Genetic Modification of Acidithiobacillus ferrooxidans for Rare-Earth Element Recovery under Acidic Conditions.

As global demands for rare-earth elements (REEs) continue to grow, the biological recovery of REEs has been explored as a promising strategy, driven by potential economic and environmental benefits. It is known that calcium-binding domains, including helix-loop-helix EF hands and repeats-in-toxin (RTX) domains, can bind lanthanide ions due to their similar ionic radii and coordination preference to calcium. Recently, the lanmodulin protein from Methylorubrum extorquens was reported, which has evolved a high affinity for lanthanide ions over calcium. Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile, which has been explored for use in bioleaching for metal recovery. In this report, A. ferrooxidans was engineered for the recombinant intracellular expression of lanmodulin. In addition, an RTX domain from the adenylate cyclase protein of Bordetella pertussis, which has previously been shown to bind Tb3+, was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb3+, Pr3+, Nd3+, and La3+) was improved by up to 4-fold for cells expressing lanmodulin and 13-fold for cells expressing the RTX domains in both pure and mixed metal solutions. Interestingly, the presence of lanthanides in the growth media enhanced protein expression, likely by influencing protein stability. Both engineered cell lines exhibited higher recoveries and selectivities for four tested lanthanides (Tb3+, Pr3+, Nd3+, and La3+) over non-REEs (Fe2+ and Co2+) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.

Gradient Architecture Design in Scalable Porous Battery Electrodes.

Because it has been demonstrated to be effective toward faster ion diffusion inside the pore space, low-tortuosity porous architecture has become the focus in thick electrode designs, and other possibilities are rarely investigated. To advance current understanding in the structure-affected electrochemistry and to broaden horizons for thick electrode designs, we present a gradient electrode design, where porous channels are vertically aligned with smaller openings on one end and larger openings on the other. With its 3D morphology carefully visualized by Raman mapping, the electrochemical properties between opposite orientations of the gradient electrodes are compared, and faster energy storage kinetics is found in larger openings and more concentrated active material near the separator. As further verified by simulation, this study on gradient electrode design deepens the knowledge of structure-related electrochemistry and brings perspectives in high-energy battery electrode designs.

Tunable Porous Electrode Architectures for Enhanced Li-Ion Storage Kinetics in Thick Electrodes.

Thick electrodes, although promising toward high-energy battery systems, suffer from restricted lithium-ion transport kinetics due to prolonged diffusion lengths and tortuous transport pathways. Despite the emerging low-tortuosity designs, capacity retention under higher current densities is still limited. Herein, we employ a modified ice-templating method to fabricate low-tortuosity porous electrodes with tunable wall thickness and channel width and systematically investigate the critical impacts of the fine structural parameters on the thick electrode electrochemistry. While the porous electrodes with thick walls show diminished capability under a C-rate larger than 1.5 C, those with thinner walls could maintain ∼70% capacity under 2.5 C. The superior capacity retention is ascribed to the fast diffusion into the thin lamellar walls compared with their thicker counterparts. This study provides deeper insights into structure-affected electrochemistry and opens up new perspective of 3D porous architectural designs for high-energy and high-power electrodes.

Glutathione Synthetase Overexpression in Acidithiobacillus ferrooxidans Improves Halotolerance of Iron Oxidation.

Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria. IMPORTANCE The use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits, including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for the use of freshwater resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.

Dispersion of sulfur creates a valuable new growth medium formulation that enables earlier sulfur oxidation in relation to iron oxidation in Acidithiobacillus ferrooxidans cultures.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig-sulfur, can be used to support the rapid antibiotic selection of plasmid-transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig-sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.

Lithium vanadium oxide (Li1.1V3O8) thick porous electrodes with high rate capacity: utilization and evolution upon extended cycling elucidated via operando energy dispersive X-ray diffraction and continuum simulation.

The phase distribution of lithiated LVO in thick (∼500 µm) porous electrodes (TPEs) designed to facilitate both ion and electron transport was determined using synchrotron-based operando energy dispersive X-ray diffraction (EDXRD). Probing 3 positions in the TPE while cycling at a 1C rate revealed a homogeneous phase transition across the thickness of the electrode at the 1st and 95th cycles. Continuum modelling indicated uniform lithiation across the TPE in agreement with the EDXRD results and ascribed decreasing accessible active material to be the cause of loss in delivered capacity between the 1st and 95th cycles. The model was supported by the observation of significant particle fracture by SEM consistent with loss of electrical contact. Overall, the combination of operando EDXRD, continuum modeling, and ex situ measurements enabled a deeper understanding of lithium vanadium oxide transport properties under high rate extended cycling within a thick highly porous electrode architecture.

