Copper solubilization strategies for mold biomachining and bioleaching from printed circuit boards

COPPER SOLUBILIZATION STRATEGIES FOR MOLD BIOMACHINING AND BIOLEACHING FROM PRINTED CIRCUIT BOARDS DEPARTAMENT OF CHEMICAL AND ENVIRONMENTAL ENGINEERING ARRATE SANTAOLALLA RAMÍREZ 2021 University of the Basque Country (UPV/EHU) Faculty of Engineering of Vitoria-Gasteiz Department of Chemical and Environmental Engineering COPPER SOLUBILIZATION STRATEGIES FOR MOLD BIOMACHINING AND BIOLEACHING FROM PRINTED CIRCUIT BOARDS Arrate Santaolalla Ramírez Vitoria-Gasteiz, 2021 Supervisors: Dra. Astrid Barona Fernández Dra. Naiara Rojo Azaceta (cc) 2021 Arrate Santaolalla Ramírez (cc by-nc-nd 4.0) Acknowledgments ACKNOWLEDGMENTS Después de dedicar los últimos casi cuatro años a esta tesis y aunque parecía no llegar nunca este momento, aquí estoy, escribiendo los agradecimientos para finiquitar así este capítulo de mi vida. Por tanto, es el momento de agradecer a todas esas personas que han hecho de este viaje una experiencia de vida en vez de un simple proyecto de investigación y que han caminado junto a mi durante esta etapa. Con el permiso de mi familia y amigos, me gustaría empezar agradeciendo de forma especial a quienes han hecho posible que yo ahora mismo este redactando estas palabras. Quiero empezar mencionando de forma especial a mis directoras de tesis, Astrid y Naiara. Gracias por confiar en mí y darme la oportunidad de hacer esta tesis con vosotras. Gracias por vuestro apoyo, por vuestro esfuerzo y por vuestra dedicación a pesar de tener otras mil cosas que hacer. Este trabajo no habría sido posible sin vuestra ayuda. A Gorka y Junkal, porque, aunque no han sido oficialmente parte de la dirección de mi tesis, siempre han estado para ayudarme y colaborarme en todo lo que he necesitado (y por esos dulces que siempre encontraba en mi taza al llegar). Me habéis dado mucho a cambio de muy poco. A Esti, que me enseñó todo lo que debía saber sobre nuestros bichos y tuvo mucha paciencia para que aprendiera todo bien. Me siento muy afortunada de haber podido formar parte del departamento de ingeniería química y del Medio Ambiente en general, y de la sección de Vitoria-Gasteiz en particular. Durante estos años he conocido gente espectacular que espero no dejen de formar parte de mi círculo. Gracias por hacerme sentir parte del departamento y por incluirme y contar conmigo siempre para todos los “rolletes” que iban surgiendo. A Zuriñe, Loli, Ainara, Jon, Irati, Nieves, y demás (lo siento si me dejo a alguien sois muchos), por esos cafés solos pero tan acompañados de interesantes conversaciones a media mañana. A los que no toman café, pero también forman parte de la sección. I would like to thank also Pr. Piet Lens for giving me the opportunity to be part of his research group during my stay in Galway. I also want to thank Arindam for guiding me in my experiments during my stay. To whole research group for the very warm welcome to the group. A Borja, por su paciencia y por ayudarme en el laboratorio (y fuera de él). Ahora, con el permiso de mis directoras y compañeros voy a dar las gracias a mi familia por haberme acompañado a lo largo de este camino. Todo lo que tengo que agradeceros es demasiado para dejarlo escrito, aunque voy a intentar resumirlo. Acknowledgments A mi padre, porque estés donde estés sé que estarás orgulloso de mí. Te echo de menos. A mi madre, por haberme permitido llegar a ser quien soy hoy. Me has enseñado a trabajar duro para conseguir lo que quiero y a dar siempre lo mejor de mí. A ti te debo todo lo que soy y todo lo que he conseguido. A mis hermanas mayores, Itziar y Leire. Por apoyarme a lo largo de este camino. Porque de vosotras he aprendido que nunca es suficiente y que siempre se puede un poquito más. Siempre habéis sido y seréis un ejemplo para mí. A Iratxe, mi gemela, mi otra mitad, porque siempre estás para mí. Gracias por aguantarme siempre todas mis chapas, por acompañarme a apagar el baño a últimas horas del día, pero sobre todo por apoyarme siempre durante este camino. Porque aun estando ocupada, siempre me has permitido estar más ocupada que tú. Si es verdad que estamos conectadas sabrás lo agradecida que te estoy. A Asier, porque aún sin saber muy bien de lo que le hablaba muchas veces, siempre me preguntaba por mis bichos. Por adaptarse siempre a mi disponibilidad (que a veces no ha sido tan amplia como nos hubiera gustado), pero sobre todo por apoyarme siempre y estar a mi lado. Porque en su compañía las cosas malas se convierten en buenas. Gracias por estar siempre ahí para mí. A mis amigas, por perdonarme los plantones y estar ahí para los buenos ratos. A mis Aitos, por rezarle siempre a Santa Rita pidiéndole que todo me fuera bien. Al resto de mi familia. No puedo nombrar a todos porque somos muchos, pero ellos ya saben lo importantes que son para mí. Siento que todas las palabras aquí escritas son insuficientes y espero algún día poder devolveros todo lo que me habéis dado. Eskerrik asko! Table of contents i SUMMARY ........................................................................................................................ vii RESUMEN ........................................................................................................................... xi LABURPENA ...................................................................................................................... xv MOTIVATIONS AND THESIS OVERVIEW ................................................................................ 1 OBJECTIVES ......................................................................................................................... 7 LITERATURE REVIEW .......................................................................................................... 11 1. MICROORGANISM-ASSISTED METAL MOBILIZATION ......................................................... 13 2. MICROORGANISMS IN MICROORGANISM-ASSISTED METAL MOBILIZATION PROCESSES . 13 2.1. Chemolithoautotrophic bacteria ................................................................................. 14 2.2. Heterotrophic bacteria ................................................................................................ 16 2.3. Fungi and yeasts .......................................................................................................... 17 2.4. Microorganism consortium ......................................................................................... 18 3. Acidithiobacillus ferrooxidans GENUS ................................................................................. 20 4. MECHANISMS OF MICROBIAL METAL MOBILIZATION ........................................................ 22 4.1. Direct mechanism ....................................................................................................... 22 4.2. Indirect mechanism ..................................................................................................... 23 4.3. Cooperative leaching/mechanism .............................................................................. 24 4.4. Thiosulfate and polysulfide mechanism ...................................................................... 24 4.5. Mechanisms according to the type of reaction .......................................................... 25 4.5.1. Redoxolysis .......................................................................................................... 25 4.5.2. Acidolysis ............................................................................................................. 26 4.5.3. Complexolysis ...................................................................................................... 26 4.5.4. Bioaccumulation .................................................................................................. 26 5. FACTORS INFLUENCING MICROORGANISM-ASSISTED METAL MOBILIZATION .................. 27 5.1. Temperature ............................................................................................................... 27 5.2. pH ................................................................................................................................ 28 5.3. Shaking speed .............................................................................................................. 28 5.4. Bacterial concentration and biomass immobilization ................................................. 29 5.5. Iron concentration....................................................................................................... 30 5.6. Presence of process inhibitors .................................................................................... 31 5.7. Precipitate formation .................................................................................................. 32 6. APPLICATIONS OF MICROORGANISM-ASSISTED MOBILIZATION OF METALS .................... 33 TABLE OF CONTENTS Table of contents ii 6.1. Biomachining ............................................................................................................... 35 6.1.1. Metal workpieces preparation ............................................................................ 36 6.1.2. Specific metal removal rate (SMRR) .................................................................... 36 6.1.3. Surface finish ....................................................................................................... 37 6.2. Electronic waste bioleaching ....................................................................................... 38 6.2.1. PCB pre-treatment .............................................................................................. 42 6.2.2. Treatment mode: single and multi-stages bioleaching ....................................... 44 6.2.3. Pulp density ......................................................................................................... 45 7. REFERENCES ........................................................................................................................ 45 MATERIALS AND GENERAL METHODS ................................................................................ 65 1. MATERIALS .......................................................................................................................... 67 1.1. Microorganisms ........................................................................................................... 67 1.2. Copper workpieces ...................................................................................................... 67 2. GENERAL METHODS ............................................................................................................ 68 2.1. Acidithiobacillus ferrooxidans bacterial growth .......................................................... 68 2.2. Copper mobilization experiments ............................................................................... 69 2.3. Determination of the bacterial concentration in the medium ................................... 69 2.4. Metal removal rate and specific metal removal rate .................................................. 71 2.5. Determination of iron species in solution ................................................................... 71 2.6. Metal content analysis (ICP and AAS) ......................................................................... 73 2.7. Scanning electron microscopy (SEM) .......................................................................... 73 2.8. Other methods ............................................................................................................ 74 3. REFERENCES ........................................................................................................................ 