[001] Introduction

[002] Global demand for rare earth elements (REEs) is at an all-time high and is steadily increasing. REEs, composed of the lanthanides (Lns), and scandium, and yttrium, are critical metals for modem clean energy, communication, advanced transportation, and consumer technologies [1,6,24]. China maintains its status as the world’s foremost producer of REEs, generating heavy reliance on a single market that can easily jeopardize REE supply and compromise national security. Simultaneously, a growing understanding of the disastrous environmental impacts of REE mining and production has led to decreased output of REEs by China [13], and an expansion of new mining and production operations worldwide. The development of technologies for domestic REE reuse and recycling has garnered interest as a means of moving towards independence from foreign importation.

[003] Microbiological REE leaching and extraction methods offer promising alternatives to current state-of-the-art methods (hydrometallurgical, pyrometallurgical, and electrometallurgical approaches) that produce large quantities of sludge, acidic wastewater, atmospheric pollution, and radioactive tailings [7,8,13,22]. In particular, microbial bioaccumulation and biomineralization is cost effective and highly efficient for dilute, low-grade, and potentially dangerous REE waste streams [3,18,19]. Bioaccumulation and biomineralization of REEs in bacteria was first shown for the model methylotroph Methylobacterium (also known as Methylorubrum) extorquens AMI [14]. Methylotrophic bacteria, organisms that thrive on one- carbon compounds such as methane and methanol, provide an attractive new approach for REE bioleaching and extraction due to their natural ability to acquire Ln from the surrounding environment [9,17]. This includes soluble and insoluble low-grade REE sources such as electronic waste (E-waste). Mesophilic methylotrophs have dedicated systems for acquisition, uptake, and intracellular storage of Lns as polyphosphate granules in lanthasome compartments, making them effective agents of bioleaching and bioaccumulation [14] without the need for high acidity or temperature. REE use by mesophilic methylotrophs was thought to be restricted to the light Ln, but recently, a genetic variant of M. extorquens AMI was isolated and characterized that can transport, store, and grow using the heavy Ln, gadolinium [5]. Detailed genetic and Ln uptake studies indicate the likely possibility of an additional system dedicated to heavy Lns. Thus, methylotrophs may already possess the biological means to separate light and heavy Lns, and have the potential to be engineered for uptake of specific Ln species from mixed, low-grade feedstocks. Further, developing strains resistant to metal toxicity will be vitally important in order to recover REEs from complex, low-grade sources like E-waste. E-waste contains many heavy metals that can generate toxicity, including cadmium, cobalt, manganese, mercury, lead, tungsten, and tellurium.

[004] Summary of the Invention

[005] The invention provides a bacterial platform that efficiently and selectively bioaccumulates and recovers rare earths elements (REEs). The invention provides optimization of growth medium conditions for leaching and strain engineering (e.g., generation of genetic variants) to enhance REE bioaccumulation using Methylorubrum/Methylobacterium extorquens, particularly but not exclusively strain AMI. REEs include scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium. Growth medium amended with REEs, including neodymium magnet swarf (NdFeB magnet), REE salts, REE oxides, REE ores (i.e. monazite, bastnasite), REE crystals, and electronic waste (cell phone, computer hard drive, speaker, etc.). The invention provides high-yield microbial growth and bioaccumulation with greater than 10% REE source pulp density. The invention provides bioleaching enhancement mediated by REE-bioligand production. The invention provides bioaccumulation enhancement by manipulation of pathways that are independent of carbon metabolism, including phosphate metabolism and REE deregulation of transport. The invention provides construction of a strain of M. extorquens AMI that is resistant to > 5% electronic waste. The invention provides bioaccumulation of REEs in mineral form by M. extorquens AMI. Recovery of pure REE after filtration, cell breakage and organic precipitation.

[006] In an aspect the invention provides a microbial platform for rare earth element (REE) bioaccumulation comprising: a culture of M. extorquens AMI and a medium comprising: methanol atl0-200 mM, inorganic phosphate at 3-10 mM, and a magnet swarf pulp at 1-10% (w/v) and comprising the REE, wherein the platform has a selective, at least a 2-fold bioconcentration of the REE in the culture.

[007] In embodiments:

[008] the methanol is at 0.01-1%, or about 50 mM.

[009] the inorganic phosphate is at 3.6-7.3 mM, or about 7 mM.

[010] the swarf pulp is 1-5% (w/v), or about 1% (w/v).

[Oil] the platform has a selective, at least a 3.6-fold bioconcentration of the REE in the culture.

[012] the culture is engineered to overexpress LCC (lanthanide chelation cluster, e.g. META1_4129 through META1_4138) in trans, particularly wherein the platform provides increased Nd bioaccumulation during growth with the insoluble sources Nd2Oa (such as 2.3- fold) and magnet swarf (such as 3.2-fold), particularly wherein NdFeB magnet swarf also contains significant amounts of the light Ln Pr and the heavy Ln Dy, and bioaccumulation of these Lns is increased (e.g. on average by 3.5-fold) with expression of the LCC in trans.

[013] the swarf comprises a low-grade REE source, that is ores monazite or bastnasite, or bastnasite crystal.

[014] the swarf comprises toxic electronic waste, that is post-consumer electronic waste, like smart phone E-waste, comprising iron, boron, copper, tellurium, manganese, mercury, lead, tungsten, lithium, neodymium, nickel, boron and/or cobalt.

[015] the culture achieves growth with 5% blended cellular phone pulp density.

[016] the M. extorquens AMI comprises a disrupted oxalate/formate antiporter, oxlT (META1_2757), particularly wherein the oxlT is disrupted by genetic change which disrupts function or expression of the antiporter, wherein the change maybe a natural mutation, or edit, e.g. cre-lox or CRISPR, particularly wherein addition of an effective amount of formate (preferably titrated for optimization, e.g. 2 mM) is used to increase E-waste resistance of the oxlT mutant.

[017] the M. extorquens AMI comprises a strain having reduced exopolyphosphatase activity to enhance REE bioaccumulation, particularly wherein the strain is selected for reduced exopolyphosphatase activity or comprises a functional deletion or disruption of a ppx gene encoding exopolyphosphatase.

[018] In aspects, the invention provides:

[019] A method for bioaccumulation of REEs comprising operating a platform herein.

[020] A method for bioaccumulation of REEs comprising operating a platform herein, wherein the same swarf batch is sequentially processed for further REE extraction in subsequent bioreactor runs.

[021] A method for bioaccumulation of REEs comprising operating a platform herein wherein the swarf batch is processed in a continuous culture setup to maximize REE extraction from a swarf batch.

[022] A method for bioaccumulation of REEs comprising operating a platform herein wherein bioaccumulation of REE from complex waste streams is effected, including high-grade sources (REE ores, REE oxides, electronic waste) and low-grade sources including REE waste, REE- contaminated waste water and medical waste.

[023] A method for bioaccumulation of REEs comprising operating a platform herein, wherein accumulated REE are purified for commercial or industrial-scale use/reuse in products.

[024] A method for bioaccumulation of REEs comprising operating a platform herein, wherein bioremediation of REE-contaminated water and waste streams is effected. [025] A method for bioaccumulation of REEs comprising operating a platform herein, wherein accumulated REE are purified for development of lanthanide (sensors)

[026] A method for bioaccumulation of REEs comprising operating a platform herein, for production of biofertilizers, or other agents promoting plant growth.

[027] In aspect, the invention provides a system, platform composition or method as described herein.

[028] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[029] Brief Description of the Drawings

[030] Fig. 1. M. extorquens AMI as a platform for Nd recovery through efficient, high- yield bioaccumulation of Nd from complex sources. M. extorquens AMI is incorporated in a microbial platform for REE recovery due to its innate ability to acquire, transport, and accumulate REE metals into polyphosphate granules.

[031] Fig. 2A-2B. Impact of phosphate and magnet swarf pulp density on strain performance. A, Growth rate of M. extorquens AMf increases with decreasing inorganic phosphate in the medium. Wild type M. extorquens AMI was grown in a microplate in Hypho minimal medium with 50 mM methanol and 2 pM NdCE. Blue, 14.5 mM phosphate (Pi). Orange, 7.3 mM phosphate /i Pi). Gray, 3.6 mM phosphate (% Pi). Box plots represent the interquartile ranges for 6 biological replicates; standard deviation is shown by error bars B, Yield of M. extorquens AMI grown on methanol with Nd is influenced by inorganic phosphate in the medium. Box plots represent the averages of 3 biological replicates with interquartile ranges. Error bars are standard deviations.

[032] Fig. 3A-3C. Bioreactor performance with NdFeB magnet swarf. M. extorquens AMI was grown in 0.75 L bioreactors with reduced phosphate Hypho medium containing 50 mM methanol and Nd magnet swarf. A, growth with 1% swarf. B, growth with 10% swarf pulp density. C, Methanol fed-batch bioreactor cultivation of M. extorquens AMI with 1% swarf pulp density to obtain higher cell density. Individual data points represent two biological replicates with less than 10% variation.

[033] Fig. 4. Selectivity for REE. M. extorquens AMI was grown in methanol medium in a benchtop bioreactor with 1% Nd magnet swarf. Cell and supernatant samples were taken once the culture reached early stationary growth phase. Metal concentrations in samples were determined by ICP-MS and normalized to the total metal uptake.

[034] Fig. 5. Strain engineering for increased REE bioaccumulation. Bioaccumulation of REE by M. extorquens AMf is enhanced by the LCC cluster when using magnet swarf. [035] Fig. 6. Magnet swarf can be reused for increased REE recovery. Nd bioaccumulation is equally efficient in two consecutive bioreactor process cycles using the same magnet swarf batch (1% pulp density). Plots show the mean of two distinct experiments, each using a single swarf batch for both process runs. Error bars indicate standard deviations.

[036] Fig. 7A-7D. Flexibility for M. extorquens AMI as a REE recovery platform from low-grade sources. M. extorquens AMI was grown in methanol medium with and without complex REE sources. A, monazite ore; B, bastnasite ore; C, bastnasite crystal. Images are of actual specimens used in growth studies. Ores shown are prior to pulverization. 2 p M LaCE was included as a positive REE source control. Cells were grown in 25 x 150 mM tubes in a 10 mL culture volume. Aliquots of 200 pL were removed at times shown and the OD 600 was read in a Spectramax M2 plate reader. Background from a no cell control was subtracted from the measurements. D, Growth rates for M. extorquens AMI with diverse low-grade REE sources. N.G., no growth.

[037] Fig. 8A-F. Toxicity of electronic waste is reduced by oxlTl deletion. Blended smartphone is a usable Ln source for methanol growth by M. extorquens AMI . A, M. extorquens AMI was grown in methanol medium with no Lns (open circles), 2 pM LaCE (black squares), or 0.5% (w/v) blended phone with battery (blue triangles). Growth shown as colony forming units per mL of culture. B, increasing blended smartphone to 5% pulp density (w/v) is toxic to M. extorquens AML M. extorquens AMI was unable to grow in methanol medium with smartphone as the only Ln source (blue triangles). Even with the addition of soluble La, 5% smartphone decreased cell viability (black squares). M. extorquens AMI cell counts did not change significantly over 36 hours when the smartphone was not added (white circles). C, E- waste resistant colonies (EWr for “E-waste resistant M. extorquens AMI”) were isolated by plating M. extorquens AMI strains on solid methanol medium with 1 % blended smartphone. D, The causative mutation for E-waste resistance was identified by genomic sequencing of EWr isolates as a deletion in oxlT. This mutation was reconstructed and allowed methanol growth with 5% smartphone. E, Addition of 2 mM oxalate increased sensitivity to E-waste in the oxlT mutant. F, Addition of 2 mM formate increased E-waste resistance of the oxlT mutant which indicates that media optimization can enhance E-waste resistance further.

[038] Description of Particular Embodiments of the Invention

[039] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes. [040] Examples

[041] M. extorquens AMI uses the inexpensive single-carbon feedstock methanol, has the innate ability to selectively extract insoluble REEs from the environment by excreting REE- binding ligands (called lanthanophores), and transports them into the cell for use in metabolism and storage in mineral form. We report optimization of growth conditions for bioaccumulation of REEs using M. extorquens AMI and NdFeB magnet swarf as an E- waste analog. M. extorquens AMI can efficiently acquire and bioaccumulate REEs from other low-grade sources including REE-rich ores and E-waste. In addition, we demonstrate enhanced REE bioaccumulation by genetically engineering M. extorquens AMI to overproduce a lanthanophore. Finally, we report a mutation that enables M. extorquens AMI to be resistant to high pulp densities when using E-waste that bypasses an important bottleneck when scaling up the process.

[042] In these examples we demonstrate an optimized method for REE uptake from neodymium magnet swarf (NdFeB) by M. extorquens AMI. We investigated the impacts of methanol substrate concentration, magnet swarf pulp density, and medium composition on Nd bioaccumulation. By optimizing these parameters we have achieved bioaccumulation of 11.5 mg Nd per gram of cell dry weight, or 1.15% dry weight, using up to 10% swarf pulp density. These data demonstrate of the economic viability using M. extorquens AMI as a platform for biomining and bioaccumulation of REEs from complex feedstocks such as magnet swarf (Fig. 1). In addition, we report efficient growth of M. extorquens AMI with unprocessed bastnasite and monazite REE ores, expanding the use of the REE recovery platform to unrefined sources. Finally, we also report the isolation of an E-waste resistant strain of M. extorquens AMI capable of growth with 5% blended cellular phone pulp density, showing adaptability of the platform for industrial scale applications.

[043] Optimization of phosphate in growth medium increases growth performance

[044] M. extorquens AMI biomineralizes REEs as intracellular phosphate granules [14]. Inorganic phosphate is an essential component of defined bacterial growth media, and REE phosphates are poorly soluble. Inorganic phosphate in the growth medium could negatively impact REE solubility and uptake through the formation of REE phosphates, which could hinder REE-dependent methanol growth. To assess the impact of inorganic phosphate on strain performance, we measured the growth rate of M. extorquens AMI cultured in 48-well microplates in Hypho minimal medium [2] with the standard amount of phosphate (14.5 mM) and compared it to the growth rates of cultures with half the standard amount (7.3 mM) and a quarter of the standard amount (3.6 mM). Growth rate and phosphate concentration exhibited an inverse relationship. The largest relative increase in growth rate (~2-fold) was observed when inorganic phosphate was decreased to 7.3 mM (Figure 2A). Growth rate increased an additional -0.2-fold when phosphate was reduced to 3.6 mM (Figure 2A). Reducing phosphate to 7.3 mM resulted in a culture density that was nearly 2-fold higher than culture with the standard phosphate (Figure 2B). Decreasing phosphate to a quarter of the standard amount increased yield as well, but only by 0.4-fold overall (Figure 2B).

[045] Bioreactor performance with optimized growth medium

[046] M. extorquens AMI was grown in reduced phosphate (7.3 mM) Hypho medium in a 0.75 L benchtop bioreactor at 30 °C with 50 mM methanol and either 1% or 10% magnet swarf. In the bioreactor, cultures reached maximum densities in -40 h (Table 1), representing a 34-50% reduction in growth cycle relative to culturing in shake flasks. Growth rates were significantly increased compared to shake flask cultures: 54% increase with 1% swarf pulp (Figure 3 A) and 26% increase with 10% swarf pulp (Figure 3B) (growth in flasks not shown). Growth with 1 % pulp density produced the highest growth rate and exhibited the shortest cycle time. For these reasons we chose 1% pulp density to investigate bioaccumulation of Nd by M. extorquens AMI. [047] Our previous work indicated that M. extorquens AMI accumulated the highest proportion of Lns during stationary phase [14]. Baseline bioaccumulation of cells in stationary phase after growth in the bioreactor with 1% magnet swarf and 50 mM methanol was determined by ICP-MS (Table 1). Bioreactor cultures were grown using reduced phosphate Hypho medium, cell and supernatant samples were collected during exponential, early stationary, and late stationary growth phases, and samples were analyzed for Nd content by ICP- MS. Relative to baseline bioaccumulation levels, bioreactor-grown M. extorquens AMI accumulated more Nd during each phase of growth. The highest bioaccumulation occurred at early stationary phase (-26 h), accounting for a nearly 400% increase over baseline and 26% above exponential growth phase samples. Bioaccumulation, normalized to dry weight, decreased by 38% into late stationary phase (50 h), though the culture density only decreased slightly (Table 1).

[048] After successful culturing and Nd bioaccumulation in the methanol-batch bioreactor, we tested M. extorquens AMI in methanol fed-batch conditions in the bioreactor to scale up culture density and total Nd yield. A culture density of 20 OD was achieved with a reduction in growth rate, corresponding to a -50 hr cycle time (Figure 3C).

[049] Efficient acquisition and bioaccumulation of REE from low-grade sources [050] Swarf composition was determined by ICP-MS to be 68.0% Fe, 26.7% Nd, 4.35% Pr, and 3.34% Dy. Fe is the primary metal component of Nd magnet swarf rendering non-selective leaching and uptake mechanisms insufficient for effective REE bioaccumulation. Metal selectivity is crucial for the development of a successful REE recovery platform. Batch bioreactor cultures with 1 % magnet swarf were grown maximum culture density with 50 mM methanol (1.8 OD), metal content in cells and supernatant were determined by ICP-MS, and normalized to the total amount of metal accumulated. Of the swarf metal contained in M. extorquens AMI cells, 96.8% was Nd, and Fe accumulation accounted for only 2.0% of the cell metal content (Fig. 4). Given that cells normally acquire Fe as a micronutrient from the growth medium, the amount of Fe obtained from the magnet swarf is likely much lower. Pr and Dy accounted for 1% combined of metal accumulation (Fig. 4). As REE is leached from the swarf for uptake, we also measured metal content in the culture supernatant. Supernatant metal content consisted of mostly Fe (95.9%), with little REE remaining (Nd, 3.6%; Pr, < 1%; Dy, < 1%) (Fig. 4). Overall, these data show highly selective 3.6-fold bioconcentration of REE from magnet swarf by M. extorquens AM 1 .

[051] The identification of the biosynthetic pathway for a REE-chelator has been previously reported (23) and was named LCC (lanthanide chelation cluster), according to the function of its predicted product. Expression of the LCC in trans significantly increased Nd bioaccumulation during growth with the insoluble sources Nd2C>3 (2.3-fold) and magnet swarf (3.2-fold). NdFeB magnet swarf also contains significant amounts of the light Ln Pr and the heavy Ln Dy, both of which have high technological value. Bioaccumulation of these Lns was increased on average by 3.5 -fold with expression of the LCC in trans.

[052] Current extraction yields based on cell density do not extract 100% of the REE present in the magnet swarf. Therefore, we tested if a single magnet swarf batch could be processed for further REE extraction in subsequent bioreactor runs. We confirmed that similar REE recover)' yields are obtained from first and second process runs using the same swarf batch. These results open the possibility for implementation of our technology in a continuous culture setup to maximize REE extraction from a swarf batch.

[053] Due to the successful, promising growth and bioaccumulation of Nd from magnet swarf, we tested the ability of M. extorquens AMI to grow with low-grade REE sources with very low solubility in the growth medium. Monazite ore, for example, contains up to -45% REEs, with Ce2O3 comprising as much as 17% of the total REEs. However, the REEs in monazite are insoluble as oxide compounds. When challenged to grow on methanol with only the ores monazite or bastnasite, or bastnasite crystal, M. extorquens AMI grew as well as with soluble LaCF (Fig. 7ABC). With each of the REE sources used, the insolubility of the metals and complexity of the source did not significantly impact growth rate or growth yield of the culture (Fig. 7D) showing the flexibility and robustness of M. extorquens AMI as a biological platform for the bioaccumulation of REE from complex sources.

[054] Generation of a strain resistant to toxic electronic waste. Cellular smartphones contain several species of REE, including yttrium, lanthanum, terbium, neodymium, gadolinium and praseodymium, making smartphone E- waste a valuable, untapped source for REE recovery. However, smartphone E-waste poses two major challenges. First, the metals in this E-waste are highly insoluble in oxide form. Further, smartphone batteries contain other metals (e.g. iron, boron, copper, tellurium, manganese, mercury, lead, tungsten, lithium, cobalt), all of which can be toxic. We first tested the feasibility of using smartphone E-waste as a REE source by assessing the capacity of M. extorquens AMI to grow on methanol. With 0.5% pulp density (w/v) of blended smartphone, M. extorquens AMI grew as well as with soluble LaCF (Fig. 8 A). Next, we assessed the toxicity of smartphone E-waste and saw that increasing pulp density to 5% was enough to eliminate growth even if soluble La was provided as an additional REE source (Fig. 8B). Using 1 % pulp density, we were able to isolate colonies that had grown on solid methanol medium, indicating the cells had acquired E-waste resistance (Fig. 8C). The genomes of the E-waste resistant strains were sequenced, and we identified a deletion in the oxalate/formate antiporter, oxlT (META1_2757), as a mutation consistent in all resistant strains. The oxlT deletion was reconstructed, and strains containing this deletion demonstrated E-waste resistance, showing it was the causative mutation (Fig. 8D).

[055] As oxalate/formate antiporters simultaneously import oxalate and excrete fomate, we investigated the effect of adding exogenous formate and oxalate to the growth medium. 2 mM oxalate increased E-waste sensitivity of the oxlT mutant (Fig. 8E) while 2 mM formate further increased resistance (Fig. 8F). These data indicate media optimization can further enhance E- waste resistance.

[056] REEs are stored in intracellular polyphosphate granules in M. extorquens AMI. Depolymerization of polyphosphate is catalyzed by exopolyphosphatase activity. We hypothesized that limiting the cellular capacity for polyphosphate depolymerization could generate higher levels of REE bioaccumulation. A ppx (encoding exopolyphosphatase) deletion strain was generated and assessed for REE bioaccumulation, showing a ~5.5-fold increase in Nd levels reaching 202 mg Nd/g DW.

[057] Table 1 Bioleaching and bioaccumulation of Nd by M. extorquens AMI during bioreactor cultivation. Nd content of cell and supernatant samples were measured by ICP-MS. Fold increase is relative to the initial bioaccumulation measured for M. extorquens AMI before optimization of the growth medium with reduced phosphate . Growth phase refers to exponential growth phase (23-29 h cultivation), early stationary phase (26-32 h cultivation), and late stationary phase (50 h cultivation).

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