Simulation assisted design for microneedle manufacturing: Computational modeling of two-photon templated electrodeposition.

Fully metallic micrometer-scale 3D architectures can be fabricated via a hybrid additive methodology combining multi-photon lithography with electrochemical deposition of metals. The methodology - referred to as two-photon templated electrodeposition (2PTE) - has significant design freedom that enables the creation of complicated, traditionally difficult-to-make, high aspect ratio metallic structures such as microneedles. These complicated geometries, combined with their fully metallic nature, can enable precision surgical applications such as inner ear drug delivery or fluid sampling. However, the process involves electrochemical deposition of metals into complicated 3D lithography patterns thicker than 500 µm. This causes potential and chemical gradients to develop within the 3D template, creating limitations to what can be designed. These limitations can be explored, understood, and overcome via numerical modeling. Herein we introduce a numerical model as a design tool that can predict growth for manufacturing complicated 3D metallic geometries. The model is successful in predicting the geometric result of 2PTE, and enables extraction of insights about geometric constraints through exploration of its mechanics.

Enhanced microbial corrosion of stainless steel by Acidithiobacillus ferrooxidans through the manipulation of substrate oxidation and overexpression of rus.

Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to the presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental impacts and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low-grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells that enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to the initial sulfur- and cell-free control experiments containing only pyrite. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to a further 85% increase in the corrosion of the steel in addition to the improvements made by changes to the process conditions. Thus, the modification of the culturing conditions and the use of rus-overexpressing cells led to a more than threefold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This study demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.

Microbially Influenced Corrosion of Stainless Steel by Acidithiobacillus ferrooxidans Supplemented with Pyrite: Importance of Thiosulfate.

Microbially influenced corrosion (MIC) results in significant damage to metallic materials in many industries. Anaerobic sulfate-reducing bacteria (SRB) have been well studied for their involvement in these processes. Highly corrosive environments are also found in pulp and paper processing, where chloride and thiosulfate lead to the corrosion of stainless steels. Acidithiobacillus ferrooxidans is a critically important chemolithotrophic acidophile exploited in metal biomining operations, and there is interest in using A. ferrooxidans cells for emerging processes such as electronic waste recycling. We explored conditions under which A. ferrooxidans could enable the corrosion of stainless steel. Acidic medium with iron, chloride, low sulfate, and pyrite supplementation created an environment where unstable thiosulfate was continuously generated. When combined with the chloride, acid, and iron, the thiosulfate enabled substantial corrosion of stainless steel (SS304) coupons (mass loss, 5.4 ± 1.1 mg/cm2 over 13 days), which is an order of magnitude higher than what has been reported for SRB. There results were verified in an abiotic flow reactor, and the importance of mixing was also demonstrated. Overall, these results indicate that A. ferrooxidans and related pyrite-oxidizing bacteria could produce aggressive MIC conditions in certain environmental milieus.IMPORTANCE MIC of industrial equipment, gas pipelines, and military material leads to billions of dollars in damage annually. Thus, there is a clear need to better understand MIC processes and chemistries as efforts are made to ameliorate these effects. Additionally, A. ferrooxidans is a valuable acidophile with high metal tolerance which can continuously generate ferric iron, making it critical to copper and other biomining operations as well as a potential biocatalyst for electronic waste recycling. New MIC mechanisms may expand the utility of these cells in future metal resource recovery operations.

Equilibria and Rate Phenomena from Atomistic to Mesoscale: Simulation Studies of Magnetite.