74 CHAPTER 1. OPERATION WITH SUSPENDED BIOMASS ......................................................... 77 1.1. OBJECTIVE ............................................................................................................................ 79 1.2. MATERIALS AND METHODS................................................................................................. 79 1.2.1. Copper pieces .......................................................................................................... 79 1.2.2. Microorganisms and culture media ........................................................................ 80 1.2.3. Metal mobilization experiments ............................................................................. 80 1.2.3.1. Effect of iron content on bacterial growth and metal mobilization ............... 80 1.2.3.2. SMRR as a function of time during the metal mobilization process ............... 81 1.2.4. Alternate process: metal mobilization + regeneration ........................................... 82 1.2.5. Analytical methods .................................................................................................. 82 1.3. RESULTS ............................................................................................................................... 82 1.3.1. Metal mobilization experiments ............................................................................. 82 Summary ix solution, precipitation and electrodeposition, rendered high metal recovery at a reasonable cost and required the preliminary oxidation and precipitation of iron, which obviously implied the additional consumption of reagents and longer operation time. Although the precipitation method was more affordable (although time-consuming), the final product obtained by the electrorecovery was more attractive for the stock market. Bearing in mind that the world demand of copper has been on the rise during the last three months and is expected to go further up, the metal recovery from the PCBs and the depleted solutions can be an entrepreneurial opportunity integrated in the circular economy. In summary, the biomachining of metallic molds and the metal bioleaching from PCBs were concluded to be two attainable bioprocesses whose future implementation on a large scale will make an important contribution to sustainable production, efficient waste management and circular economy. Summary x Resumen xi RESUMEN Actualmente la biotecnología desempeña un papel fundamental en el desarrollo sostenible. En este ámbito, los fundamentos de los procesos de solubilización de metales empleando microorganismos, que tradicionalmente han sido empleados en la extracción de metales de minerales, están siendo aplicados a otras áreas, como la fabricación de microestructuras (biomecanizado) y la recuperación de metales presentes en residuos (biolixiviación). El imparable aumento de la demanda mundial de componentes a escala micrométrica hace que la tecnología del micromecanizado esté en constante desarrollo con el fin de encontrar alternativas más económicas y sostenibles a los procesos físicos-químicos tradicionales. En este sentido, el biomecanizado se convierte en una prometedora alternativa que utiliza el potencial de los microorganismos para mejorar el proceso de grabado de microestructuras, resultando ser sostenible y respetuoso con el medio ambiente por su bajo consumo energético y su bajo coste operacional. Por otro lado, el interés en avanzar en la recuperación de metales de residuos de aparatos eléctricos y electrónicos (RAEE) se debe a la posibilidad de devolver los metales a la cadena de valor y a la oportunidad de gestionar de manera sostenible estos residuos, cuya generación ha aumentado exponencialmente en los últimos años. A pesar de que estas aplicaciones son prometedoras y presentan ventajas frente a otras tecnologías, todavía existe la necesidad de estudiar ciertos aspectos operacionales que permitan diseñar e implantar procesos mejorados a escala productiva. El principal objetivo de esta tesis es investigar el proceso de solubilización de cobre en un medio bacteriano, con el fin de proponer estrategias que permitan mejorar el rendimiento de cara a dos aplicaciones concretas: el biomecanizado de moldes para la fabricación de dispositivos microfluídicos y la recuperación de metales a partir de placas de circuito impreso de teléfonos móviles en desuso. La conocida bacteria extremófila Acidithiobacillus ferrooxidans fue seleccionada por su resistencia en medios ácidos y su nivel de bioseguridad. Antes de aplicar el proceso de movilización biológica de metales a los dos usos descritos, se estudiaron algunos aspectos relacionados tanto con el crecimiento microbiano, como con la solubilización del cobre y la operatividad del proceso propuesto. Dado que el rendimiento de solubilización del cobre depende de la concentración de oxidante, inicialmente se estudió la influencia de esta variable en el medio biológico. La concentración de hierro (Fe2+ para el crecimiento microbiano o Fe3+ para la solubilización del metal) más eficaz resultó ser 9 g L-1 (medio 9K). La velocidad de biooxidación del Fe2+ en el medio 9K fue 2.8 veces superior que en otros medios y la actividad bacteriana Resumen xii contribuyó a que la cantidad de cobre solubilizada fuera 25% más elevada que en un medio abiótico. A continuación, teniendo en cuenta que la máxima tasa específica de eliminación (TEE) se alcanzó durante la primera hora de tratamiento, se diseñó un proceso que alternaba una etapa de solubilización del metal de 3 horas con la pieza metálica sumergida en la disolución y una etapa de bioregeneración del oxidante sin la pieza. Esta alternativa demostró ser efectiva para reducir el tiempo necesario para solubilizar una determinada cantidad de metal, así como para extender el tiempo de vida de la disolución de tratamiento. Posteriormente, se llevó a cabo el estudio de la inmovilización de la biomasa sobre varios materiales de soporte, como una estrategia para favorecer la operatividad del sistema. Tras un ensayo con varias alternativas, se seleccionó la celulosa bacteriana por sus adecuadas propiedades mecánicas, estabilidad química, y estructura porosa. Asimismo, se optimizaron las condiciones de operación que permitieron una oxidación más rápida del Fe2+. Este soporte presentó las ventajas de inmovilizar de manera satisfactoria la biomasa, de no interferir en el crecimiento bacteriano, de poder ser almacenada a 4 °C en estado activo y de tener capacidad para oxidar el Fe2+ en presencia de cantidades elevadas de Cu2+. Asimismo, presentó mejor comportamiento que otro material también testado, el polivinil alcohol. En relación a las aplicaciones, se estudió el biomecanizado como alternativa para la generación de moldes metálicos para la fabricación de estructuras microfluídicas. En este proceso es totalmente necesario proteger adecuadamente la superficie metálica que no se desea mecanizar (o grabar). Para ello se seleccionó una combinación de una laca roja y un adhesivo de PSA que no afectaron a la actividad microbiana. Un aspecto a destacar de esta aplicación es la repetitividad del proceso empleando tanto la disolución fresca como la disolución regenerada (varios ciclos). Esto permitió establecer las ecuaciones matemáticas que posibilitan predecir el tiempo necesario para obtener un molde con una altura definida (en dos tramos de tratamiento: 0-1 h y 1-7 h). Los tiempos de regeneración fueron más cortos cuando la biomasa estaba inmovilizada en biocelulosa, aunque los rendimientos de mecanizado fueron similares a los de la suspensión. La creciente acumulación de cobre disuelto en el medio tuvo un efecto negativo en la regeneración de la disolución de biomecanizado, aunque no fue importante para concentraciones inferiores a 3 g Cu2+ L-1. El empleo de la biomasa inmovilizada en BC presentó además la ventaja de la facilidad operativa a la hora de reemplazar el medio bacteriano. En la aplicación de biolixiviación de cobre a partir de placas de circuito impreso de móviles en desuso, la heterogeneidad de estos materiales en términos de tamaño, composición o estructura obligó a diseñar una etapa previa de adecuación. Se decidió Resumen xiii eliminar la cubierta epoxi y tratar las piezas enteras o parte de ellas, pero sin trituración previa. La biolixiviación se llevó a cabo en dos pasos (biooxidación y biolixiviación por separado). Esta estrategia contribuyó al aumento de la movilización de los metales de las placas, siendo la eficacia de la lixiviación en medio bacteriano significativamente más elevada en comparación con la lixiviación química. El empleo de placas sin triturar permitió trabajar con un medio “más limpio” y favorable para el crecimiento de la biomasa. Las dos aplicaciones descritas generan disoluciones agotadas con alto contenido de metales que deben ser tratadas antes de su vertido. La recuperación de cobre de esas disoluciones devuelve este metal a la cadena de valor, mitiga el impacto ambiental, y contribuye a la viabilidad económica de ambas aplicaciones. En este estudio se ha trabajado con una disolución sintética agotada y se han aplicado dos sencillos tratamientos: precipitación química fraccionada y electrólisis. Ambas propuestas fueron eficaces en términos de recuperación del cobre de la disolución residual, aunque el proceso electrolítico da como resultado un producto más atractivo (Cu0) desde el punto de vista de mercado. En resumen, el mecanizado de moldes metálicos y la extracción de metales de placas de circuitos impresos en medio bacteriano son dos aplicaciones prometedoras cuya implantación futura a mayor escala supondrá una importante contribución a la producción sostenible, la gestión integrada de residuos y la economía circular. Resumen xiv Laburpena xv LABURPENA Gaur egun bioteknologiaren aplikazioek funtsezko garrantzia dute garapen jasangarria lortzeko. Arlo horretan, mikroorganismoak erabiltzen dituen solubilizazio prozesua aspalditik erabili izan da meatzaritzan, mineraletatik metalak erauzteko. Bioprozesu horren oinarriak beste alor batzuetan ere aplikatu dira azken urteotan, hala nola, mikroegituren fabrikazioan (biomekanizatuan) eta hondakinetan dauden metalen berreskurapenean (biolixibiazioan). Mikroegituren fabrikazioari dagokionez, eskala mikrometrikoko osagaien eskaera asko igo da mundu mailan azken urteotan. Eskaera horrek, mikromekanizazio teknologiaren garapen etengabea bultzatu du, ohiko prozesu fisiko-kimikoak baino alternatiba ekonomikoagoak eta jasangarriagoak aurkitzea premiazkoa delako. Aukeren artean, biomekanizazioa deritzon teknika aipa daiteke bereziki, hau da, mikroorganismoek lagunduta, mikroegiturak grabatzeko teknika jasangarria. Energia-kontsumo baxua eta operazio-kostu moderatua dira beraren aldeko ezaugarrietako batzuk. Bestaldetik, Tresna elektriko eta elektronikoen hondakinak (TEEH)gero eta kantitate handiagotan pilatzen dira munduan, eta horien kudeaketa arazoa oso larria da gaur egun. Hondakin horien kudeaketa eraginkorraren eta jasangarriaren helburu nagusia, metalak balorizatu eta balio-katera (lehengaien merkatura) bueltatzea da, metalen iturri naturalen agorpena saihesteko. Balorizazio hori, mikroorganismoek lagundutako biolixibiazioaren bidez burutu daiteke. Aipatutako prozesu biek (biomekanizazioak eta biolixibiazioak) abantaila nabarmenak dituzte betidanik erabili diren teknologiekin alderatuta. Hala ere, zenbait aspektu operatibo sakon aztertu behar da hobetutako prozedurak diseinatu eta horiek ekoizpen eskalan (eskala handian) inplementatu ahal izateko. Tesi honen helburu nagusia mikroorganismoek lagunduriko kobrearen solubilizazio prozesua ikertzea izan da, eta horretarako adierazitako aplikazio bien errendimendua hobetzeko estrategiak bilatu eta