) and (c) The surface of hydrogel is covered with a high density of recovered Pd as indicated by the observed round particles confirmed by EDX. (b) Pd particles on PV-4 are about 100 nm in size, while as can be seen in (c) Pd particles on B-rGO are tens of nm. (d) The internal part of the hydrogels is composed of low density of PV-4 and B-rGO and has a smooth surface. This image suggests that a minimum amount of Pd is recovered at the internal part of the hydrogels.

Figure 9. EDX characterization of a hydrogel after Pd recovery (a) the EDX analysis, of which recovered Pd (grey) is mainly coated on the surface of the hydrogels while carbon (green) and oxygen (blue) are uniformly distributed over the entire hydrogels, (b) the corresponding SEM image.

Figure 10. Design and operation of flow reactor (a) the design of flow reactor. The arrows indicate the flow directions, (b) image of flow reactor with hydrogel loaded (scale bar: 10 mm) and (c) images of the operational setup, of which, the flow reactor was connected with a peristaltic pump to circulate the Pd solution for continuous treatment.

Figure 11. Simulated in-line Pd recovery: (a) Operational setup showing an individual flow reactor connected with a peristaltic pump to circulate the Pd solution for continuous treatment, (b) Sequential treatment using two flow reactors, where the first flow reactor was replaced by a duplicate flow reactor after a 48-hour operation, further improved treatment efficiency to 87%, which suggests that the fresh surface of hydrogel is critical in Pd recovery.

Figure 12. Scaling-up the packed bed bioreactor for the implementation of hydrogels in Pd recovery: (a) flow chart of the scale-up design. In the design, the nutrient solution and leachate are mixed before being introduced into the bioreactor. Multiple parallel bioreactors can be set up in the process based on the quantity of leachate, (b) Image of the bioreactor prototype connected with a peristaltic pump to circulate the Pd solution for continuous treatment.

Figure 13. Pd recovery efficiency (dot/solid line) and rate (circle/dash line) of the scale-up packed bed bioreactor with 100 hydrogels loaded. The amount of recovered Pd is calculated based on the initial concentration and quantity of simulated e-waste leachate at 100 ppm and IL, respectively.

Figure 14. Oxygen-limited thermal post-treatment: (a) The thermogravimetric analysis was performed under nitrogen flow of 90 mL/min and a heating rate of 10 °C/min to 600 °C to carbonize the organic contents, (b) The SEM-EDX analysis of the nanoPd coated active carbon after oxygen-limited thermal post-treatment showed a 90% of surface coverage.

DETAILED DESCRIPTION OF THE INVENTION

Bioreduction is a modem, desirable approach to extracting valuable metals from leaching solutions because this method can directly transform soluble metal ions into recoverable solid metals while minimizing energy consumption and environmental impact. These “living hydrogels” are self-sustainable, and can spontaneously evolve into living networks (i.e biofilms) that are capable of long-term operations with easy operation and extremely low energy consumption. The application of naturally derived biofilms is commonly hindered by inefficient mass transport.

Biocarriers can combine the advantages of biofilm- and planktonic-based bioreduction by growing biofilm on an mm-to-cm scale 3-D polymer architecture with a large surface area that can float inside a bioreactor. The high specific surface area increases the biomass loading and mass transfer of attached biofilm is actively promoted as biocarriers are suspended and flow within the metal-contained water streams by agitation during a whole reduction process. Currently, physiochemical mismatch between the microorganism and the polymer-based biocarrier eventually causes both the failure of metal recovery and significant secondary contamination due to the detached microorganisms.

To overcome these limitations a bottom-up assembling strategy is disclosed herein to construct a hydrogel composed of a seamlessly integrated bacterial network, which can serve as free-standing yet fully active biocarrier. In this living hydrogel, Shewanella loihica PV-4 (PV-4) can be selected as an exemplary electrochemically active bacteria for metal recovery. PV-4 is capable of reducing a diverse set of metal ions into solid-state nanomaterials through their unique ability in cross-membrane transport of metabolic electrons. The living hydrogel can be directly implemented as a filter without any passive structural support.

Living hydrogel formulation and fabrication

According to the disclosure herein, hydrogels can be synthesized from a precursor solution comprising an electrochemically active bacteria, such as Shewanella loihica or Geobacter spp., a nutritional solution, and graphene oxide (GO) under anaerobic culture as shown in Figure 1. GO can be provided with an initial oxygen content, for example > 36%, present as terminal functional groups. The nutritional solution can include nutrients necessary for the bacteria PV-4 to thrive as reported previously (Proc Natl Acad Sci, 2006; 103(30), 11358-11363; Proc Natl Acad Sci 2009, 106(23), 9535).