Batteries are dynamic devices composed of multiple components that operate far from equilibrium and may operate under extreme stress and varying loads. Studies of isolated battery components are valuable to the fundamental understanding of the physical processes occurring within each constituent element. When the components are integrated into a full device and operated under realistic conditions, it can be difficult to decouple the physical processes that occur across multiple interfaces and multiple length scales. Thus, the physical processes studied in isolated components may change in a full battery setup or may be irrelevant to performance. Simulation studies on many length scales play a key role in the analysis of experiments and in the elucidation of the relevant physical processes impacting performance. In this Account, we aim to highlight the use of modeling on multiple length scales to identify rate limiting phenomena in lithium-ion batteries. To illustrate the utility of modeling, we examine lithium-ion batteries with nanostructured magnetite, Fe3O4, as the positive electrode active material against a solid Li0 negative electrode. Due to continuous operation away from equilibrium, batteries exhibit highly nonideal behavior, and a model that aims to reproduce behavior under realistic operating conditions must be able to capture the physics occurring on the length scales relevant to the performance of the system. It is our experience that limiting behavior in lithium-ion batteries can be observed on the atomic scale and up through the electrode scale and thus, predictive models must be capable of integrating and communicating physics across multiple length scales. Magnetite is studied as an electrode material for lithium-ion batteries, but it is found to suffer from slow solid-state transport of lithium, slow reaction kinetics, and poor cycling. Magnetite (Fe3O4) is a material capable of undergoing multiple electron transfers (MET), and can accept up to eight lithium per formula unit (Li8Fe3O4). Magnetite, (Fe8a3+)[Fe3+Fe2+]16dO4,32e2-, has a close-packed inverse spinel structure and undergoes intercalation, structural rearrangement, and conversion reactions upon full lithiation. (1) To overcome solid-state transport resistances, magnetite can be nanostructured to decrease Li+ diffusion lengths, and this has been shown to increase capacity. Additionally, unique architectures incorporating both carbon and Fe3O4 have shown to alleviate transport and cycling issues in the material. (2) Here, we solely address traditional composite electrodes, in which Fe3O4 is synthesized as nanoparticles and combined with additives to fabricate the electrode. In the case of nanoparticulate magnetite, it has been found that the electrode fabrication process results in the formation of micrometer-sized agglomerates of the Fe3O4 nanoparticles, introducing a secondary structural motif. The agglomerates may form in one or more fabrication processes, and their elimination may not be straightforward or warranted. Here, we highlight the impact of these secondary formations on the performance of the Fe3O4 lithium-ion battery. We illustrate how simulations can be used to design experiments, prioritize research efforts, and predict performance.

Size dependent behavior of Fe3O4 crystals during electrochemical (de)lithiation: an in situ X-ray diffraction, ex situ X-ray absorption spectroscopy, transmission electron microscopy and theoretical investigation.

The iron oxide magnetite, Fe3O4, is a promising conversion type lithium ion battery anode material due to its high natural abundance, low cost and high theoretical capacity. While the close packing of ions in the inverse spinel structure of Fe3O4 enables high energy density, it also limits the kinetics of lithium ion diffusion in the material. Nanosizing of Fe3O4 to reduce the diffusion path length is an effective strategy for overcoming this issue and results in improved rate capability. However, the impact of nanosizing on the multiple structural transformations that occur during the electrochemical (de)lithiation reaction in Fe3O4 is poorly understood. In this study, the influence of crystallite size on the lithiation-conversion mechanisms in Fe3O4 is investigated using complementary X-ray techniques along with transmission electron microscopy (TEM) and continuum level simulations on electrodes of two different Fe3O4 crystallite sizes. In situ X-ray diffraction (XRD) measurements were utilized to track the changes to the crystalline phases during (de)lithiation. X-ray absorption spectroscopy (XAS) measurements at multiple points during the (de)lithiation processes provided local electronic and atomic structural information. Tracking the crystalline and nanocrystalline phases during the first (de)lithiation provides experimental evidence that (1) the lithiation mechanism is non-uniform and dependent on crystallite size, where increased Li+ diffusion length in larger crystals results in conversion to Fe0 metal while insertion of Li+ into spinel-Fe3O4 is still occurring, and (2) the disorder and size of the Fe metal domains formed when either material is fully lithiated impacts the homogeneity of the FeO phase formed during the subsequent delithiation.

Characterization of endogenous promoters for control of recombinant gene expression in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an important iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is used extensively in metal extraction and refining, and more recently in the bioproduction of chemicals. However, a lack of genetic tools has limited the further development of this organism for industrial bioprocesses. Using prior microarray studies that identified genes, which may express differentially in response to the availability of iron and sulfur, the cycA1 and tusA promoter sequences have been characterized for their ability to drive green fluorescent protein expression. The promoters exhibited opposite control behavior, where the cycA1 sequence was repressed and the tusA promoter was induced by the presence of sulfur in the growth medium. Sulfur was found to be the dominant signal. The sulfur IC50 for cycA1 was 0.56 mM (18 mg/L), whereas the sulfur EC50 of tusA was 2.5 mM (80 mg/L). Together these sequences provide two new tools to selectively induce or repress gene expression in A. ferrooxidans. Acidithiobacillus ferrooxidans is an important industrial organism; however, genetic tools for control of gene expression do not exist. Here, we report the identification of promoter sequences that allow for the development of control of gene expression for engineering this organism.

Enhancing isobutyric acid production from engineered Acidithiobacillus ferrooxidans cells via media optimization.

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has previously been genetically modified to produce isobutyric acid (IBA) from carbon dioxide while obtaining energy from the oxidation of ferrous iron. Here, a combinatorial approach was used to explore the influence of medium composition in both batch and chemostat cultures in order to improve IBA yields (g IBA/mol Fe(2+)) and productivities (g IBA/L/d). Medium pH, ferrous concentration (Fe(2+)), and inclusion of iron chelators all had positive impact on the IBA yield. In batch experiments, gluconate was found to be a superior iron chelator because its use resulted in smaller excursions in pH. In batch cultures, IBA yields decreased linearly with increases in the final effective Fe(3+) concentrations. Chemostat cultures followed similar trends as observed in batch cultures. Specific cellular productivities were found to be a function of the steady state ORP (Oxidation-reduction potential) of the growth medium, which is primarily determined by the Fe(3+) to Fe(2+) ratio. By operating at low ORP, chemostat cultures were able to achieve volumetric productivities as high as 3.8 ± 0.2 mg IBA/L/d which is a 14-fold increase over the previously reported value.

Engineering the iron-oxidizing chemolithoautotroph Acidithiobacillus ferrooxidans for biochemical production.

There is growing interest in developing non-photosynthetic routes for the conversion of CO2 to fuels and chemicals. One underexplored approach is the transfer of energy to the metabolism of genetically modified chemolithoautotrophic bacteria. Acidithiobacillus ferrooxidans is an obligate chemolithoautotroph that derives its metabolic energy from the oxidation of iron or sulfur at low pH. Two heterologous biosynthetic pathways have been expressed in A. ferrooxidans to produce either isobutyric acid or heptadecane from CO2 and the oxidation of Fe(2+). A sevenfold improvement in productivity of isobutyric acid was obtained through improved media formulations in batch cultures. Steady-state efficiencies were lower in continuous cultures, likely due to ferric inhibition. If coupled to solar panels, the photon-to-fuel efficiency of this proof-of-principle process approaches estimates for agriculture-derived biofuels. These efforts lay the foundation for the utilization of this organism in the exploitation of electrical energy for biochemical synthesis.

Addition of citrate to Acidithiobacillus ferrooxidans cultures enables precipitate-free growth at elevated pH and reduces ferric inhibition.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is important in biomining and other biotechnological operations. The cells are able to oxidize inorganic iron, but the insolubility and product inhibition by Fe(3+) complicates characterization of these cultures. Here we explore the growth kinetics of A. ferrooxidans in iron-based medium in a pH range from 1.6 to 2.2. It was found that as the pH was increased from 1.6 to 2.0, the maintenance coefficient decreased while both the growth kinetics and maximum cell yield increased in the precipitate-free, low Fe(2+) concentration medium. In higher iron media a similar trend was observed at low pH, but the formation of precipitates at higher pH (2.0) hampered cell growth and lowered the specific growth rate and maximum cell yield. In order to eliminate ferric precipitates, chelating agents were introduced into the medium. Citric acid was found to be relatively non-toxic and did not appear to interfere with iron oxidation at a maximum concentration of 70 mM. Inclusion of citric acid prevented precipitation and A. ferrooxidans growth parameters resumed their trends as a function of pH. The addition of citrate also decreased the apparent substrate saturation constant (KS ) indicating a reduction in the competitive inhibition of growth by ferric ions. These results indicate that continuous cultures of A. ferrooxidans in the presence of citrate at elevated pH will enable enhanced cell yields and productivities. This will be critical as these cells are used in the development of new biotechnological applications such as electrofuel production.

Overexpression of quorum sensing genes in Acidithiobacillus ferrooxidans enhances cell attachment and covellite bioleaching.

Cell adhesion is generally a prerequisite to the microbial bioleaching of sulfide minerals, and surface biofilm formation is modulated via quorum sensing (QS) communication. We explored the impact of the overexpression of endogenous QS machinery on the covellite bioleaching capabilities of Acidithiobacillus ferrooxidans, a representative acidophilic chemolithoautotrophic bacterium. Cells were engineered to overexpress the endogenous qs-I operon or just the afeI gene under control of the tac promoter. Both strains exhibited increased transcriptional gene expression of afeI and improved cell adhesion to covellite, including increased production of extracellular polymeric substances and increased biofilm formation. Under low iron conditions, the improved bioleaching of covellite was more evident when afeI was overexpressed alone as compared to the native operon. These observations demonstrate the potential for the genetic modulation of QS as a mechanism for increasing the bioleaching efficiency of covellite, and potentially other copper sulfide minerals.

The Reductive Leaching of Chalcopyrite by Chromium(II) Chloride for the Rapid and Complete Extraction of Copper.

A hydrometallurgical process is developed to lower the costs of copper production and thereby sustain the use of copper throughout the global transition to renewable energy technologies. The unique feature of the hydrometallurgical process is the reductive treatment of chalcopyrite, which is in contrast to the oxidative treatment more commonly pursued in the literature. Chalcopyrite reduction by chromium(II) ion is described for the first time and superior kinetics are shown. At high concentrate loadings of 39, 78, and 117 g L-1 , chalcopyrite reacted completely within minutes at room temperature and pressure. The XRD, SEM-EDS, and XPS measurements indicate that chalcopyrite reacts to form copper(I) chloride (CuCl). After the reductive treatment, the mineral products are leached by iron(III) sulfate to demonstrate the complete extraction of copper. The chromium(II) ion may be regenerated by an electrolysis unit inspired by an iron chromium flow battery in a practical industrial process.

Engineering Polyhistidine Tags on Surface Proteins of Acidithiobacillus ferrooxidans: Impact of Localization on the Binding and Recovery of Divalent Metal Cations.

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies.Acidithiobacillus ferrooxidansis an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans. The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.

Chromatography of bis-(3-sulfopropyl) disulfide and its breakdown products by HPLC coupled with electrochemical detection.

A chromatographic method for the detection of bis-(3-sulfopropyl) disulfide (SPS), a common additive in acidic copper plating baths, and its breakdown products is demonstrated. The detection scheme involves a combination of solid-phase extraction for sample pre-treatment, C(18) reversed-phase high-performance liquid chromatography column for separation, and electrochemical sensor for detection of all non-fully oxidized sulfur-containing compounds. We were able to achieve an effective separation and accurately assign chromatographic peaks to all detectable species. Owing to a high sensitivity of the utilized electrochemical detector, detection in low parts per billion range was possible. This can prove crucial for plating bath control, since minute amounts of certain by-products significantly affect the bath performance.

Drug delivery device for the inner ear: ultra-sharp fully metallic microneedles.

Drug delivery into the inner ear is a significant challenge due to its inaccessibility as a fluid-filled cavity within the temporal bone of the skull. The round window membrane (RWM) is the only delivery portal from the middle ear to the inner ear that does not require perforation of bone. Recent advances in microneedle fabrication enable the RWM to be perforated safely with polymeric microneedles as a means to enhance the rate of drug delivery from the middle ear to the inner ear. However, the polymeric material is not biocompatible and also lacks the strength of other materials. Herein we describe the design and development of gold-coated metallic microneedles suitable for RWM perforation. When developing microneedle technology for drug delivery, we considered three important general attributes: (1) high strength and ductility material, (2) high accuracy and precision of fabrication, and (3) broad design freedom. We developed a hybrid additive manufacturing method using two-photon lithography and electrochemical deposition to fabricate ultra-sharp gold-coated copper microneedles with these attributes. We refer to the microneedle fabrication methodology as two-photon templated electrodeposition (2PTE). We demonstrate the use of these microneedles by inducing a perforation with a minimal degree of trauma in a guinea pig RWM while the microneedle itself remains undamaged. Thus, this microneedle has the potential literally of opening the RWM for enhanced drug delivery into the inner ear. Finally, the 2PTE methodology can be applied to many different classes of microneedles for other drug delivery purposes as well the fabrication of small scale structures and devices for non-medical applications. Graphical Abstract Fully metallic ultra-sharp microneedle mounted at end of a 24-gauge stainless steel blunt syringe needle tip: (left) Size of microneedle shown relative to date stamp on U.S. one-cent coin; (right) Perforation through guinea pig round window membrane introduced with microneedle.