aztertu ziren. Aplikazio horiek ondokoak izan ziren: gailu mikrofluidikoak ekoizteko molde metalikoen biomekanizazioa, eta sakeleko telefonoen zirkuitu inprimatuetan dauden metalen berreskurapena. Acidithiobacillus ferrooxidans bakteria extremofilo ezaguna aukeratu zen ikerketan, ingurune azidoetan hazteko duen gaitasun aipagarriagatik eta maneiatzeko duen biosegurtasun maila altuagatik. Metalen mobilizazio biologikoa adierazitako bi erabileratara aplikatu baino lehen, mikroorganismoen hazkuntzan, kobrearen solubilizazioan eta proposatutako prozesuaren eraginkortasunean eragina duten hainbat faktore aztertu ziren. Lehenik, oxidatzailearen kontzentrazioaren eragina aztertu zen, kobrearen solubilizazioaren etekina horren menpekoa baita. Kobre kontzentrazio eraginkorrena Laburpena xvi (Fe2+ hazkuntza-fasean eta Fe3+ metalaren solubilizazioan) 9 g L-1 izan zen (9K hazkuntzamedioa). Burdin(II) espeziearen biooxidazio-abiadura 9K hazkuntza-medioan, beste kulturatan baino 2.8 aldiz handiagoa izan zen, eta bakterioen aktibitateari esker solubilizatutako kobre kantitatea ingurune abiotikoan baino % 25 altuagoa izan zen. Operatibitateari dagokionez, metalaren solubilizazio-prozesua eta oxidatzailearen birsortze-prozesua txandaka (banatuta) aplikatzeko estrategia diseinatu zen, ezabatzetasa espezifiko maximoa lehenengo orduan lortu zela kontuan hartuta. Estrategia hau oso egokia izan zen, metal kantitate jakina solubilizatzeko behar den denbora murrizteko eta disoluzioaren erabilgarritasuna luzatzeko. Biomasaren immobilizazioaren eraginkortasuna aztertzeko, hainbat material hautatu ziren lehendabizi. Guztien artean, zelulosa bakterianoa aukeratu zen osteko esperimentuetan erabiltzeko, ezaugarri mekaniko bikainak, egonkortasun kimiko altua, eta egitura porotsu egokia zituelako. Gainera, Fe2+-aren oxidazio azkarragoa ahalbidetzen zuela eta ez zuela hazkuntza mikrobiarra kaltetzen ondorioztatu zen. Mikroorganismoak itsatsita zituen zelulosa bakterianoak (euskarri aktiboak) hainbat onura zituen suspentsio zelularrarekin alderatuta, hala nola: biomasa efektiboki immobilizatzen zuen, 4 °Can biltegian gordetzeko egokia izan zen, eta Cu2+ kontzentrazio handien presentzian Fe2+-a oxidatzeko ahalmena mantendu zuen. Lehenengo aplikazioari dagokionez, biomekanizazioa ikertu zen. Prozesu horretan, mekanizatu (grabatu) nahi ez den azalera egoki babestu behar da derrigorrez eta, ikerketa honetan, mikroorganismoen aktibitatean kalterik eragiten ez zuten laka eta PSA pegatina aukeratu ziren helburu horretarako. Nabarmentzekoa da prozesua errepikakorra izan zela, bai disoluzio oxidatzaile prestatu berria eta baita birsortutako disoluzioa erabili zirenean. Horri esker, eta datu esperimentaletan oinarrituta, altuera jakina duen moldea sortzeko behar den denbora aurresateko ekuazio matematiko bi proposatu ziren bi tratamendu-tarte bitan: 0-1 h eta 1-7 h tarteetan. Biomekanizazioaren etekina antzekoa izan zen biomasa zelulosa bakterianoan immobilizatu zenean eta bakteriak esekiduran (suspentsioan) erabili zirenean. Aitzitik, disoluzio oxidatzailearen birsortzea azkarragoa izan zen euskarri aktiboa erabili zenean. Disolbatutako kobre kontzentrazioaren igoera progresiboak eragin negatiboa izan zuen disoluzio oxidatzailearen birsortzean. Hala ere, 3 g Cu2+ L-1 baino kontzentrazio baxuagotan eragina ez zen larria izan. Zelulosa bakteriano aktiboa erabiltzearen beste abantaila bat medio biologikoa errazago eta azkarrago ordezkatzeko aukera ematen zuela zen. Sakeleko telefonoen zirkuitu inprimatuek duten kobrearen berreskurapenari dagokionez, lehenbizi, laginen egokitze etapa diseinatu egin behar izan zen, zirkuitu hauek oso heterogenoak zirelako. Epoxi estalkia kentzea eta piezak osorik (edo horien partea) tratatzea erabaki zen, birrinketarik gabe. Biolixibazioa bi pausuetan burutu zen: Laburpena xvii biooxidazioa lehenengo eta ostean biolixibiazioa, zirkuitua mediotik aterata. Estrategia horrek metalen mobilizazioan eragin positiboa izan zuen eta, ondorioz, lixibiazioaren etekina handiagoa izan zen medio bakterianoan ingurune kimikoan baino. Zirkuituak birrindu gabe erabiltzeari esker, biomasaren hazkuntzarako egokiagoa izan zen medio “garbiagoa” erabili zen. Deskribatutako bi aplikazioek, metal kontzentrazio altuko disoluzio agortuak sortu zituzten eta, ondorioz, hondakin likido hori kudeatzeko modu egokia aurkitu behar izan zen. Disolbaturik zegoen kobrea berreskuratzea irtenbide erakargarria izan zen, metal preziatu hori balio-katera (merkatura) bueltatzeko negozio-aukera zelako, bioprozesuen ingurumen-inpaktua murrizten zelako eta bi aplikazioen bideragarritasun ekonomikoan inpaktu positiboa izan zuelako. Ikerketa-lan honetan disoluzio agortu sintetikoa erabili zen eta bi tratamendu aplikatu zitzaizkion: hauspeatze zatikatua eta elektrolisia. Bi prozesuak eraginkorrak izan ziren eta kobrearen berreskurapen-maila altua lortu zen, nahiz eta prozesu elektrolitikoaren bidez salmentarako produktu erakargarriagoa lortu. Laburbilduz, ikerketa honetan aztertu diren bi bioprozesuak (metalezko moldeen biomekanizazioa eta zirkuitoen inprimatutik metalen bioerauzketa edo biolixibiazioa) eraginkorrak izateaz gain, ekoizpen jasangarriaren, hondakinen kudeaketa integratuaren eta ekonomia zirkularrean aldeko bultzada emateko etorkizun handiko tresnak dira. Laburpena xviii OBJECTIVES Objectives 9 OBJECTIVES The main objective of this thesis is to improve the efficiency of the microorganismassisted solubilization of copper by proposing strategies that can be applied in the following processes: the biomachining of copper pieces for engraving microstructures and the recovery of that metal from disused mobile telephones. Both applications have been studied using the same bacterium (A. ferrooxidans) as it is a very versatile and resistant microorganism with a low biosafety risk. The secondary objectives are: o To contribute to the proposal of a semi-continuous metal mobilization process with suspended biomass that allows the maintenance of the maximum removal rate of copper while minimizing the amount of depleted solution. The continuous regeneration of the oxidant will allow the process to work on as long as the biomass is active. o To explore the potential of using a support material for immobilizing the biomass. That material should be easily biosynthesized in the laboratory, affordable and sustainable. o To assess the benefits of using immobilized biomass for improving the efficiency of the metal solubilization process, in comparison to suspended biomass. o To design a process for engraving microstructures on copper pieces with the final objective of manufacturing molds for microfluidic devices. This new application of the biomachining has not been explored so far, and it is an innovative use of that bioprocess with manufacturing perspectives. o To study the metal extraction efficiency when the printed circuit boards from obsolete mobile telephones are bioleached. The timely pretreatments of the heterogenous PCBs before metal extraction is a challenge to be faced. o To assess the technical and economic viability of two alternatives for treating the waste solutions obtained in the copper biomachining: chemical precipitation and electrochemical method. Objectives 10 LITERATURE REVIEW Literature review 13 1. MICROORGANISM-ASSISTED METAL MOBILIZATION The hydrometallurgical extraction of metals from minerals and the subsequent precipitation and recovery is an ancient technology that was first used in China as early as 100-200 B.C. (Ehrlich, 2001). In the past, the mobilization of the metals in minerals was attributed to an abiotic process and it was only in the 1950s that the first acidophilic iron and sulfur oxidizing bacteria were isolated and identified from acid mining drains, and the on-going research clarified the basic mechanisms of the biosolubilization of the metals (Colmer et al., 1950; Mishra et al., 2005). Copper extraction from its ores was introduced in Spain by the Arabs in the mines of Rio Tinto around the 18th century. Previously, during the 17th century, more than 2 million tons of copper were obtained by bioleaching in the deposits of the Falun mine (Sweden) (Ehrlich,2001). It was not until the middle of the 20th century when this process began to be scientifically studied and the presence of the involved bacteria was discovered (Bosecker et al., 1997). Bacteria A. ferrooxidans (formerly Thiobacillus ferrooxidans) and Acidithiobacillus thiooxidans (A. thiooxidans) were reported to be responsible for the bioleaching process, being the most studied ones to date. Nowadays, biohydrometallurgy (or bioleaching) is considered an environmental friendly technology that uses the activity of microorganisms for the recovery metals from minerals, concentrates and recycled or residual materials (Mishra et al., 2005; Rawlings and Johnson, 2007; Gumulya et al., 2018; Kaksonen et al., 2018; Habibi et al., 2020). The microbial role is the continuous bio-regeneration of the oxidizing agent (Fe3+) responsible for the chemical dissolution of the metal. Theoretically, the oxidant is never depleted and the extraction continues as long as the microbial performance is maintained. Consequently, the oxidizing agent does not have to be continuously supplied by chemical addition, with the consequent economic and environmental benefits (Barona et al., 2018) The most important application of bioleaching in the last century has been mineral extraction, but other biotechnological alternatives based on the same principle have also been studied in recent years. Two of these applications are the biomachining of metallic pieces (the machining of metal pieces by biological methods) and the recovery of metals from waste electrical and electronic equipment (WEEE). Both of them will be detailed in the last sections of this literature review. 2. MICROORGANISMS IN MICROORGANISM-ASSISTED METAL MOBILIZATION PROCESSES The most distinctive characteristic of any microorganism-assisted metal mobilization process is the use of microorganisms. The main microbial groups involved in the process Literature review 14 are chemolithoautotrophic prokaryotes, heterotrophic bacteria and fungi. In addition, microbial consortiums have also been studied. 2.1. Chemolithoautotrophic bacteria The group of autotrophic chemolithotrophic bacteria are the most studied microorganisms both in the biomachining of metallic pieces and in the bioleaching of PCBs. This type of organisms presents a high tolerance to heavy metals, being a crucial characteristic that makes them suitable for these applications (Orell et al., 2010). chemolithotrophic bacteria obtain the energy required for growing from the oxidation of some inorganic compounds such us as sulfides, elemental sulfur (S0), ferrous ions, and, some of them, even from hydrogen ions (Hedrich and Johnson, 2013). In biohydrometallurgy, the most employed chemolithotrophic bacteria are acidophilic bacteria that grow preferably at pH values from 1.5 to 4 under aerobic conditions. Among the acidophilic ones, the most studied bacterium is A. ferrooxidans, due to its ability to oxidize both soluble and non-soluble inorganic substrates (Uno et al., 1993; Wang et al., 2009; Liang et al., 2010; Hocheng et al., 2012b; Hocheng et al., 2012c; DíazTena et al., 2014; Xenofontos et al., 2015; Nie et al., 2015a; Muhammad et al., 2015; Singh et al., 2018). However, the suitability of other microorganisms has also been evaluated both for their application in biomachining and in WEEE bioleaching. Figure 1 shows two common chemolithotrophic microorganisms used in bioleaching processes. Figure 1. SEM micrograph: A. thiooxidans (published by Quatrini et al., 2017) (a) and Leptospirillum ferrooxidans (published by Vrdoljak and Spiller, 2005) (b). Table 1 summaries some chemolithotrophic microorganisms reported in bibliography. Literature review 15 Table 1. Some chemolithotrophic microorganisms used in microorganism-assisted metal mobilization processes reported in literature. Name Type pH T (°C) Reference Acidithiobacillus ferrooxidans M. 1.5-4.0 28-35 Wang et al., 2009; Liang et al., 2010; Hocheng et al., 2012c; Díaz-Tena et al., 2014; Xenofontos et al., 2015; Muhammad et al., 2015; Nie et al., 2015a; Singh et al., 2018; Benzal et al., 2020 Acidithiobacillus thiooxidans M. 2.0-3.5 28-30 Brandl et al., 2001; Chang et al., 2008; Wang et al., 2009; Liang et al., 2013; Isildar et al., 2016; Marra et al., 2018; Naseri et al., 2019; Lee et al, 2020 Acidithiobacillus ferrivorans M. 1.9-3.4 27-32 Isildar et al., 2016; Peng et al., 2019 Sulfobacillus thermosulfidooxidans M.T. 1.9-2.4 40-60 Deveci et al., 2004; Ilyas et al., 2007; Díaz-Tena et al., 2018 Acidithiobacillus caldus M.T. 2.0-2.5 42-45 Zhou et al., 2007; Fu et al., 2008; Wang et al., 2012 Sulfobacillus sibiricus M.T. 2.0 50 Zhang et al., 2015 Leptospirillum ferriphilum M.T. 1.5-1.8 45–50 Fu et al., 2008; Gu et al., 2013; Zhao et al., 2015 Acidianus manzaensis E.T. 1.0-5.0 60–90 He et al., 2009; Zhu et al., 2011; Liu et al., 2016 Sulfolobus metallicus E.T. 1.3-1.7 65-80 Vilcáez et al., 2008; Plumb et al., 2008 Acidianus brierleyi E.T. 2.0 65 Konishi et al., 1998; Bharadwaj and Ting, 2013; Samadzadeh et al., 2020 Acidianus copahuensis E.T. 3.0 75 Castro and Donati, 2016 Metallosphaera sedula E.T. 2.0-4.5 65-80 Mikkelsen et al., 2007; Yu et al., 2019 Sulfolobus solfataricus E.T. 2.0-4.0 80 Roshani et al., 2017 Acidithiobacillus albertensis M. 2.0-4.5 28-50 Xia et al., 2007 Acidianus ambivalens E.T. 2.0-5.0 80 Roshani et al., 2017 Metallosphaera hakonensis E.T. 3.0 70 Krok et al., 2013 Sulfolobus acidocaldarius E.T. 1.0-6.0 55-85 Lindström et al., 1993 Metallosphaera prunae E.T. 2.0-3.0 75 Stott et al., 2003 Acidianus infernus 1.0-5.5 65-96 Mikkelsen et al., 2006 Thiobacillus prosperus M. 1.0-4.5 23-41 Huber and Stetter, 1989 Ferroplasma cupricumulans M.T. 1.0-1.2 22-63 Hawkes et al., 2006 M.: Mesophilic; M.T.: Moderate thermophilic; E.T.: extreme thermophilic. Literature review 16 The thermophiles group can be classified into moderately thermophilic bacteria and extremely thermophilic bacteria according to their optimal growing temperature range. Moderately thermophilic bacteria have an optimal growth temperature between 40-60 °C and comprise strains such as Sulfobacillus thermosulfidooxidans (Devici et al., 2004; Ilyas et al., 2007), Leptospirillum ferriphilum (Gu et al., 2013, Zhao et al., 2015) or Acidithiobacillus caldus (Zhou et al., 2007; Wang et al., 2012). There is an increasing interest in studying thermophilic bacteria, especially for application in the chalcopyrite bioleaching, due to their unique metabolic characteristics and their tolerance to high temperatures, which can enhance the bioleaching kinetics (Deveci et al., 2004; Vilcáez et al., 2008; Zhao et al., 2019). Surprisingly, this group of bacteria apparently are more sensitive to high pulp densities and exhibit a lower tolerance to metal concentration (Panda et al., 2015). The extremely thermophilic bacteria that are able to grow at higher temperatures (between 60 and 90 °C) are scarce in literature. As an example, Acidianus manzaensis (He et al., 2009; Zhu et al., 2011) or Sulfolobus metallicus (Vilcáez et al., 2008; Plumb et al., 2008) strains can be mentioned. 2.2. Heterotrophic bacteria Heterotrophic bacteria obtain the energy from the oxidation of organic compounds such as lipids, alcohols, sugars or hydrocarbons and, under appropriate conditions, they produce certain organic amino acids and other metabolites which are responsible for the metal dissolution in the processes (indirect mechanism) (Bosecker, 1997; Brandl et al., 2008; Shabani et al., 2013; Barnett et al., 2018). The ability of metal bioleaching from WEEE by heterotrophic microorganisms has also been reported in literature (Hassanien et al., 2014; Shin et al., 2015; Barnett et al., 2018). The genus Bacillus and Pseudomonas have been described as the most effective heterotrophic bacteria for metal solubilization (Bosecker et al., 1987; Groudev, 1987). Figure 2 shows two examples of these genus, particularly Pseudomonas aeruginosa and Pseudomonas putida. Figure 2. SEM micrograph: Pseudomonas aeruginosa (published by Kasuga et al., 2011) (a) and Pseudomonas putida (published by Merum et al., 2017) (b). Literature review 23 Although the exact mechanism used by A. ferrooxidans to colonize surfaces has not been clarify yet, these bacteria can cover surfaces with a dense biofilm and a correlation between pilus expression and strong attachment has been found (Li et al., 2010). The EPSs produced by this microorganism are composed of neutral sugars, fatty acids and uronic acids. They mediate attachment to the surface and concentrate Fe2+ ions by complexation through uronic acids and other metabolites (Li et al., 2010) The following direct or contact mechanism for iron leaching has been proposed (for A. ferrooxidans bacteria) (Sand et al., 2001): FeS2 + 3.5 O2 + H2O → Fe2+ + 2 H+ + 2 SO42- (Eq. 1) 2 Fe2+ + 0.5 O2 + 2 H+ → 2 Fe3+ + H2O (Eq. 2) Or the general global equation shown below can be used to summarize the direct leaching mechanism from metal sulfides, where it can be noted that the final product is the metal sulphate (Bosecker et al., 1997): (Eq. 3) 4.2. Indirect mechanism The indirect bioleaching process takes place without physical contact between the bacteria and the solid (Sand et al., 2001; Kumar and Yaashikaa, 2020). Therefore, it is also called non-contact mechanism (Tributsch, 2001). The metal is oxidized by the action of the ferric ion (oxidizing agent). Thus, the ferric iron is capable of oxidizing metals while being reduced to ferrous iron. The latter is microbially oxidized again into Fe3+, leading to a cyclic oxidation process (Hamidian, 2011; Mishra and Rhee, 2014) (Figure 4b). Indirect bioleaching mechanism for the oxidation from metal sulfides can be described as follows (for A. ferrooxidans bacteria) (Sand et al., 2001): FeS2 + 14 Fe3+ + 8 H2O → 15 Fe2+ + 16 H+ + 2 SO42- (Eq. 4) MS + 2 Fe3+ → M2+ + S0 + 2 Fe2+ (Eq. 5) S0 + 1.5 O2 + H2O → 2 H++ SO42- (Eq. 6) For the specific case of pure metal leaching the equations are as follows: Chemical process for metal dissolution M0 (s) + 2 Fe3+ (ac) → M2+ (ac) + 2Fe2+ (ac) (Eq. 7) MeS + 2 O 2 MeSO 4 Bacteria Literature review 24 Biochemical regeneration of the oxidant Fe3+ Fe2+ (ac) + O2 (g) + 4H+ (ac) → Fe3+ (ac) + 2H2O (ac) (Eq. 8) 4.3. Cooperative leaching/mechanism It has been reported that cooperative leaching between attached and planktonic cells is possible (Rojas-Chapana et al., 1998). In this case, the bacteria attached to mineral surfaces supply Fe2+ and S to the planktonic cells as their energy source. Then the ferrous ion is oxidized by free cells, supplying the oxidant which is further used in indirect leaching (Tributsch, 2001; Li et al., 2013). Consequently, it is therefore a combination between the direct and indirect mechanism, or contact and non-contact mechanisms (Zhang et al., 2018) (Figure 4c). 4.4. Thiosulfate and polysulfide mechanism Thiosulfate mechanism is based on the oxidation of acid-insoluble sulfides FeS2, MoS2, and WS2 (pyrite, molybdenite, and wolframite) by the Fe3+ ions with the thiosulfate as the main intermediate and sulfate as the end-product (Hamidian, 2011). In the polysulfide mechanism, mineral solubilization such as sphalerite, galena, or chalcopyrite (ZnS, PbS, CuFeS2, respectively) occurs through a combined attack of ferric iron and protons. In this case, the main intermediates are polysulfides and elemental sulfur (Figure 5). The generated elemental sulfur can be oxidized to sulfate in the presence of sulfur-oxidizing microbes as shown in Equation 13. The two mechanisms are summarized by the following equations (Sand et al., 2001): Thiosulfate mechanism: FeS2 + 6 Fe3+ + 3 H2O → S2O32- + 7 Fe2+ + 6 H+ (Eq. 9) S2O32 + 8 Fe3+ + 5 H2O → 2SO42- + 8 Fe2+ + 10 H+ (Eq. 10) Polysulfide mechanism: MS + Fe3+ + H+ → M2+ + 0.5 H2Sn + Fe2+ (n ≥ 2) (Eq. 11) (Eq. 12) (Eq. 13) 0.5 H2Sn + Fe3+ 0.125 S8 + Fe2+ + H+ Bacteria 0.125 S8 + 1.5 O2 + H2O SO4- + 2 H+ Bacteria Literature review 25 Figure 5. Schematic diagram of thiosulfate (a) and polysulfide (b) mechanisms in bioleaching (adapted from Rohwerder et al., 2003). 4.5. Mechanisms according to the type of reaction Microbial metal mobilization can also be described according to the type of reaction involved. Thus, it can take place by acidolysis (formation of organic or inorganic acids), redoxolysis (oxidation-reduction process), complexolysis (formation of complexes and chelates) and, to a lesser extent, bioaccumulation (Bosshard et al., 1996; Wu and Ting, 2006; Okoh et al., 2018). 4.5.1. Redoxolysis Redoxolysis mechanism is divided into direct and indirect mechanism (Watling, 2006). Thus, metals can be dissolved both directly by the direct contact with bacterial cells or with their extracellular compounds (direct mechanism or “contact” mechanism) or indirectly by the metabolic products generated by the biomass (indirect or non-contact mechanism) (Isildar et al., 2016; Cornu et al., 2017). Microorganisms playing a role in redoxolysis processes obtain energy from solid minerals and the iron oxidizing A. ferrooxidans or sulfur oxidizing A. thiooxidans bacteria can be particularly highlighted. Ferric iron (Fe3+) ion is one of the most common redoxolysis agents in leaching systems as it is a strong oxidant with a standard reduction potential of +0.770 V(SHE) (Diaz-Tena et al., 2016; Isildar et al., 2016). Redoxolysis process is based on the previously described non-contact mechanism where a cyclic oxidation process takes place. In addition, some microbial strains are capable of oxidizing S0 under anaerobic conditions, reducing Fe3+ to Fe2+ (Rawlings, 2005). Literature review 26 In the redoxolysis mechanism, the redox potential is an essential parameter controlling the process since it is related to the [Fe3+]/[Fe2+] concentration ratio. This value increases as long as the ferrous iron is being oxidized into ferric iron and it is related to the formation of sulfuric acid. Thus, the redox potential is a quick and convenient parameter to determine the biomachining/bioleaching progress (Rohwerder et al., 2003; Diaz-Tena et al., 2016). 4.5.2. Acidolysis In this case, the metal solubilization is achieved by the action of organic (malic, oxalic, gluconic, acetic, citric) and inorganic (H2SO4) acids produced by microorganisms. The protons becoming from the acids synthesized by these microorganisms, are capable of weakening the bond of metal ions rendering the dissolution of the metal (Burgstaller et al., 1992; Brandl and Faramarzi, 2006). The oxygen atoms that cover the metal surface are quickly protonated. Then the protons and the oxygen combine with water and, therefore, the metal is detached from the surface (Amriri, 2012; Bahaloo-Horeh et al., 2018; Xia et al., 2018). Although this mechanism can be performed by autotrophic sulfur oxidizer microorganisms (A. thiooxidans), acidolysis is the principal mechanism in metal mobilization processes involving heterotrophic fungal strains (Anjum, 2009, Vakilchap et al., 2016). The following equation represents the mechanism of acidolysis, MeO + 2H+ → Me2+ + H2O (Eq. 14) (MeO, being the metal oxide) 4.5.3. Complexolysis Unlike acidolysis, the organic acids produced by certain microorganisms (Aspergillus niger, Penicillium spp., or Rhizopus spp., among others) act as chelating agents which are capable of leaching metals through the formation of complexes (Mishra and Rhee, 2014; Horeh et al., 2016). Complexolysis process is slower than acidolysis, although both mechanisms can occur jointly (Okoh et al., 2018). Apart from organic acids, other metabolites, such as siderophores can form complex and solubilize metals such as the ferric iron (Shenker et al., 1999, Gadd, 2004; Osman et al., 2019). 4.5.4. Bioaccumulation Bioaccumulation consists of the accumulation of organic or inorganic pollutants inside living microorganisms (Barron et al., 1995; Chatterjee et al., 2020). However, there is a Literature review 27 controversy about the “bioaccumulation” concept when the pollutant accumulation occurs in dead cells (biosorption) (Chojnacka, 2007; Velásquez and Dussan, 2009). Several microorganisms such as bacteria, fungi, yeast, and some algae are able to accumulate heavy metals by bioaccumulation and by biosorption (Aksu and Karabayır, 2008; Park et al., 2010). For instance, Aspergillus niger, Aspergillus foetidus, Aspergillus nomius, Trichoderma harzianum, Aspergillus lentulus and Yarrowia lipolytica are reported to be metal bioaccumulators (Dursun et al., 2003; Ge et al., 2011; Mishra Malik, 2012; Zotti et al., 2014; Chatterjee et al., 2019). The main drawback of this process is the toxicity of high concentrations of the metal in the medium which could cause the death of bioaccumulator strains (Chojnacka, 2007; Ge et al., 2011). 5. FACTORS INFLUENCING MICROORGANISM-ASSISTED METAL MOBILIZATION As every biological system, this process is affected by several factors such as bacterial concentration, temperature, pH, shaking speed or the presence of inhibitors, among others (Díaz-Tena et al., 2016). In this type of processes, the iron concentration (oxidant) in the medium is also a relevant variable. The studies carried out to date have concluded that the optimal operating conditions (optimal values for temperature, pH, Fe2+ concentration, and others) are dependent on the strain used in each specific case. Therefore, the influence of these factors is common to many biotechnological applications, although specific features have to be considered in each particular case. The main factors that affect the microorganism-assisted metal mobilization process are described below. 5.1. Temperature In order to maintain optimal bacterial growth conditions, it is necessary to establish a suitable temperature range during the bioleaching process. Each microorganism requires different optimal temperature conditions. Thus, mesophilic microorganisms (the most typical ones in bioleaching/biomachining processes) grow at temperatures between 25 and 40 °C (Rawlings, 1997; Leahy et al., 2007; Kumar and Yaashikaa, 2020), while thermophiles grow at temperatures above 40 °C (Plumb et al., 2008; Dopson and Johnson, 2012, Donati et al., 2016). For example, A. ferrooxidans, the most common microbe associated with bioleaching/biomachining processes, has its optimum growing temperatures at 30 °C, and it loses its activity at temperatures below 10 °C or o above 40 °C (Modak et al., 1996, Mousavi et al., 2007, Karimi et al., 2010). Literature review 28 Temperature is also a determining parameter for increasing oxidation reaction rates, as deduced from the Arrhenius equation (Levenspiel, 2005; Franzmann et al., 2005; Leahy et al., 2007). 5.2. pH In addition to being related to the microbial growth, the pH value is closely related to the metal dissolution efficiency and to the formation of precipitates in the culture medium. The pH in the 2.0 and 3.0 range is the optimal value for bacteria to oxidize ferrous iron (Kumar and Yaashikaa, 2020). The metal solubilization process involves acid consuming reactions, which causes the pH increase thorough the process (Vilcaez et al., 2008) (Equation 8). If the pH exceeds the value of 2.2, the precipitation of metals and the formation of jarosite are clear inconvenients (Xenofontos et al., 2015). Therefore, working at pH values as low as possible (depending on the bacterial strain employed) can increase the efficiency of the metal mobilization since most of the metals are usually solubilized under low pH values (Ilyas et al., 2007; Xiang et al., 2010). 5.3. Shaking speed An optimal shaking speed provides the appropriate conditions for the microbial growth, as well as an efficient contact between the material to be treated and the culture medium. In addition, shaking speed is a parameter that affects the bioprocesses such as biomachining and bioleaching of PCBs. As far as the biomachining of metallic workpieces is concerned, some authors have concluded that the specific metal removal rate (SMRR) when using A. ferrooxidans strains keeps to a minimum if agitation is not provided, and that it considerably increases when increasing the shaking speed (Jadhav et al., 2013; Xenofontos et al., 2015; DíazTena et al., 2016). However, this increase is not linear at high shaking speeds (Xenofontos et al., 2015). Jadhav et al. (2013) concluded that the optimal shaking speed is 150 rpm under the tested conditions (treatment of copper pieces at 30 °C using the supernatant from a culture of A. ferrooxidans (13823) strain with an initial iron concentration of approximately 3.6 g Fe2+ L-1). These authors obtained the highest SMRR value (15.9 ± 1.3 mg Cu h-1 cm-2) at 150 rpm, in comparison to the results obtained when shaking more vigorously. In the case of bioleaching treatment of electronic waste, many experiments have been reportedly carried out at shaking speeds of 150 rpm (Brandl et al., 2001; Shah et al., 2015; Isildar et al., 2016; Marra et al., 2018; Wang et al., 2018), although, in a lesser Literature review 29 extent, lower speeds (120 rpm) (Xiang et al., 2010) and higher ones (up to 200 rpm) (Ilyas et al., 2007; Liang et al., 2010) have also been studied. 5.4. Bacterial concentration and biomass immobilization The bacterial density plays an important role in the process efficiency, since it determines the time required by bacteria to re-oxidize all the Fe2+ to Fe3+ (Zhu et al., 2017). In general, a constant cell concentration is recommended during the process in order to avoid the reduction in the dissolution rate of metals (Díaz-Tena et al., 2016). One of the alternatives proposed by some authors to increase the cell density thorough the process is the immobilization of the microorganisms on suitable support materials that allows growth on their surface. Some of the materials proposed by different authors for the immobilization of the A. ferrooxidans bacterium are shown in Table 5. Table 5. Different support materials reported in bibliography for A. ferrooxidans immobilization. Support material Operating conditionsa Reference Nickel alloy fibre pH 1.8, 10 % inoc., 200 rpm, 30 °C, 600 mL Gomez et al., 2000 Ceramic beads pH 1.6, 10 % inoc., air, 30 °C, 500 mL Junfeng et al., 2007 Hemp fibres pH 1.6, 10 % inoc., 180 rpm, 30 °C, 50 mL Akhlaghi, 2019 Monolithic particles pH 1.6, 30 °C, air Kahrizi et al., 2008 Chitosan beads pH 1.8, air, 30 °C, 100 mL Giaveno et al., 2008 Cotton gauze Activated carbon Zeolite pH 2.0, 10 % inoc., air, 30 °C, 500 mL Zhu et al., 2017 Cotton gauze pH 2.0, 10 % inoc., air, 30 °C, 500 mL Nie et al., 2015b Foam material pH 1.5-2.0, 10 % inoc., 240 rpm, 30 °C Jaisankar and Modak, 2009 SulfSDVB-GAC SulfSDVB PUF pH 1.8, 10 % inoc., 200 rpm, 35 °C, 200 mL Koseoglu-Imer and Keskinler, 2013 Biocellulose pH 1.8, 5 % inoc., 170 rpm, 31 °C, 100 mL Santaolalla et al., 2021 aProcesses that do not specify shaking speed is because are shaken by air injection, (indicated as air). Literature review 30 Most of the studies have been carried out with the A. ferrooxidans bacterium, due to its natural tendency to immobilization (Nemati et al., 1998; Giaveno et al., 2008; Jaisankar et al., 2009). These studies have focused on investigating the immobilization process and the capacity of the immobilized biomass to oxidize Fe2+. On the contrary, little data is available about the activity of the immobilized biomass during the metal leaching stage or the influence of the increasing metal concentration throughout the process. The encapsulation of the biomass in a matrix is another alternative for biomass immobilization. In addition to ensuring adequate cell density, this option has a protective effect against shaking turbulences (Vermeulen and Nikolay, 2017). Likewise, Giaveno et al. (2008) have concluded that a packed reactor with chitosan beads in which A. ferrooxidans had previously been grown could operate with a medium flow rate up to eight times higher than a reactor with free cells culture. Another advantage of biomass encapsulation is the greater tolerance to increasing copper concentrations, in comparison to the cell suspension mode (Vermeulen and Nikolay, 2017). 5.5. Iron concentration Iron is the main actor in the bioleaching and biomachining process when the A. ferrooxidans bacterium is employed, since it contributes to the microbial growth and the metal dissolution process. As far as the growth stage of A. ferrooxidans bacteria is concerned, it is affected by the concentration of iron in the medium in different ways. First, the concentration of Fe2+ has an impact on the time required by the culture to oxidize all Fe2+ to Fe3+, with longer times being necessary as the initial Fe2+ concentration increases (Jadhav et al., 2013). Second, the cell concentration also seems to be affected by the initial concentration of Fe2+ in the culture medium. In a study carried out by Hocheng et al. (2012c) using A. ferrooxidans (BCRC 13820) strain, these authors reported that the cell concentration practically doubled when the concentration of Fe2+ in the medium increased from 11 to 22 g Fe2+ L-1 while the increase was less noticeable when the concentration of Fe2+ was 3 and 4 times higher. Kawabe et al. (2003) concluded that excessively high concentrations of Fe3+ can inhibit the oxidation of Fe2+ by A. ferrooxidans bacterium and that the degree of inhibition is a function of the strain used. Thus, the T23-3 strain is capable of oxidizing Fe2+ with concentrations of 26 g Fe3+ L-1 in the medium, while the activity of the ATCC19859 strain is completely inhibited at 16 g Fe3+ L-1. In the biomachining process, the increasing concentrations of Fe3+ enforces the dissolution of the metal (copper in most studies) and allows a higher specific metal removal rate (SMRR) to be achieved. However, too high concentrations of this ion are not recommended for industrial application, since the final surface quality of the biomachined piece would be very poor, the sulfuric acid consumption would increase Literature review 31 and uncontrolled jarosite precipitation would take place (Díaz-Tena et al., 2016). Jadhav et al. (2013) have observed a linear tendency in the increment of SMRR with iron concentration (in the range 3.6-14.8 g L-1, approximately), although the surface deterioration was considerably greater when the highest concentrations were tested (at 30 °C using the supernatant of A. ferrooxidans (13823) culture). In the WEEE bioleaching process, Yang et al. (2009a) have concluded that the higher the concentration of Fe3+ in the medium, the faster the copper in the PCBs dissolved. Thus, the time required to solubilize all the copper present in the PCB was reduced from 96 to 36 h when the concentration was increased from 2.4 to 6.7 g Fe3+ L-1 (A. ferrooxidans culture at 30 °C and 165 rpm). In a process in which the growth of microorganisms and the solubilization of metals took place simultaneously, Xiang et al. (2010) have found that the rate of metal leaching increased with the concentration of Fe2+ up to 9 g Fe2+ L1, but it decreased with the presence of 12 and 15 g Fe2+ L-1, which was attributed to the jarosite precipitation and the consequent passivation of the surface to be treated. Xiang et al. (2010) and Khatri et al. (2018) have jointly concluded that an initial concentration of 9 g Fe2+ L-1 is the adequate one that allows obtaining a higher solubilization of metals when using two consortiums of microorganisms with different origins. 5.6. Presence of process inhibitors The presence of a variety of metals in the leaching medium can exert a toxicological effect on microbial growth. As an example, copper is essential for the metabolic activity of A. ferrooxidans, since it serves as an electron donor during the microbial growth process and it has been found in the structure of Rusticyanin, a protein that acts as an electron transporter (Lilova et al., 2007; Mykytczuk et al., 2011; Cornu et al., 2017). Conversely, high concentrations can induce the denaturation of proteins and nucleic acids, causing the biomass death (Valix, 2017). Therefore, the presence of certain concentrations of metals in solution, which are continuously produced during the process, can inhibit bacterial activity (Lilova et al., 2007). Nevertheless, some authors have suggested that the tolerance of microorganisms to different metals can be increased if they are adapted to the metal presence during their activation (Ilyas et al., 2007; Liang et al., 2013; Ravindra et al., 2015; Pourhossein and Mousavi, 2018). In the particular case of the WEEEs bioleaching, toxic elements including a large number of metals (Ag, Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn) can be dissolved, as well as other organic components and epoxy resins (Ongondo et al., 2011; Isildar et al., 2016). In order to increase the tolerance of the microorganisms to the toxic presence, three main strategies have been studied: a) the pre-acclimation of the microorganisms to heavy metals´ presence before carrying out the leaching stage (Ilyas et al., 2007; Liang et al., 2010), b) the use of consortia of microorganisms (Ilyas et al., 2007; Ilyas et al., Literature review 32 2013b) and c) cellular adaptation by treating the wastes in several stages (Xiang et al., 2010). The metal concentration values with toxicological effects reported in bibliography vary depending on the bacterial strain and the operating conditions. As an example, in the case of the culture of bacteria A. ferrooxidans isolated from Río Tinto (Huelva, Spain), Cabrera et al. (2005) concluded that this strain was able to tolerate the following metal concentrations: 0.4 g Cr3+ L-1, 10 g Cu2+ L-1, 10 g Cd2+ L-1, 30 g Zn2+ L-1 and 30 g Ni2+ L-1. These limit values varied when a consortium of heterotrophic Acidiphilium bacteria was added to the culture, obtaining in this case limit values of 0.4 g Cr3+ L-1, 4 g Cu2+ L-1, 10 g Cd2+ L-1, 40 g Zn2+ L-1 and 15 g Ni2+ L-1. Das et al. (1998) used an A. ferrooxidans strain isolated from the Malanjkhand copper mine (India) and it was found to survive in the presence of 20 g Cu2+ L-1 in a single culture stage. Tolerance was significantly increased in a study by Vermeulen and Nikolay (2017), in which the encapsulation of A. ferrooxidans (DSM 11477) in polyvinyl alcohol allowed to obtain an oxidation rate of Fe2+ to Fe3+ in the presence of 40 g Cu2+ L-1 2-3 times higher than that obtained by the free cells. 5.7. Precipitate formation When Fe3+ accumulates in the leaching medium and the pH of the solution increases, Fe3+ may precipitate as jarosite, which can lead to a loss of 77 % of the iron available in the solution (Xiang et al., 2010; Wang et al., 2018). Likewise, precipitates can cover the surface to be treated, thus preventing the dissolution of the metals (Pradhan et al., 2008; Xiang et al., 2010). Therefore, the formation of precipitates has an influence both on the process efficiency and cost. In the case of a bioleaching/biomachining solution containing Fe3+, the main precipitates are iron hydroxides and jarosite (MFe3 (SO4)2 (OH)6, where M = K+, Na+, NH4+, Ag+ or H3O+). The formation of these precipitates is related to pH, temperature, cation availability or SO42− concentration (Wang et al., 2018). In addition, the joint presence of a high variety of metals (such as in PCB bioleaching solutions) can result in a formation of more complex precipitates. Ilyas et al. (2013b) analyzed the precipitate obtained in a PCB bioleaching process using a bacterial consortium including Sulfobacillus thermosulfidooxidans and Thermoplasma acidophilum thermophilic bacteria. The precipitate was determined to be composed of PbSO4, Ag2SO4, SnO2, AgFe3(SO4)2 (OH)6 and H2SnO3, in the presence of Al 0.5±0.04 %, Pb 17±0.9 %, Sn 8.5±0.034 %, Zn 0.08±0.004 %, Fe 0.8±0.05 %, Cu 0.5±0.05 %, and Ag 0.003±0.002 %. Literature review 39 Thus, the proper management of discarded electrical and electronic equipment (EEE) has become one of the main environmental concerns in developed countries due to serious effects their components generate both for human health and for the environment (Ortuño et al., 2013; Jadhao et al., 2016; Horlgersson et al., 2017). Regarding the environmental impact of WEEE, the European Union (EU) attempted to limit the use of hazardous substances in the manufacturing of these equipment through the enactment of the RoHS Directive 2002/95/EC (Kumar et al., 2017). As far as the Directive 2012/19/EU of the European Parliament on WEEE is concerned, these residues are classified in the following categories: 1. Temperature exchange equipment; 2. Screens, monitors, and equipment containing screens having a surface greater than 100 cm2; 3. Lamps; 4. Large equipment (any external dimension more than 50 cm); 5. Small equipment (no external dimension more than 50 cm); 6. Small IT and telecommunication equipment (no external dimension more than 50 cm). Among these categories, the total amount of WEEE generated in 2019 was basically made up of small equipment (17.4 Mt), large equipment (13.1 Mt), temperature exchange equipment (10.8 Mt) and screens (6.7 Mt). Lamps and small telecommunication equipment, with 0.9 Mt and 4.7 Mt respectively, represented smaller fractions (Figure 8) (Nithya et al., 2020). It has been predicted that the generation of wastes from temperature exchange devices and small and large devices will register the highest growth rates among all the categories. Conversely, screen waste is expected to decrease in the coming years due to the replacement of heavy cathode-ray tube (CRT) screens by flat alternatives (Baldé et al., 2017). Figure 8. Total WEEE generation in 2019 per category (adapted from Nithya et al., 2020). Despite regulations in force, the effective management of most WEEE is still scarce. In Europe, approximately only 35 % of that waste is recycled, while the rest is dumped, transferred to developing countries, or “simply lost” (Isildar et al., 2019). However, WEEE is an important secondary source of high-value metals, so its sustainable recovery 0 5 10 15 20 Lamps Small IT Screens and monitors Heat exchanging devices Large equipment Small equipment WEEE generation in 2019 (Mt) Literature review 40 is mandatory to avoid the depletion of natural sources, mitigate the pollution that causes its disuse and achieve its efficient recycling within the application of the circular economy principles. Due to its high content of valuable materials, several studies on WEEE recycling have been carried out as an attempt to recover those valuable materials (Mizero et al., 2018; Priya and Hait, 2020; Roy et al., 2021). In addition to legal regulations, the “waste principle” was included in the Waste Framework Directive, Directive 2008/98/EC of the European Parliament and Council, in 2008. Thus, the commonly known waste hierarchy pyramid (Figure 9) is composed of five measures: prevention, preparing for reuse, recycling, recovery (including energy recovery), and disposal. This waste hierarchy pyramid gives the highest priority to the prevention and reduction of waste generation, and, if generated, it gives priority to direct reuse and recycling methods (Directive 2008/98 / CE). Figure 9. Waste hierarchy according to directive 2008/98/CE. Among the different WEEE types, mobile phones (included in category 6) significantly contribute to the waste amount to be managed (Hira et al., 2017). In 2016, about 435 Kt of disused mobile phones were generated worldwide (Baldé et al., 2017). The average weight composition of a standard mobile phone is approximately 50 % plastics, 15 % glass and ceramic, 15 % metals, and the rest other materials (Moltó et al., 2011; Tesfaye et al., 2017). It is used as a support for electronic components and as a basis for connecting those using conductive pathways (Hadi et al., 2015). The main environmental impact of this type of waste is attributed to the printed circuit boards (PCBs), which have a high metal content. Figure 10 shows the average weight composition of a standard mobile phone PCB (Yamane et al., 2011; Palmieri et al., 2014). Literature review 41 Figure 10. Average weight composition of a standard mobile phone PCB. A mobile phone can contain up to 40 different elements, such as commonly used metals (copper and tin), precious metals (gold and silver), and others such as platinum and palladium (Kaya, 2016). Between 65 and 80 % of the materials in a mobile phone are recyclable (Moltó et al., 2011) but, recycling PCBs can be particularly difficult due to the wide variety of components. The extraction of the metals contained in the PCBs (so called urban mining) is an undoubted economic opportunity and a priority measure to protect the environment and public health. It is estimated that the value of the raw materials contained in discarded mobile phones in 2016 was € 9,400 million (Baldé et al., 2017). The traditional techniques used for the recycling of PCBs are grouped into two main types: hydrometallurgical processes (based on the extraction of metals using aqueous and organic liquid solutions) and pyrometallurgical processes (based on heating). These treatments are responsible for several environmental problems such as the formation of brominated and chlorinated di-benzo furans, the generation of dioxins or the toxicity of the employed reagents (Ning et al., 2017; Khatri et al., 2018; Liu et al., 2020). Conversely, the bioleaching process is an environmentally friendly recycling alternative, as it has advantages over current technologies: it has a low treatment cost, and its environmental impact is moderate. However, some technical issues require further research for improving process performance and subsequent high scale implementation (Isildar et al., 2019; Arya and Kumar, 2020). As far as the techno-economic assessment of hydrometallurgy and biohydrometallurgy is concerned, Isildar (2018) and Baniasadi et al. (2019) calculated the treatment cost per kg PCB and the contribution of each method to the climate change. They concluded that the most affordable and environmentally friendly process is the biological alternative (Table 8, reproduced from Sodha et al., 2020). Literature review 42 Table 8. Economical and environmental assessment comparison of hydrometallurgy and biohydrometallurgy methods for PCB treatment (Sodha et al., 2020). Technique Cost (€ kg-1 PCB) Climate change contribution Investment Operational Total cost kg CO2 kg-1 PCB Biohydrometallurgy 0.457 0.159 0.616 8.26 Hydrometallurgy 0.446 0.224 0.670 14.6 The main drawbacks that must be overcome for the industrial implementation of this technology are: the complex nature of some PCB materials (Brandl et al., 2001; Xiang et al., 2010), the possible toxicity of the non-metallic fraction of PCBs (Ilyas et al., 2007; Shah et al., 2015; Isildar et al., 2019), the inhibition of bacteria by the presence of high metal concentrations in the solution (Ilyas et al., 2013b; Arshadi and Mousavi, 2015a), and the lack of a standardized process for the recovery of the metals present in the depleted solutions. The previous dismantling or removal of components from PCBs is an additional problem that can be overcome by a novel application of bioleaching: biodismantling. MonneronEnaud et al. (2020) presented the biodismantling (dismantling using bioleaching) as a novel feasible application for removing the PCB components. It can be implemented as a new unit operation in the recycling process. The specific key aspects for the successful bioleaching of mobile phone PCB such as the pre-treatment of the PCBs (entire pieces or crushed PCB), the treatment of the material in multi-stages or the amount of material to be treated per volume of medium cultivation (pulp density) are described below. 6.2.1. PCB pre-treatment The materials used in the manufacturing of mobile phone PCBs are divided in two main fractions: non-metallic and metallic components. The non-metallic components are fiberglass (inorganic), epoxy resin (organic) and brominated flame retardants. The other fraction, made up of metallic elements, contains a single layer or multilayer of Cu, lead solder, tin solder, and other metals such as nickel, iron, lead, cobalt, aluminium, gold, indium, or antimony (Arshadi and Mousavi, 2015b). The recycling of the non-metallic fraction has not received much attention in literature because it is not profitable to date (Ilyas et al., 2007; Adhapure et al., 2014). Two types of PCB pre-treatments have been proposed: removal of the non-metallic fraction and size reduction. Literature review 43 The non-metallic fraction (epoxy resin) covering the boards is toxic to microorganisms (Isildar et al., 2019) and it hinders the interaction between the metallic surface and the bioleaching solution. For this reason, some authors separate the metallic and nonmetallic fraction before immersing the waste into the bioleaching medium (Jujun et al., 2015; Wu et al., 2018). The removal of the non-metallic materials by washing the PCB dust samples with a saturated solution of NaCl has proved to improve the process (Ilyas et al., 2007; Shah et al., 2015). Senophiyah-Mary et al. (2018) have studied the efficiency of other different pre-treatment methods for epoxy coating removal from PCBs. These authors have tested different solutions containing NaOH and solvents like ethanol, acetone, tween80, carbinol, benzyl alcohol, and acids like sulfuric acid, nitric acid and hydrochloric acid. No epoxy removal has obtained with solvents while complete epoxy cover removal was achieved with sodium hydroxide solutions and with sulfuric and nitric acids. However, they selected the NaOH as the best pre-treatment method since the acid attack dissolved many components simultaneously. Moyo et al. (2020), have also reported the best results when employing NaOH for epoxy cover removal, and Adhapure et al. (2014) concluded that immersing the PCBs in 10 M NaOH overnight was the optimal pretreatment for eliminating the epoxy covering. Once the epoxy resin is removed, the entire PCB (without crushing) can also be treated. This last alternative of entire pieces is very interesting for the industrial scale application, since it simplifies the pre-treatment (no crushing is required) and, facilitates the extraction of the depleted piece from the solution. Regarding size reduction, particle size obviously determines the contact surface area between the medium and the material to be treated so that, when the particle size decreases, the mass transfer increases accordingly. On the contrary, when particle size is high, the collisions between the microorganisms and the PCB particles can damage the bacterial cells (Arshadi and Mousavi, 2015a). Most of the PCB bioleaching studies have been carried out using pulverized samples (Liang et al., 2010; Arshadi and Mousavi, 2015a; Arshadi et al., 2016; Isildar et al., 2016; Wang et al., 2018; Khatri et al., 2018). The usual equipment for crushing the boards is a metal crusher, hammer or ball mill. Initially, the PCB grinding step can be carried out in a hammer mill to grind the coarser particles and subsequently a ball mill to grind the fine ones (Kasper et al., 2011). The main difficulties of these pre-treatments are the loss of material (which can be up to 40 %), the high energy consumption and the formation of a fine mixture of metallic or non-metallic particles that can be dangerous to health and difficult to separate (Kumar et al., 2017). Once the material is crushed, sieves are used to classify the particles according to particle size. Literature review 44 Most authors employ particles smaller than 200 μm, although there are studies that use larger particle sizes (Table 9). Table 9. Pulp densities (g L-1) and particle sizes (µm) reported in literature. Migroorganism Particle size Pulp density Metalsa Reference A. ferrooxidans 37-150 1-20 (optimal: 8.5) Cu, Ni Arshadi and Mousavi., 2015b Bacillus megaterium 37-150 1-20 (optimal: 8.1) Cu, Au Arshadi et al., 2016 S. thermosulfidooxidans 50-150 10 Cu, Ni, Al, Zn Ilyas et al., 2007 A. ferrooxidans and A. thiooxidans 100200 4-12 Cu, Zn, Ni Liang et al., 2010 A. ferrivorans and A. thiooxidans ≤ 500 5-50 (optimal: 10) Cu, Ag Isildar et al., 2016 P. putida and P. fluorescens ≤ 500 5-50 (optimal: 10) Cu, Ag Isildar et al., 2016 Enriched mixed culture (origin: active sludge) ≤ 180 50 Cu Wang et al., 2018 Microorganism consortium dominated by L. ferriphilum ≤ 200250 10-100 (optimal: 10) Cu, Fe, Zn, Ni, Pb, Cd, Au, Ag, Co Khatri et al., 2018 A. thiooxidans <75 10-50 (optimal: 30) Li, Co, Mn Naseri et al., 2019 A. ferrooxidans and A. acidophilum 751000 7.5−15 (optimal: 7.5) Cu, Zn. Ni, Pb, Ag, Au, Sc, Ce, La, Nd Priya and Hait, 2020 A. ferrooxidans < 100 5-100 Co, Li Roy et al., 2021 aanalyzed dissolved metals 6.2.2. Treatment mode: single and multi-stages bioleaching The bioleaching experiments with PCBs can be carried out in one (Arshadi et al., 2016; Khatri et al., 2018) or two stages (Brandl et al., 2001; Shah et al., 2015; Isildar et al., 2016; Khatri et al., 2018; Marra et al., 2018). In the first case, the extraction process takes place in the presence of the microorganisms (biotic medium), while in the twostage process the metal extraction is carried out by immersing the PCB in the filtered supernatant (abiotic medium) that is obtained in a previous stage where the microbial oxidation of Fe2+ to Fe3+ occurs. The two-stage operation allows a better control of the Literature review 45 process, since the parameters corresponding to the biotic stage and the abiotic stage can be optimized separately. 6.2.3. Pulp density The pulp density is the ratio between the amount of material to be treated (usually powder or dust) and the volume of leaching medium (Xin et al., 2012). This is another relevant parameter because high values of pulp density can be detrimental for the extraction if the samples are not previously pretreated. In this case, the amount of dissolved organic material in the medium can be too high, which can definitively inhibit microbial activity (Ilyas et al., 2007; Vestola et al., 2010; Zhou et al., 2013; Valix, 2017). Obviously the higher the pulp density, the higher metal extraction, which would quickly increase toxicity of the medium. Likewise, a low material:medium ratio requires large reactors and higher investment costs (Valix, 2017), as well as a greater energy requirement for heating and mixing. Some of the pulp density values tested by different authors are shown in Table 9. The optimal concentration reported by most authors is equal to or lower than 10 g PCB L-1. However, Liang et al. (2010) proposed a procedure based on the addition of the PCB powder in three steps (4 g L-1 after 48 h of culture, 6 g L-1 after 96 h and 8 g L-1 after 144 h). A total concentration of 18 g L-1 was treated and the leaching results were 93, 89, 91 and 86 % for Cu, Ni, Zn and Pb, respectively. Similarly, Khatri et al. (2018) reported that it is possible to obtain a 95 % solubilization of Cu and more than 50 % of Ni by using a PCB pulp density of 100 g L-1 (10 %), although the values obtained were lower than those registered with lower pulp densities. 7. REFERENCES Abraham, J., Chatterjee, A., Sharma, J., 2020. Isolation and Characterization of a New Bacillus licheniformis Strain for Bioleaching Heavy Metals. Journal of Applied Biotechnology Reports. 7, 139-144. Adhapure, N.N., Waghmare, S.S., Hamde, V.S., Deshmuck, A.M., 2013. Metal solubilization from powdered printed circuit boards by microbial consortium from bauxite and pyrite ores. Applied Biochemistry and Microbiology. 49, 256-262. Adhapure, N.N., Dhakephalkar, P.K., Dhakephalkar, A.P., Tembhurkar, V.R., Rajgure, A.V., Deshmuck, A. M., 2014. Use of large pieces of printed circuit boards for bioleaching to avoid precipitate contamination problem’ and to simplify overall metal recovery. MethodsX. 1, 181-186. Akhlaghi, N., 2019. Hemp fibres as novel green support material for immobilization of Acidithiobacillus ferrooxidans. International Journal of Engineering. 32, 1225-1230. Literature review 46 Aksu, Z., Karabayır, G., 2008. Comparison of biosorption properties of different kinds of fungi for the removal of Gryfalan Black RL metal-complex dye. Bioresource Technology. 99, 77307741. Amiri, F., Mousavi, S.M., Yaghmaei, S., Barati M., 2012. Bioleaching kinetics of a spent refinery catalyst using Aspergillus niger at optimal conditions. Biochemical Engineering Journal. 67, 208-217. Anjum, F., Bhatti, H.N., Ghauri, M.A., Bhatti, I.A., Asgher, M., Asi, M.R., 2009. Bioleaching of copper, cobalt and zinc from black shale by Penicillium notatum. African Journal of. Biotechnology. 8, 5038-5045. Annamalai, M., Gurumurthy, K., 2019. Enhanced bioleaching of copper from circuit boards of computer waste by Acidithiobacillus ferrooxidans. Environmental Chemistry Letters. 17, 1873-1879. Arshadi, M., Mousavi, S., 2015a. Enhancement of simultaneous gold and copper extraction from computer printed circuit boards using Bacillus megaterium. Bioresource Technology. 175, 315-324. Arshadi, M., Mousavi, S.M., 2015b. Multi-objective optimization of heavy metals bioleaching from discarded mobile phone PCBs: Simultaneous Cu and Ni recovery using Acidithiobacillus ferrooxidans. Separation and Purification Technology. 147, 210-219. Arshadi, M., Mousavi, S.M., Rasoulnia, P., 2016. Enhancement of simultaneous gold and copper recovery from discarded mobile phone PCBs using Bacillus megaterium: RSM based optimization of effective factors and evaluation of their interactions. Waste Management. 57, 158-167. Arshadi, M., Yaghmaei. S., 2020. Advances in bioleaching of copper and nickel from electronic waste using Acidithiobacillus ferrooxidans: evaluating daily pH adjustment. Chemical Papers. 74, 2211-2227. Arya, S., Kumar, S., 2020. Bioleaching: urban mining option to curb the menace of e-waste challenge. Bioengineered. 11, 640-660. Badurdeen, F., Jawahir, I.S., 2017. Strategies for value creation through sustainable manufacturing. Procedia Manufacturing. 8, 20-27. Bahafid, W., Joutey, N.T., Asri, M., Sayel, H., Tirry, N., El Ghachtouli, N., Sayel, N.T.H., 2017. Yeast Biomass: An Alternative for Bioremediation of Heavy Metals. Yeast-Industrial Applications, 2017. Bahaloo-Horeh, N., Mousavi, S. M., Baniasadi, M., 2018. Use of adapted metal tolerant Aspergillus niger to enhance bioleaching efficiency of valuable metals from spent lithiumion mobile phone batteries. Journal of Cleaner Production. 197, 1546-1557. Bajestani, M.I., Mousavi, S.M., Shojaosadati, S.A., 2014. Bioleaching of heavy metals from spent household batteries using Acidithiobacillus ferrooxidans: statistical evaluation and optimization. Separation and Purification Technology. 132, 309-316. Balde, C. P., Forti, V., Gray, V., Kuehr, R., Stegmann, P., 2017. The Global e-Waste Monitor 2017: Quantities, Flows and Resources (United Nations University, International Telecommunication Union and International Solid Waste Association). Literature review 47 Baniasadi, M., Vakilchap, F., Bahaloo-Horeh, N., Mousavi, S.M., Farnaud, S., 2019. Advances in bioleaching as a sustainable method for metal recovery from e-waste: A review. Journal of Industrial and Engineering Chemistry. 76, 75-90. Bankar, A., Winey, M., Prakash, D., Kumar, A.R., Gosavi, S., Kapadnis, B., Zinjarde, S., 2012. Bioleaching of fly ash by the tropical marine yeast, Yarrowia lipolytica NCIM 3589. Applied Biochemistry and Biotechnology. 168, 2205-2217. Barnett, M., Palumbo-Roe, B., Gregory, S., 2018. Comparison of heterotrophic bioleaching and ammonium sulfate ion exchange leaching of rare earth elements from a madagascan ion-adsorption clay. Minerals. 8, 236. Barona, A., Etxebarria, M.B., Rojo, N., Santaolalla, A., Otegi, J.R., Díaz-Tena, E., Gallastegui, G., 2018. A perspective on a white biotechnology under development: biomachining. Current Topics in Biotechnology. 9, 45-54. Barron, J.L.C., Lueking, D.R., 1990. Growth and maintenance of Thiobacillus ferrooxidans cells. Applied and Environmental Microbiology. 56, 2801-2806. Barron, M.G., Galbraith, H., Beltman, D., 1995. Comparative reproduction and developmental toxicology of birds. Comparative Biochemistry and Physiology. 112c, 1-14. Benzal, E., Cano, A., Solé, M., Lao-Luque, C., Gamisans, X., Dorado, A.D., 2020. Copper recovery from PCBs by Acidithiobacillus ferrooxidans: Toxicity of bioleached metals on biological activity. Waste Biomass Valorization. 11, 5483-5492 Beolchini, F., Fonti, V., Ferella, F., Centofanti, M., Veglio, F., 2009. Bioleaching of nickel, vanadium and molybdenum from spent refinery catalysts. Advanced Materials Research Institute. 71-73, 657-660. Beolchini, F., Fonti, V., Dell’Anno, A., Rocchetti, L., Veglio, F., 2012. Assessment of biotechnological strategies for the valorization of metal bearing wastes. Waste Management. 32, 949-956. Bharadwaj, A., Ting, Y.P., 2013. Bioleaching of spent hydro treating catalyst by acidophilic thermophilic Acidinaus brierleyi: leaching mechanism and effect of decoking. Bioresource Technology. 130, 673-680. Bigham, J.M., Jones, F.S., Özkaya, B., Sahinkaya, E., Puhakka, J.A., Tuovinen, O.H., 2010. Characterization of jarosites produced by chemical synthesis over a temperature gradient from 2 to 40 °C. International Journal of Mineral Processing. 94, 121-128. Birloaga, I., De Michelis, I., Ferella, F., Buzatu, M., Vegliò, F., 2013. Study on the influence of various factors in the hydrometallurgical processing of waste printed circuit boards for copper and gold recovery. Waste Management. 33, 935-941. Bolaños-Benítez, V., Hullebusch, E.D.V., Lens, P.N.L., Quantin, C., Vossenberg, J.V.D., Subramanian, S., Sivry, Y., 2018. (Bio)leaching Behavior of Chromite Tailings. MineralsBasel. 8, 261. Boon, M., 2001. The mechanism of ‘direct’ and ‘indirect’ bacterial oxidation of supplied minerals. Hydrometallurgy. 62, 67-70. Bosecker, K., 1987. Microbial recycling of mineral waste products. Acta Biotechnologica. 7, 487-497. Bosecker, K., 1997. Bioleaching: metal solubilisation by microorganisms. FEMS Microbiology Reviews. 20, 591-604. Literature review 48 Bosshard, P.P., Bachofen, R., Brandl, H., 1996. Metal leaching of fly ash from municipal waste incineration by Aspergillus niger. Environmental Science and Technology. 30, 3066-3070. Bosshard, R., Wegmann, M., 2001. Computer-munching microbes: metal leaching from electronic scrap by bacteria and fungi. Hydrometallurgy. 59, 319-326. Brandl H., Faramarzi M. A., 2006. Microbe-metal-interactions for the biotechnological treatment of metal-containing solid waste. China Particuology. 4, 93-97. Brandl, H., Lehmann, S., Faramarzi, M. A., Martinelli, D., 2008. Biomobilization of silver, gold and platinum from solid waste materials by HCN-forming microorganisms. Hydrometallurgy. 94, 14-17. Burgstaller W., Stxaser H., Shinner F., 1992. Solubilization of zinc oxide from filter dust with Penicillium simplicissimum: bioreactor, leaching and stoichiometry. Environmental Science and Technology. 26, 340-346. Burgstaller W., Schinner F., 1993. Leaching of metals with fungi. Journal of Biotechnology. 27, 91-116. Cabrera, G., Gómez, J.M., Cantero, D., 2005. Influence of heavy metals on growth and ferrous sulphate oxidation by Acidithiobacillus ferrooxidans in pure and mixed culture. Process Biochemistry. 40, 2683-2687. Castro, C., Donati, E., 2016. Effects of different energy sources on cell adhesion and bioleaching of a chalcopyrite concentrate by extremophilic archaeon Acidianus copahuensis. Hydrometallurgy. 162, 49-56. Chakankar, M., Su, C.H., Hocheng, H., 2019. Leaching of metals from end-of-life solar cells. Environmental Science and Pollution Research. 26, 29524-29531. Chang, J.H., Hocheng, H., Chang, H.Y., Shih, A., 2008. Metal removal rate of Thiobacillus thiooxidans without pre-secreted metabolite. Journal of Materials Processing Technology. 201, 560-564. Chatterjee, A., Das, R., Abraham, J., 2019. Bioleaching of heavy metals from spent batteries using Aspergillus nomius JAMK1. International journal of Environmental Science and Technology, 17, 49-66. Chi, T.D., Lee, J.C., Pandey, B.D., Yoo, K., Jeong, J., 2011. Bioleaching of gold and copper from waste mobile phone PCBs by using a cyanogenic bacterium. Minerals Engineering. 24, 12191222. Choi, M.S., Cho, K.S., Kim, D.S., Kim, D.J., 2004. Microbial recovery of copper from printed circuit boards of waste computer by Acidithiobacillus ferrooxidans. Journal of Environmental Science and Health. 39(1112), 2973-2982. Chojnacka, K., 2007. Bioaccumulation of Cr (III) ions by Blue-Green alga Spirulina sp. Part I. A comparison with biosorption. American Journal of Agricultural and Biological Sciences. 2, 218-223. Colmer, A.R., Temple, K.L., Hinkle, M.E., 1950. An iron-oxidizing bacterium from the acid drainage of some bituminous coal mines. Journal of Bacteriology. 59(3), 317-328. Cornu, J.Y., Huguenot, D., Jezequel, K., Lollier, M., Lebeau, T., 2017. Bioremediation of copper-contaminated soils by bacteria. World Journal of Microbiology and Biotechnology. 33, 26. Literature review 55 Lixandru, A., Venkatesan, P., Jönsson, C., Poenaru, I., Hall, B., Yang, Y., Gutfleisch, O., 2017. Identification and recovery of rare-earth permanent magnets from waste electrical and electronic equipment. Waste Management. 68, 482-489. Ma F., Huang H., Cui C., Mater J., 2020. Biomachining properties of various metals by microorganisms. Process Technology. 278, 116512. Mahmoud, A., Cézac, P., Hoadley, AFA., Contamine, F., D’Hugues, P., 2017. A review of sulfide minerals microbially assisted leaching in stirred tank reactors. International Biodeterioration and Biodegradation. 19, 118-146. Malki, M., González-Toril, E., Sanz, J.L., Gómez, F., Rodríguez, N., Amils, R., 2006. Importance of the iron cycle in biohydrometallurgy. Hydrometallurgy. 83, 223-228. Marra, A., Cesaro, A., Rene, E.R., Belgiorno, V., Lens, P.N.L., 2018. Bioleaching of metals from WEEE shredding dust. Journal of Environmental Management. 210, 180-190. Merum, P., Chu, Y., Shi, F., Cao, Y., 2017. Biological effects in Pseudomonas species by the non-thermal atmospheric pressure plasma conditions. Advances in Engineering Research. 143, 828-833. Mikkelsen, D., Kappler, U., McEwan, A.G., Sly, L.I., 2006. Archaeal diversity in two thermophilic chalcopyrite bioleaching reactors. Environmental Microbiology. 8, 2050-2056. Mikkelsen, D., Kappler, U., Webb, R.I., Rasch, R., McEwan, A.G., Sly, L.I., 2007. Visualisation of pyrite leaching by selected thermophilic archaea: Nature of microorganism-ore interactions during bioleaching. Hydrometallurgy. 88, 143-153. Mishra, D., Kim, D.J., Ahn, J.G., Rhee, Y.H., 2005. Bioleaching: a microbial process of metal recovery; A review. Metals and Materials International. 11, 249-256. Mishra, A., Malik, A., 2012. Simultaneous bioaccumulation of multiple metals from electroplating effluent using Aspergillus lentulus. Water Research. 46, 4991-4998. Mishra, D., Rhee, Y.H., 2014. Microbial leaching of metals from solid industrial wastes. Journal of Microbiology. 52, 1-7. Mizero, B., Musongo, T., Rene, E.R., Battes, F., Lens, P.N., 2018. Optimization of process parameters for the chemical leaching of base metals from telecom and desktop printed circuit boards. Process Safety and Environmental Protection. 120, 14-23. Modak, J.M., Natarajan, K.A., Mukhopadhyay, S., 1996. Development of temperaturetolerant strains of Thiobacillus ferrooxidans to improve bioleaching kinetics. Hydrometallurgy. 42, 51-61. Mohanty, B.K., Mishra A.K., 1993. Scanning electron micrograph study of Bacillus licheniformis during bioleaching of silica from magnesite ore. Journal of Applied Bacteriology. 74, 284-289. Moltó, J., Egea, S., Conesa, J.A., Font, R., 2011 Thermal decomposition of electronic wastes: Mobile phone case and other parts. Waste Management. 31, 2546-2552. Monneron-Enaud, B., Wiche, O., Schlömann, M., 2020. Biodismantling, a Novel Application of Bioleaching in Recycling of Electronic Wastes. Recycling. 5, 22. Mousavi, S. M., Yaghmaei, S., Jafari, A., 2007. Influence of process variables on biooxidation of ferrous sulfate by an indigenous Acidithiobacillus ferrooxidans. Part II: Bioreactor experiments. Fuel. 86, 993-999. Literature review 56 Moyo, T., Chirume, B.H., Petersen, J., 2020. Assessing alternative pre-treatment methods to promote metal recovery in the leaching of printed circuit boards. Resources, Conservation and Recycling. 152, 104545. Muhammad, I., Sana Ullah, S. M., Sup Han, D., Jo Ko, T., 2015. Selection of optimum process parameters of biomachining for maximum metal removal rate. International Journal of Precision Engineering and Manufacturing-Green Technology. 2, 307-313. Mykytczuk, N. C. S., Trevors, J. T., Ferroni, G. D., Leduc, L. G., 2011. Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans. Microbiological Research. 166, 186-206. Nasab, M.H., Noaparast, M., Abdollahi, H., Amoozegar, M.A., 2020. Indirect bioleaching of Co and Ni from iron rich laterite ore, using metabolic carboxylic acids generated by P. putida, P. koreensis, P. bilaji and A. niger. Hydrometallurgy 193, 105309. Naseri, T., Bahaloo-Horeh, N., Mousavi, S.M., 2019. Environmentally friendly recovery of valuable metals from spent coin cells through two-step bioleaching using Acidithiobacillus thiooxidans. Journal of Environmental Management. 235, 357-367. Natarajan, G., Ting, Y-P., 2014. Pretreatment of e-waste and mutation of alkali-tolerant cyanogenic bacteria promote gold biorecovery. Bioresource Technology. 152, 80-85. Nemati, M., Harrison, S.T.L., Hansford, G.S., Webb, C., 1998. Biological oxidation of ferrous sulphate by Thiobacillus ferrooxidans: a review on the kinetic aspects. Biochemical Engineering Journal. 1, 171-190. Nguyen, V. K., M. H. Lee, H. J. Park, and J. U. Lee., 2015. Bioleaching of arsenic and heavy metals from mine tailings by pure and mixed cultures of Acidithiobacillus spp. Journal of Industrial and Engineering Chemistry. 21, 451-458. Nie, H.C.Y., Zhu, N., Wu, P., Zhang, T., Zhang, Y., Xing, Y., 2015a. Isolation of Acidithiobacillus ferrooxidans strain Z1 and its mechanism of bioleaching copper from waste printed circuit boards. Journal of Chemical Technology and Biotechnology. 90, 714-721. Nie, H., Zhu, N., Cao, Y., Xu, Z., Wu, P., 2015b. Immobilization of Acidithiobacillus ferrooxidans on cotton gauze for the bioleaching of waste printed circuit boards. Applied Biochemistry and Biotechnology. 177, 675-688. Ning, C., Lin, C.S.K., Hui, D.C.W., McKay, G., 2017. