Microbial biominers: Sequential bioleaching and biouptake of metals from electronic scraps

Abstract Electronic scraps (e‐scraps) represent an attractive raw material to mine demanded metals, as well as rare earth elements (REEs). A sequential microbial‐mediated process developed in two steps was examined to recover multiple elements. First, we made use of an acidophilic bacteria consortium, mainly composed of Acidiphilium multivorum and Leptospidillum ferriphilum, isolated from acid mine drainages. The consortium was inoculated in a dissolution of e‐scraps powder and cultured for 15 days. Forty‐five elements were analyzed in the liquid phase over time, including silver, gold, and 15 REEs. The bioleaching efficiencies of the consortium were >99% for Cu, Co, Al, and Zn, 53% for Cd, and around 10% for Cr and Li on Day 7. The second step consisted of a microalgae‐mediated uptake from e‐scraps leachate. The strains used were two acidophilic extremotolerant microalgae, Euglena sp. (EugVP) and Chlamydomonas sp. (ChlSG) strains, isolated from the same extreme environment. Up to 7.3, 4.1, 1.3, and 0.7 µg by wet biomass (WB) of Zn, Al, Cu, and Mn, respectively, were uptaken by ChlSG biomass in 12 days, presenting higher efficiency than EugVP. Concerning REEs, ChlSG biouptake 14.9, 20.3, 13.7, 8.3 ng of Gd, Pr, Ce, La per WB. Meanwhile, EugVP captured 1.1, 1.5, 1.4, and 7.5, respectively. This paper shows the potential of a microbial sequential process to revalorize e‐scraps and recover metals and REEs, harnessing extremotolerant microorganisms.

possible source of contamination of the atmosphere, sediments, and water streams. On the other hand, they contain high amounts of valuable materials, including copper (Cu), iron (Fe), gold (Au), or rare earth elements (REEs), even in higher values than natural ores (Cao et al., 2016;Cucchiella et al., 2015;Tan et al., 2015;Wibowo & Deng, 2015), making them a source of raw materials.
The change in focus of many countries toward waste-to-resource policies promotes mining from solid waste, instead of exploiting new sources (European Waste Electrical and Electronic Equipment Directive [2012/19/EU]). There are already examples of the harnessing of scraps, such as the regeneration of lithium electrode scraps to obtain new cathodes for lithium-ion batteries , or the recovery of copper and other precious metals from circuit boards scraps through a two steps method consisting of a combination of mechanical and electrometallurgical process (Mecucci & Scott, 2002;Veit et al., 2006), to name a few. The biotechnological approach has also been employed with this objective, and in fact, there are several publications about these applications. For example, the recovery of gold and palladium from e-scraps with a sulfate-reducing bacterium (Creamer et al., 2006) and biorecovery of gold from e-scrap material through a mutated cyanogenic bacterium Natarajan & Ting, 2014). So, there is an increasing effort to develop new technologies for the valorizing of e-scraps. However, there are no studies that string together microbial e-scraps leaching and elements recovery processes. One of the challenges encountered is the extreme pH values (3.6) yielded from the bioleaching process. This drawback may be overcome by employing biomass. But this option is limited by the amount of biomass used and mediated by passive accumulation (Drexler & Yeh, 2014;He & Chen, 2014). Another alternative is to employ extremotolerant microorganisms.
In this study, we investigate the bioleaching and/or accumulation abilities of different groups of microorganisms for the revalorization and metal extraction from e-scraps. We propose a two-phase process, the first stage of bioleaching from solid e-scraps mediated by a bacteria consortium, followed by the second step of capture with microalgae.
Specifically, for the first bioleaching phase, we made use of a bacteria consortium mainly composed of Acidiphilium multivorum and Leptospirillum ferriphilum isolated from a uranium mining site. For the second step of biocapture, we evaluated microalgae-mediated metals recovery from leachate of e-scraps. More specifically, Chlamydomonas sp. and Euglena sp. extremotolerant microalgae, natural inhabitants of uranium acid drainage tailings (Baselga-Cervera et al., 2018García-Balboa et al., 2013). The novelty of this work is the potential synergic effect from the combination of bioleaching and bio-uptaking to revalorize and extract valuable metals from e-scraps.

| E-scraps material
E-scraps raw material was supplied by LYRSA-Derichebourg, a Spanish company specializing in the recycling and management of industrial waste and e-scraps. We employed as e-scraps a residual powder, the fraction resulting from the recycling process after shedding and mechanical separation. E-scraps powder was composed of solids of 1-3 mm of particle size. The characteristic materials composition of the e-scraps is detailed in Appendix Table A1 (corresponds to "E-scraps metallic content").

| Experimental microorganism isolation and culture conditions
Liquid samples of acid mine drainage were collected for the recovery of chemolithotrophic bacteria at the uranium mining sites of Saelices, and Villavieja in Salamanca (Spain) (García-Balboa et al., 2013). Selection of a Fe-oxidant and S-oxidant chemolithoautotrophic bacteria consortium was performed culturing 10 ml of mine samples (Villavieja) in 90 ml of 9k medium (Silverman & Lundgren, 1959) (Baselga-Cervera et al., 2020) were isolated from the uranium mine acid drainage at Saelices and Villavieja, respectively. Both strains were cultured in filtered mine water enriched with BG-11 standard broth (Sigma-Aldrich). Mine waters used to prepare the media were: Saelices water for ChlSG and Villavieja water for EugVP, with final pH of 3.6 ± 0.2, and 2.5 ± 0.3, respectively (mine water physicochemical characteristics can be consulted in (García-Balboa et al., 2013). For regular maintenance, cultures were grown in 50-250 ml cell culture flasks (Greiner; Bio-one Inc.), under continuous light conditions and at 80 µm m −2 ·s −1 over the waveband 400-700 nm and 22°C ± 2°C temperature. Both strains are deposited in the culture collection of the research group Albiotox, Universidad Complutense de Madrid, Spain. Chlamydomonas sp. ChlSG strain has been described and characterized by the research group (ChlSG strain [Baselga-Cervera et al., 2018).

| Bioleaching tests and leaching ability determination
Bioleaching was carried out in 250 ml Erlenmeyer flasks with 100 ml final volume of 9k medium pH (2.05) without any additional source of energy (Fe or S) and 1% e-scraps w/v. Two flasks were inoculated with the bacterial consortium described previously and two flasks without bacterial inoculum were used as control. Bioleaching trials were maintained in agitation (250 rpm) and at 30°C for 15 days.
Bacterial inoculum consisted of 20 ml of a saturated culture of the selected bacterial consortium. During the biolixiviation experiment, the bacterial consortium thrived using e-scraps as a primary source of energy.
Samples were collected on days 7 and 15 since the inoculum.
The whole volume of each Erlenmeyer flask was sampled. Samples were filtered with a sterile filter of 0.45 microns of pore size to separate the liquid and solid phases. The liquid phase, leachate, and pellet were analyzed for 45 elements concentration and preserved for the next biouptake experiment. The solid phase was also recovered to analyze the elemental composition and compare it with the starting material.
Leaching ability (L i ) of each element i was obtained as in the following equation (Savvilotidou et al., 2015): where L i is the leaching ability for element i (mg/g), C ie the con- The extraction efficiency (E i ) for each element was thus calculated by referring the leaching ability to the initial concentration in the e-scraps powder, as given in the following equation (Chen et al., 2015): where C in is the concentration of element i in the e-scraps powder.

| Bio-uptake studies
Metal bio-uptake capacity from e-scraps leachate-recovered liquid phase-was measured in the ChlSG and EugVP strains. Cells from both strains were grown for 20 days to the stationary phase before exposure. Twelve Greiner flasks with a final volume of 10 ml were established: four with ChlSG, four with EugVP, and four without cells.
Each Greiner contained 9 ml of the filtered phase from the 15 day leachate and 1 ml of the microbial culture (grown in BG-11 culture medium) or fresh BG-11 media in the controls. Starting pH values of the study and control dissolutions were 3.55. The initial inoculum consisted of~3.5 ± 1.1 × 10 5 cells for the ChlSG strain, and~2.1 ± 0.5 × 10 3 cells for the EugVP strain.
The whole volume of one vial of each one of the three groups Cellular densities were directly counted using a hemocytometer under the microscope (Hoshaw & Rosowski, 1973). Later, the whole volume of each culture was centrifuged (4000 rpm for 15 min), supernatants were discarded, and pellets were preserved for metal analysis.

| Analytical methods
Multiple metal screening was conducted in the two-phase processbioleaching and bio-uptake-to extract metals from e-scraps. Metal analysis of solid samples required digestion in aqua regia solution (

| RESULTS AND DISCUSSION
3.1 | Metal composition of e-scraps E-scraps were chemically characterized to identify the metals concentrations, presenting a complex composition. E-scraps metallic content consisted mainly of zinc (7.3%), aluminum (5.9%), titanium (5.2), magnesium (4.6%), and important amounts of barium (3.35%), manganese (3.3%), lead (1.6%), and copper (1%), among others (a complete description of the metals analyzed can be found in Appendix Table A1). The other 75% of the scraps comprised other non-tested metals and non-metal content, probably plastic matter, and other organic components.
| 3 of 14 The metal composition of e-scraps varies considerably depending on age, manufacturer, and composition, as previously reviewed by Cui and Zhang (2008). There is not a generic composition, and the content of precious metals decreases due to modern manufacturing.
In contrast, the content of REE increases because of the intensive use of these elements in electric and electronic components. Different compositions may demand different microbial approaches and render different yields. For example, for printed circuit boards composition range of 10%-27% Cu, 8%-38% Fe, 2%-19% Al, 0.3%-2% Ni (Cui & Forssberg, 2007;Ilyas et al., 2007).

| Bacterial consortia composition
As expected, the majority of the OTUs presented low abundance and were "rare" (see Supporting Information at https://doi.org/10.5281/ zenodo.5819060). For the present study, we considered the role of the rare bacteria in the bioleaching process as negligible. The two major species identified in the consortium were A. multivorum (66% of the OTUs) an acidophilic chemoorganotrophic bacterium (Wakao et al., 1994), and L. ferriphilum (representing 31% of the OTUs) another acidophilic bacteria known to use the ferrous iron as electron donor (Li et al., 2020;Smith & Johnson, 2018).
Leptospirillum spp. is one of the most used bacteria in commercial biomining operations that require aerobic conditions and can grow with low concentrations of soluble iron (Smith & Johnson, 2018).
This suggests that both bacteria are oxidizing and reducing iron species, creating a circle that previously has been reported with other consortia-containing bacterial species from the same genus (Smith & Johnson, 2018). Previous studies using microbial consortia isolated from REE ore materials (Reed et al., 2016) and Kombucha (Hopfe et al., 2017(Hopfe et al., , 2018 have addressed the microbial potential for recovery REE from e-scraps powders. Their studies suggest that REE solubilization might be influenced not just by the species that compose the consortium, but also by the organic acid production of the consortia. Similar results have been also observed in fungal species (Mouna & Baral, 2019).

| Bioleaching efficiency of the bacterial consortium
In this study, the results are presented for a 1% w/v e-scraps concentration, using a bacterial consortium isolated from mine acid drainage and selected for iron-oxidizing and sulfur-oxidizing bacteria.
E-scraps presented a small particle size (0-3 mm) which increased the surface area and improved the oxidative capacity of bacteria (Ilyas et al., 2013). It is important to highlight that e-scraps are assimilable and more complex materials than minerals. Although Fe was not directly added, it was already present.
Different leaching efficiency (E i ) (%)-amount of the metal solubilized/initial metal concentration in the e-scraps material-were calculated among the investigated metals after 7 and 15 days of exposure (Table 1). The consortium used, after the seventh day, presented an efficiency of more than 99% Cu, 99% Co, 99% Al, 53%, Cd, and 98% Zn, whereas Cr and Li efficiencies were below 11%. The bacterial consortium studied, leached out significant amounts of demanded metals, as well as significant amounts of other critical metals, such as or U, and even precious metals like Au (Table 1).
The main economic driver of bioleaching is the recovery of precious metals, such as Pd, Ag, or Au, and critical metals like Cu, Al, Ni, Zn, Li, or U and REEs. Many researchers have investigated the processes of bioleaching of metals from e-scraps (as previously reviewed by Lee, 2014, andGu et al., 2016). Leaching efficiency depends on the source (Brandl et al., 2001;Qu & Lian, 2013;Tran et al., 2011), microorganisms (Brandl et al., 2001;Ilyas et al., 2013;Vestola et al., 2010), and leaching conditions. Metal bioleaching from e-scraps is complex due to the relation between the abovementioned factors that affect the efficiency of the process.   (Arshadi et al., 2016;Kumar et al., 2018;Pradhan & Kumar, 2012).
Varied amounts of REEs were also transferred from e-scraps to the dissolution phase: Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Sm, Tb, Tm, and Yb (Table 1). REEs were not found in the control solution, indicating that REE mobilization was mediated by the consortium activity. REE microbial mobilization has mainly focused on the extraction from native minerals (Desouky et al., 2016;Işıldar et al., 2019;Shin et al., 2015).
Moreover, the mechanisms of chemical and biochemical interaction among microorganisms with REEs are still not well-understood (Barmettler et al., 2016), but it can be assumed that general mechan- (SO4) 2 (OH) 6 . These minerals are common precipitates in the conditions of these types of reactions (Daoud & Karamanev, 2006). Ferric hydroxy sulfate minerals can form reactive surfaces and diffusion barriers, slowing down the flux from reactants to products (Nemati et al., 1998;Vestola et al., 2010). Thus, jarosite formation could be used as a signal to establish the optimal moment to stop the reaction and recover the enriched solution.
The metallic composition of the solid phase was also analyzed, before the jarosite formation at day 7, and the end of the experiment at day 15 (Table A2). The percentage of metals solubilized was more than 50% Cu, 55% Ni, 34% Al, 44% Li, and 94% Zn. The solid phase after the bioleaching step consisted of the remaining e-scraps, consortium cells, and precipitates. Our interest in analyzing the composition of the solid phase after bioleaching is related to its disposal. The final disposal of the residue, either landfilling or energetic valorization at a cement plant, is related to its composition. Cement plant admission requirements are highly influenced by the metal content of the solid deposits (Joseph et al., 2018). It is evident from these data that the bacterial consortium oxidation activity reduced the concentration of metals of the solid phase of all the main elements analyzed. It is particularly remarkable the significant decline in the case of Al, Ba, Mg, Ti, and Zn after 7 days. An extra benefit is not obtained if cultures are maintained longer because of the reactions of precipitation, as described just above. From day 7 to day 15, due to the precipitation reactions described above, the solid phase is enriched in metals such as Al, Cu, Ni, or Zn, and further depleted in Cd, Mn, P, and Ti.

| Microalgae metals and REEs removal from leachate media
In this context, we have capitalized on the advantages of two extremotolerant microalgae, ChlSG and EugVP strains (Figure 1), that evolved tolerance to acid mine drainages and have been thriving in these extreme anthropogenic environments ever since. Both strains Note: Values (value ± analytical error) represent the metals lixiviated in the leachates (at 7 and 15 days) and control from e-scraps mediated by a bacterial consortium. Leaching ability was presented as L i on day 7.
are tolerant of high metals concentrations and acid pH values below 3.5, making them tailored to the leachate conditions. We exposed  Table A2).
It is not surprising to observe differences between the strains given that both strains, despite being single cell flagellated eukaryotes grouped as microalgae, are completely different organisms that have structural and functional differences (Figure 1). In terms of structure, ChlSG is microscopic unicellular oval shape biflagellate  (Table A3).
To compare across the strain efficiency over time, we also estimated the amount of element uptake by cell, and by estimated cell Euglena spp. pellicle consists of a complex proteinaceous layer underlain by microtubules, with a high concentration of charged and polar aminoacid and sugar residues (Nakano et al., 1987). However, those polar

| CONCLUSIONS
We harvested e-scraps using a sequential microbial-mediated process developed in two stages: bioleaching using an acidophilic ferrous iron-oxidizing bacteria consortium and the biouptake of metals from the leachate by extremophilic microalgae strains. Results showed that this biotechnological methodology can be used to recover metals from e-scraps. Recovery values of the bioleaching stage were nearly