Under these conditions, PV-4 can reduce GO to bio-reduced GO (B-rGO) using selfgenerated metabolic electrons. The nutritional solution can include nutrients that are electron donors such as lactate, formate, pyruvate, hydrogen, Glucose, Luria-Bertani (LB) media, and acetate. In one non limiting example, a precursor solution with an initial concentration of 40 mM lactate as the electron donor can be used in the hydrogel synthesis. It is contemplated that concentrations such as 20 mM lactate or 30 mM lactate can be used in the hydrogel synthesis. The living hydrogels can form spontaneously when the precursor solutions are cultured under anaerobic conditions. For example, culturing a precursor solution for 48 hours under anaerobic conditions to initiate the self-assembly of PV-4 and B-rGO. As shown in Figure 2, hydrogels can self-assemble from precursor solutions having GO and PV-4 present in a range of concentrations. For example, hydrogels can form from precursor solutions having a bacteria concentration of 4xl0 ⁸ cell/cm ³ and a GO concentration of 0.5, 0.75, or 1 mg/mL. In another example, hydrogels can form from precursor solutions having a bacteria concentration of l lxlO ⁸ cell/cm ³ and a GO concentration of0.5, 0.75, or 1 mg/mL. In yet another example, hydrogels can form from precursor solutions having a bacteria concentration of 13xl0 ⁸ cell/cm ³ and a GO concentration of 0.75, 1.0 or 1.3 mg/mL. In yet another example, hydrogels can form from precursor solutions having a bacteria concentration of 21xl0 ⁸ cell/cm ³ and a GO concentration of 0.75, 1.0 or 1.3 mg/mL.

Physical and electrical characteristics of living hydrogels

As shown in Figure 3, the hydrogels can be formed in a variety of shapes. The shape of the hydrogel is at least partially determined by the shape of the container into which the precursor solution is placed. For example, a container with a cylindrical shape results in a hydrogel having a cylindrical shape. Hydrogels have been grown in a variety of example containers, such as star, triangular prism or pillar array resulting in hydrogels of these shapes. In one example, container can be elongated, having a height-to-base aspect ratio of greater than 1. In another example, the container can be slab-like, having a height-to-base aspect ratio of less than 1.

The PV-4 bacteria self-assemble with GO into the living hydrogel. As the GO is reduced, it loses oxygen functional groups and becomes more hydrophobic. The hydrophobicity of B-rGO allows it to integrate with the outer membrane of the bacteria. (ACS Appl. Bio Mater. 2020, 3, 11, 7376-7381) Since the B-rGO integrates with the outer membrane as the bacteria multiply in the culture within the container, the PV-4/B-rGO hydrogel takes the shape of the container. The shape of the hydrogel is maintained when the hydrogel is removed from the container. The B-rGO imparts enough rigidity to the hydrogel such that the hydrogel is free-standing. A free-standing hydrogel may be characterized by a height-to-base aspect ratio of greater than one. For purposes of determining the height-to-base aspect ratio, the ratio of the longest base dimension to the highest dimension may be compared. For example, a cylindrical hydrogel has a height-to-base aspect ratio determined by the height of the cylinder to the diameter of the base or a square prism has a height- to-base aspect ratio determined from height of the prism to the diagonal of the base. The hydrogels formed in the manner described herein are rigid enough that additional cross-linking materials such as alginate are not needed for mechanical support. The hydrogels are sturdy enough to be handled by tweezers with minimal damage as shown in Figure. 3. The sturdiness of the hydrogel allows for the hydrogel to maintain its shape for prolonged periods when subjected to a flowing liquid, such as a metal containing leachate. Furthermore, the hydrogels are self-supporting against gravity.

The hydrogel not only demonstrates adequate structural integrity but electrical conductivity as well. During the molding process of forming the hydrogel, PV-4 is capable of producing 2 electrons for every 1 lactate molecule consumed. As shown in Figure 4A, electrical measurements of the hydrogel show a high conductivity/charge transfer efficiency, specifically a conductivity of 0.4 mS/cm.

The bioactivity and metabolic rate of PV-4 are maintained even when encapsulated within the hydrogel. For example, the electrical capability of the hydrogel can be characterized by the reduction of an azo dye (i.e., methyl orange (MO)) as PV-4 is capable of reducing the nitrogennitrogen double bond of MO. In one example, hydrogels decolorized a solution of MO indicating a complete reduction of MO (Figure 4B, left). The MO decolorization was quantified using UV- vis spectroscopy as MO has a broad absorption peak centered at 464 nm. The absorption at 464 nm decreased dramatically after 4 h. (Figure 4B, right) Based on this result, the volumetric electron generation rate of these hydrogels is estimated as 1.8 x 10 ¹⁹ electron/hr/mL, based on the fact that 2 electrons are consumed in order to reduce one MO molecule.

Metal recovery - efficiency and dynamics

The hydrogels as disclosed herein can be used to remove metal ions from a solution by reduction. The hydrogel is conductive, as there are conductive proteins in the PV-4 outer membranes and B-rGO is conductive. Therefore, when metal ions in solution contact the hydrogel surface, they can be reduced to the elemental form. For example, Pd ions in solution can be reduced to Pd metal at the surface of the hydrogel according to the reaction:

Pd ²⁺ + 2e Pd°

The metal recovery efficiency of the hydrogels is affected by mass transport of the metal ions in the solution. In one example, a solution of 100 ppm Pd was combined with 50 hydrogels, or approximately 2 x 1021 cells/cm ³ and 100 mM lactate. As can be seen in Figure 5, the concentration of Pd in solution decreased dramatically over the first 6 hours but then reached a plateau at about 30 ppm Pd remaining at 24 h. At 48 h, another 100 ppm Pd was added to the solution. At 96 h, about 30 ppm Pd remained. These results indicate that the metal ion recovery is limited by mass transport of the ions in solution.

Increasing the surface area of the hydrogel improves the efficiency and speed of metal recovery. In one example, hydrogels of differing surface areas were exposed to Pd-containing solutions. (Figure 6) For example, hydrogels of cylinder and square prism shapes were compared. The Pd content of the solutions was monitored over time and it was found that the hydrogels with larger surface area recovered a greater amount of the Pd from the solution after 3 hours and after 24 hours.

Hydrogels can be formed with or without an inactive cross-linked material in addition to the GO. A cross-linked material can be, for example, alginate which cross-links upon addition of calcium ions. In one example PV-4/GO hydrogels were formed as microcables with and without alginate and were cultured in the presence of Pd to evaluate Pd recovery ability. (Figure 7) The alginate-containing PV-4/GO microcables (grey bars) and PV-4/GO microcables (black bars) were exposed to Pd for continuous Pd recovery. While both types of microcables lost biocatalytic activities after 4 days, the alginate-containing PV-4/G0 microcables achieve 60% Pd recovery rate while PV-4/GO microcables achieve 80% Pd recovery. While the inactive cross-linked material, such as alginate, did not improve the Pd recovery performance of the PV-4/GO hydrogels in this example, other types of inactive cross-linked materials may be contemplated that provide improved mechanical properties, increased surface area, or improved mass transport at the surface.

The hydrogels can recover metal ions from a solution by reduction of the metal ions on the surface of the hydrogel. For example, there are conductive proteins in the PV-4 outer membranes. As can be seen in Figure 8, Pd metal particles build up on the surface of the PV-4/GO hydrogels during exposure to a Pd solution indicating that the Pd ions are reduced by the hydrogel. Interestingly, the image in Figure 8 (b) shows that Pd on PV-4 presents larger in size (100 nm) as compared with Pd on B-rGO (tens of nm) as shown in Figure 8 (c). Pd particles nor traces of Pd were found on the interior of the hydrogels after Pd recovery as demonstrated by the EDX images in Figure 9. The EDX analysis shows the recovered Pd (grey) is mainly coated on the surface of the hydrogels while carbon and oxygen are uniformly distributed over the entire hydrogel.

Bioreactor design and Scale-up

Metal recovery by the hydrogel can be improved by controlling the mass transport of the metal in solution. To improve mass transport to the hydrogel surface, the hydrogels can be placed within a flow reactor 100 such that the Pd solution can be flowed over the surface of the hydrogel. One example of the flow reactor is shown in Figure 10. The flow reactor can have a chamber 102 to hold the hydrogels and the Pd solution. The chamber can have at least one inlet 104 and at least one outlet 106. The inlet 104 shown in Figure 10 (a) can spread the flow of Pd solution such that is does not forcefully impact the hydrogel. Further, the chamber can include a fluid-permeable platform 108 to support the hydrogel and allow solution to flow within the chamber. Additionally the chamber may be covered to preserve anaerobic conditions needed to support the culture of PV- 4. The flow reactor 100 can be operated with a pump 110 directing the flow of the Pd solution. The inlet can be fluidly coupled to a source of the Pd solution to allow Pd solution to flow into the chamber and over the hydrogel. The outlet can be fluidly coupled to the inlet exterior to the chamber such that the Pd solution can flow continuously through the chamber.

In one example shown in Figure 11, the flow reactor 100 can be operated with a pump 110 directing the flow of the Pd solution. In this configuration, the Pd solution can continuously recirculate through the chamber for any desired length of time, allowing the hydrogels to become saturated with recovered Pd. Additionally, and alternatively, the outlet can direct the solution away to a separate container for storage or disposal.

Hydrogels can be replaced or exchanged in the flow reactor 100 to improve Pd recovery. In one example shown in Figure 11 (b), the flow reactor was exchanged after continuously running for 48 h, at which time the Pd content of the solution has plateaued. Introducing a fresh hydrogel improved the Pd recovery to 87%.

To improve and scale up metal recovery using multiple reactors, a bioreactor to support the hydrogels is contemplated as shown in Figure 12. The bioreactor can have a leachate tank holding a leachate solution, or a metal containing solution from a waste stream or contaminated source. The bioreactor can include a nutrition tank for holding a nutritional solution to nourish the PV-4 bacteria. The particular components of such a nutritional solution have been previously reported (Deng, Pu et al. ACS Appl. Bio Mater. (2020) 3, 11, 7376-7381). The bioreactor can further include a mixing tank fluidly coupled to the leachate tank and the nutrition tank such that the leachate solution and the nutritional solution can be combined in the mixing tank to form a combined solution. A pump can be included to direct the solutions throughout the bioreactor.

The bioreactor can have at least one cartridge to house the hydrogel and preferably more than one cartridge is installed in the bioreactor. The cartridge can have an inlet and an outlet. The cartridge can further include a semi-permeable membrane to cover the inlet and the outlet. The mixing tank can be fluidly coupled to the inlet of the cartridge such that the combined solution can enter the at least one cartridge. Specifically, the combined solution can be directed to flow from the mixing tank to the inlet of the cartridge where it contacts the hydrogel. The combined solution can be directed to flow through the cartridge and exit through the outlet as an effluent. Multiple cartridges can be installed in the bioreactor in a parallel configuration as shown in Figure 12 (a) such that the combined solution enters each of the cartridges at the inlets and exits through the outlets where the solution is recombined at the recycling tank.

The recycling tank can be fluidly coupled to the mixing tank such that the combined solution can be returned through the cartridges in the bioreactor to repeatedly contact the hydrogel in order to improve metal recovery. The recycling tank can be fluidly coupled to the mixing tank such that the pump can direct the effluent to return to the mixing tank from the outlets. Additionally, and alternatively, the recycling tank can direct the combined solution to a separate container for storage or disposal. The metal content of the circulating solution can be determined by sampling the solution in the mixing tank at the recycling tank.

A leachate solution from an e-waste stream may contain other metals such as such as Fe, Ni, Cu, or Zn. These or other ions may interfere with the hydrogel capture and reduction of other metals. It is contemplated that pre-treatment of the leachate solution can remove some of these ions, such as by chemical means (e.g., pH-induced precipitation). The bioreactor can optionally include an ion exchange resin to capture these interference ions.

The cartridge can be removably coupled to the bioreactor, for example by including valves at the inlet and outlet. By closing the inlet and outlet valves, the cartridge can be removed from the bioreactor. For example, a used cartridge can thus be replaced or exchanged with a new cartridge such that fresh hydrogel is introduced into the flow path for metal recovery. In this configuration, the bioreactor can be continuously operated to recover metal from a waste stream.

In operation, the Pd reduction efficiency of hydrogels can be evaluated by continuously circulating a volume of leachate solution. Figure 13 shows the overall efficiency for 100 hydrogels and IL leachate solution (closed circles/the solid line) at 48 hours was 12%. The Pd recovery rate of this reactor can also characterized. In this example, , the results showed that the recovery rate decreased significantly between 24-48 hours (open circles/dashed line).

Post-treatment of hydrogels for metal recovery

To isolate a usable product from the hydrogel metal recovery system, a thermal treatment process such as carbonization can be implemented. More specifically, the hydrogels can be subjected to carbonization temperatures, such as a temperature between 300 °C and 900 °C, in the absence of air. An inert gas can be used, for example, nitrogen or argon, to ensure the conditions are reducing. For example, carbonization of the hydrogels can be carried out at 600 °C under a flow of nitrogen gas. Thermogravimetric analysis shows that once the hydrogel reaches 600 °C, weight loss ceases. (Fig. 14a) indicating that the organic content was transformed into inert carbon. The carbonized residue as characterized by SEM-EDX (Fig. 14b) shows surfaces of the residue are covered by Pd (>90%). This high degree of Pd coverage can imbue the carbon residue with desirable electrochemical or catalytic activity.

Miscellaneous

Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

EXAMPLES

Materials and Methods

Living hydrogel formulation and fabrication

The culture of S. loihica PV-4 was based on documented protocol (Deng, Pu et al. ACS Appl. Bio Mater. (2020) 3, 11, 7376-7381). In short, S. loihica were cultured from -80 °C glycerol stock and inoculated in 50 mL of Luria-Bertani broth (Sigma L3522) with gentle shaking (100 rpm) in the air for 48 h at 25 °C. After centrifugation at 4000 rpm for 20 min, the isolated bacteria were redispersed in mineral media (MM) containing 30 mM sodium lactate to tune the bacteria concentration to 3.2 x 10 ¹⁹ cell/mL. The formulation of MM was reported previously(Deng, Pu et al. ACS Appl. Bio Mater. (2020) 3, 11, 7376-7381). The S. loihica solution was cultured for another 24 hours and then incorporated with graphene oxide (GO) solution (4mg/mL; Sigma Aldrich 777676) in volume ratio 2: 1 (bacteria: GO) and vortexed until homogenous to be applied as the precursor solution for living hydrogel fabrication. A series of precursor solutions of GO (GO concentration ranging between 0.25 mg/mL and 1.50 mg/mL) and PV-4 (PV-4 concentration ranging between 5xl0 ⁸ cell/cm ³ and 25xl0 ⁸ cell/cm ³ ) were prepared and evaluated for selfassembly and the results are shown in Figure 2.

The living hydrogels can form spontaneously after the precursor solutions were cultured for 48 hours under the anaerobic condition to initiate the self-assembly of PV-4 and B-rGO. The biohybrid structure can be further modulated through a “molding” process. Specifically, the PV- 4/GO solutions were filled into a container with customized 3-D profiles prior to culture, where the synthesized biohybrid can replicate the 3-D profiles and grow into desired structures such as cylinder, star, triangular prism and pillar array by using containers with corresponding shapes. After fabrication, the unit bacteria density can be estimated as 2.12 x 10 ²¹ cell/mm ³ , calculated based on the initial bacteria concentration of 2.12 x 10 ¹⁹ cell/mL in the precursor solution and a 50% volume reduction after transforming from the precursor solution to the living hydrogel.

Physical characteristics of living hydrogels - Scanning electron microscope (SEM) imaging

Samples for SEM imaging were prepared as follows: After DI water rinsing, the samples were dehydrated in a series of ethanol solutions (50, 75, 90, 95, 100%, and 100%, 10 min each). Dehydrated samples were then critical point dried (Leica EM CPD300) and sputtered with Pd/Pt alloy. Images were acquired by Zeiss Ultra 55 SEM.

Electrical characteristics of living hydrogels

The direct current I~V measurements (Figure 4A) were performed on Biological SP200 Potentiostat. To establish electrical contact, one living hydrogel with a cross-section area of hl mm ² was deposited onto a pair of gold electrodes. The measurements were performed after the hydrogel was rinsed with DI water and 100% ethanol.

The living hydrogel are capable of generating metabolic electrons. The capacity for the living hydrogel can depend on the bacterial density, dissolved oxygen, temperature, age of bacteria, concentration, or any combination thereof. For example, higher bacterial density should improve metabolic electron generation. In some embodiments, the hydrogel has a metabolic electron generation rate greater than 1 x 10 ¹⁹ electron/hr/mL.The bioactivity of as-synthesized living hydrogels is characterized by the azo dye (i.e., methyl orange (MO)) as PV-4 is capable to reduce the nitrogen-nitrogen double bond of MO and hence decolor the MO solution. Specifically, the living hydrogels were cultured in HEPES buffer with pH=6.8 for 24 hours prior adding desired amount of MO solution to final concentration of 4 mg/L (= 12 mM). (Fig. 4B) As shown in Fig. 4B, the living hydrogels can effectively decolor 4 mg/L of MO within 2 hours, which suggest the synthesis process of these living hydrogels did not damage the bioactivity of PV-4. The generation rate of metabolic electron of these living hydrogels is estimated though the MO decolor test as 1.44 x 10 ²² electron/hr based on the assumption of 4 electrons consumed to reduce one MO molecule (Journal of Saudi Chemical Society (2018) 22, 322).

The living hydrogel can be characterized by a charge transfer efficiency. Suitably, the living hydrogel has a charge transfer efficiency of at least 0.1 S/cm, 0.2 S/cm, 0.3 S/cm, or 0.4 S/cm. The as-prepared Examples demonstrated a charge transfer efficiency of about 0.4 S/cm. Performance characterization of living hydrogels

To perform the simulated metal recovery, 100 ppm (0.56 mM) of palladium (Pd) was introduced into the hydrogel-contained mineral media together with 30 mM sodium lactate as a food/electron donor. The concentration of Pd is tested periodically using atomic absorption spectroscopy (Thermo Solaar S4) equipped with a palladium hollow cathode lamp (Thermo Scientific 942339020461) to evaluate the treatment efficiency of each living hydrogel filter. Standard Pd solutions were prepared by dissolving the desired amount of palladium chloride (Sigma-Aldrich 520659) in a MilliQ/H SCh mixture (pH=l).

In addition, the Biosciences UltrospeclO cell density meter (Amersham) was used to measure the OD-600/ planktonic bacteria density. In detail, a 3 mL sample from the bioreactor was loaded in the crystal sample holder for measuring OD-600, with Milli-Q water as a control to get the blank value. Each measurement was repeated three times with different samples from the same population and the results were averaged to get the final OD-600 value.

Pd recovery experiments

A Falcon 50 mL conical centrifuge tube (Fisher Scientific Catalog No.14-432-22) was applied to simulate a packed-bed bioreactor. After being cleaned with isopropyl alcohol and DI water, the bioreactor was loaded with living hydrogel filters and connected to a peristaltic pump (Watson Marlow 101F) to circulate the Pd solution. For the treatment experiments, a 100 mL of Pd solution with a concentration of 0.56 mM Pd was first filled into the main reservoir and circulated through the bioreactor. During the treatment, the Pd sample was continuously circulated between the bioreactor and the reservoir for 48 hours. The concentrations of Pd were periodically measured to determine the treatment efficiencies. The OD-600 was measured at the end of treatment to evaluate the leakage of S. loihica by Biosciences UltrospeclO cell density meter (Amersham).

Biocatalytic efficiency of a living hydrogel filter without alginate is enhanced in comparison to a hydrogel filter containing alginate prepared as previously reported (Deng, Pu et al. ACS Appl. Bio Mater. (2020) 3, 11, 7376-7381). Alginate is considered an “inactive” material in the biocatalyzation reaction driven by PV-4 metabolic electron transfer. The bacteria density and GO concentration were precisely modulated to obtain the free-standing living hydrogels without the inactive alginate while maintaining desired structural integrity. The results indicated that these living hydrogels could achieve 80% of Pd recovery before plateaued on the 4 ^th day of culture in the Pd-containing media (black bars in Figure 7). These results suggest that hydrogel filters made of “fully active” PV-4 and B-rGO can perform metal recovery from e-waste leaching streams with efficiency superior to alginate-containing hydrogel filters. Results and Discussion

Fabrication and characterization of living hydrogels

In this work, living hydrogels are synthesized from a precursor solution composed of PV- 4 and graphene oxide (GO) mixed with desired ratio (2: 1). Under anaerobic culture, PV-4 can continuously reduce GO using self-generated metabolic electrons transferred through their conductive membrane proteins and protein assemblies. The lactate with an initial concentration of 40 mM was applied as the nutrient/electron donor through the hydrogel synthesis process. In this process, PV-4 was capable of producing 2 electrons after consuming every 1 lactate molecule. Previous research [[REF? ACS Appl. Bio Mater. 2020, 3, 11, 7376-7381]] has demonstrated that this bio-reduced GO (B-rGO) can be connected with PV-4 both structurally and electrically to form microscale B-rGO/PV-4 biohybrids. After this initial bioreduction process, these biohybrids start a self-assembling process driven by hydrophobic attraction, and DLVO interactions. Eventually, these biohybrid materials can form an integrated 3-D living hydrogel with adequate structural integrity and electrical conductivity after a 2-day anaerobic culture. As shown in the results, the structural integrity of this living hydrogel allows the direct manipulation using a medical tweezer without any noticeable damage. This free-standing living hydrogel is capable of preventing the detachment of PV-4 during metal recovery. Moreover, the electrical measurement shows a high conductivity/charge transfer efficiency of this living hydrogel at 0.4 S/cm. These structural- and electrical- properties provided the unique potential for this living hydrogel in the development of free-standing biocarriers for metal recovery.

The bioactivity of as-synthesized living hydrogel is characterized by the azo dye (i.e., methyl orange (MO)) as PV-4 is capable of reducing the nitrogen-nitrogen double bond of MO; hence decoloring the MO solution. In this experiment, living hydrogels were cultured in a glass vial containing 20mL of HEPES media with pH=6.8 for 24 hours before concentrated MO being added to a final concentration of 0.8 mg/mL (2.4 mM) to initiate the decolorization test. Four hours after the test initiation, the dark orange to transparent color change indicated a complete reduction of MO (Fig 1c). The completion in MO decolorization was further validated using UV-vis spectroscopy as MO has a broad absorption peak centered at 464 nm. These results confirmed that the biohybrid living hydrogels can well preserve the bioactivity of encapsulated PV-4. Based on this result, the volumetric electron generation rate of these living hydrogels is estimated as 1.8 x 10 ¹⁹ electron/hr/mL based on the fact that 2 electrons are consumed for reducing one MO molecule. This estimation indicates that the metabolic rate of encapsulated PV-4 can be well preserved in these living hydrogels.

Efficiency and dynamics of metal recovery

Palladium (Pd) is selected as the model metal for evaluating the performance of this living hydrogel in metal recovery due to its high-value, versatility, and scarcity. The Pd recovery was initiated by introducing Pd ions with a final concentration of 100 ppm (=0.56 mM) into a HEPES buffer containing 50 living hydrogels (containing around 2.12xl0 ²¹ cells/mm ³ ). The lactate with an initial concentration of 100 mM was applied as the nutrient/electron donor during the Pd recovery process. This Pd recovery was monitored periodically using atomic absorption spectroscopy over 48 hours. First, the low turbidity of the treated solution (Fig.5 insert, of which, OD 600 approaches 0) suggested the structural integrity of this living hydrogel that can encapsulate PV-4 in its matrix through hours of operation. This property is considered superior to conventional biocarriers that the detachment of microorganisms remains one of their major limitations. In terms of efficiency, the living hydrogels demonstrated high efficiency in the first 6 hours, which were able to recover 70% (70 ppm) of Pd ions. However, the recovery rate plateaued between 6 to 24 hours, which suggested the decrease in Pd ²⁺ concentration (from 100 ppm to 30 ppm) can detain the mass transport and eventually limit the further enhancement in Pd recovery. To test this hypothesis, a separate experiment is performed, of which another 100 ppm of Pd ions were introduced on the 48th hour after sampling to recover the mass transport. The Pd concentration was monitored daily throughout the whole treatment process over 96 hours. The results showed that the living hydrogels were able to recover another 70% of Pd ions two days after the Pd replenishment (total of 140 ppm). This observation confirmed that mass transport of Pd ions is one of the factors that affects the performance of the living hydrogels, which can be actively promoted through modulating the flow dynamic in a bioreactor.

To investigate the dynamic of Pd reduction, the living hydrogels were analyzed under the scanning electron microscope (SEM) after a 48-hour Pd recovery. (Fig. 8) Interestingly, the SEM images have revealed a distinct morphological difference in recovered Pd between the PV-4 and B-rGO on the surface of the living hydrogels. As shown in the SEM images (Fig. 8b), a high density of PV-4 was observed on the surface of these living hydrogels with closely connected clusters made of 100 nm Pd nanoparticles. In contrast, B-rGO on the surface was uniformly coated by Pd nanoparticles with diameters in tens of nanometer ranges. (Fig. 8c) This phenomenon can be attributed to a limited number of conductive proteins on the PV-4 outer membranes. As Pd reduction was accomplished through the extracellular electron transfers mediated by the conductive proteins, these proteins served as nucleation sites at the initial stage of the formation of Pd nanoparticles. The low density of Pd nuclei, therefore, led to the formation of larger Pd nanoparticles since consecutively Pd reduction would only occur on these nuclei. In terms of B- rGO, this material holds uniform conductivity, which resulted in even nucleation on its surface; therefore, resulted in the formation of high-density Pd nanoparticles with smaller particle sizes.

Surface of living hydrogels and their influence on metal recovery

In addition, the SEM also revealed that minimum-to-no Pd can be found in the internal part of the living hydrogels where the density of PV-4 is also significantly lower as compared to the surface. (Fig. 9) This observation was further validated using the energy-dispersive X-ray spectroscopy (EDX) compositional analysis. As presented in the EDX, the recovered Pd are mainly coated on the surface of the hydrogels while carbon and oxygen were uniformly distributed over the entire hydrogels, which were the major elements that constructed both PV-4 cell bodies and B- rGO. (Fig. 9). This discovery suggested that the mass transport is ineffective in the internal part of these living hydrogels, which restricted the access of PV-4 to both electron donors (i.e., nutrient) and electron acceptors (i.e., Pd ions) and eventually damage the viability of PV-4. This phenomenon may originate from the small pore size of the living hydrogels, which can restrict the mass transport at the center of the living hydrogel. Such limitation in diffusion constantly limited the amount of reduced Pd while eventually damaging the viability of PV-4. Besides, it has been shown that GO is hydrophilic due to its ample functional groups including carbonyl- and, hydroxyl- groups. These functional groups may be depleted during the transformation to B-rGO contributing to the increase in hydrophobicity, which also contributes to the reduced mass transport at the internal parts of living hydrogels.

Since the reduction of Pd nanoparticles mainly occurs on the surface of hydrogels, the surface area is considered a factor for the efficiency of Pd recovery. Toward optimizing the performance of hydrogels, a “molding” technology was developed to modulate the surface area. Specifically and interestingly, the 3-D profiles of the hydrogels were determined by both (i) the boundaries of molds (i.e., cultured container) (in x-y axis) and (ii) the height of precursor solution (z-axis) during the cultures of PV-4/G0 mixtures. Therefore, the shape of the hydrogel can be precisely engineered by controlling these two parameters. As presented in Fig. 3a and Fig. 6 different sizes of cylindrical- as well as cuboidal- hydrogels were synthesized by using molds with corresponding shapes. Furthermore, a separate experiment demonstrated that the thickness of hydrogel can be precisely tuned from 0.1 to 3 mm by controlling the height of precursor solution; whereas, the formation of hydrogel is minimal when solution height is below 0.3 mm. This phenomenon may be attributed to the effect of oxygen diffusion into the thin solution layer as oxygen can serve as a soluble electron acceptor to restrict the reduction of GO.

To understand the influence of surface areas in Pd recovery, both cylindrical hydrogels (CH) and rectangular cubical hydrogel (RH) were synthesized from the same amounts (30 mL) of identical PV-4/GO precursor solutions. Consequently, a total of 20 CH and 98 RH can be obtained, of which individual CH hosts a diameter of 3 mm and height of 5 mm (total surface areas of 1225 mm ² ) while individual RH hosts a length/width of 1 mm and height of 5 mm (total surface areas of 2156 mm ² ). Both hydrogels were then applied in Pd recovery by batch-cultured in HEPES with 100 ppm Pd ions added. During Pd recovery, the Pd concentrations were measured periodically using a UV-VIS spectrophotometer and the results were presented in Fig. 6b. As shown in the results, the RH can recover 60% after 24 hours while CH can only achieve 35%. The superior efficiency from RH can be attributed to the high total surface area that promoted the interplay between Pd (electron acceptor), the nutrient (electron donor), and PV-4. In addition, 37% of Pd can be recovered within the first hour by RH; whereas noticeable Pd recovery can only be observed 3 hours after initiating the test using CH. These results suggested that a reduction in the surface area also delays the reaction start-up time. Overall, these results indicated that the solution/hydrogel contact dominated the overall dynamic of Pd recovery, and increasing the surface area can enhance PV-4 metabolism and the reaction rate, which can improve the Pd recovery.

Bioreactor Design, Scale-up, and Interference test

Next, a prototype of the reactor is created based on the principle of a packed bed reactor to demonstrate the potential of these hydrogels in integrating into existing e-waste recycling infrastructures. (Fig. 10-11) This reactor possessed a customized flow dynamic design for advancing the performance of these hydrogels through actively promoting the mass transfer using flows. A two-stage reactor design was applied to maximize the Pd recovery rate.

To start the Pd recovery, 100 rectangular cubical hydrogels with total surface areas of 2200 mm ² were loaded into both reactors and a peristaltic pump was connected with the reactor to circulate the Pd solution with a volume of 100 mL (Fig. 11). The results showed that 66% of Pd were recovered after the first 12-hour treatment; however, the Pd recovery plateaued between 12 to 48 hours. To increase the treatment efficiency, the replacement of the second-stage reactor with an identical design boosted Pd recovery to 87% after another 24 hours of operation, which suggested that the efficiency of hydrogels may be reduced when processing solution with a large amount of metal that hydrogels can be fully covered by reduced metals. (Fig. 11).

Adapting from the design shown in Fig. 11, the scale-up reactor was further investigated and the prototype was presented in Fig. 12. First, the maximum Pd recovery efficiency of a scaled- up hydrogel was characterized in this reactor to determine the packing density of the hydrogels. Specifically, 20 hydrogels (L:W: H = 10 mm: 5 mm: 5 mm) were loaded in the reactor and cultured anaerobically for 24 hours before a 100 mL of 100 ppm Pd contained simulated e-waste leachate was introduced. Fresh Pd- and nutrient- solutions were replenished periodically (i.e., every 12 hours) to ensure the metabolic activity of the bacteria in hydrogels. After a one-day operation, the total reduced Pd was measured using atomic absorption spectrometry (AA) at 11.6 mg; therefore, the Pd reduction capacity of a single hydrogel can be estimated as 0.58 mg/day. Based on this result, around 776 hydrogels are required to achieve a 90% Pd recovery from IL leachate during a 48-hour treatment.

To verify this estimation while investigating the feasibility of scaling up this design, the Pd reduction efficiency of 100 hydrogels was evaluated by continuously circulating a IL simulated e-waste leachate in the hydrogel -loaded packed-bed reactor.(Fig. 12) As shown in the results, the overall treatment efficiency (the dot/solid line) at 48 hours was 12% (= 120 mg), which is agreed with the estimation based on the maximum Pd reduction capacity of signal hydrogel (0.58 (mg/day) x 100 (hydrogels) ^x 2 (days) = 116 mg) (Fig. 13). This result supported the feasibility of the scale-up approach. The Pd recovery rate of this reactor was also characterized (the circle/ dash line), the results showed that the rate decreased significantly between 24-48 hours, which can be considered in optimizing the operation of this reactor., It is believed that both increasing the amount of hydrogel loading (for example, to 200 units) and a parallel operation of, for example, five reactors will be investigated in order to achieve a 90% Pd recovery to meet the industrial expectation.

In addition, a simulated e-waste stream was tested to identify and overcome the potential challenges in the implementation of this technology in the e-waste mining industry; hence facilitating the commercialization of this living filter. Specifically, the simulated leachate stream was prepared by introducing interference ions presented in the fourth leaching step of the industrial hydrometallurgical process of the waste printed circuit boards (Self-Assembled Living Hydrogel for Valuable Metal Recovery from Electronic Waste, AICHE conference, Phoenix, AZ). The interference ions include Fe (419 ppm), Ni (98.8 ppm), Cu (83 ppm), and Zn (74.7 ppm). The pH value of this solution was also adjusted to 1.5 to mimic the e-waste leachate. The AA result indicated that the hydrogels were able to recover 80% (80 ppm) of Pd under this interference and low pH condition, which presented the feasibility of applying this technology in the e-waste mining process. These interference ions can be removed, for example, by commercially available ion exchange resin (e g., Amberlite™ ZRC-748 and Purolite® S930Plus) before the Pd recovery process to eliminate any negative impact these ions may introduce to the efficiency of hydrogels.

Post-treatment of hydrogels

To improve the economic benefit of this technology, a thermal treatment process was implemented. This process was validated using TGA under nitrogen flow of 90 mL/min and a heating rate of 10°C/min to 600°C to carbonize the organic contents. As shown in the results, a notable weight loss of the sample was initiated at a temperature over 200°C which was attributed to the carbonization of the organic contents in the hydrogel (Fig. 14a) while the isothermal step at 600°C cannot induce further weight loss. This observation suggested that the organic content was transformed into inert carbon. Finally, 52% of the weight was retained, which suggested that the Pd content in the nanoPd doped active carbon was around 15% (estimated based on an 8% pure Pd content (measured from oxygen-rich TGA and data not shown)). After TGA, the residual was then characterized using the SEM-EDX (Fig. 14b). Most surfaces of the residual were covered by Pd (>90%), which can potentially benefit the electrochemical -/catalytical- activities of these materials; hence increasing the value of this final product. Overall, the TGA and SEM-EDX studies indicated that the nanoPd doped active carbon material can be successfully obtained after this thermal treatment. The value of nanoPd doped active carbon is estimated at 160 dollars per 1 gram of recovered Pd (based on 10% w/v Pd concentration on carbon), which is double the profit as compared with pure palladium (83.62 dollars/gram), the final product of the most conventional process. The upgrade from pure Pd to nanoPd on active carbon may improve the economic benefit of this technology; hence paving the way for industrial implementation.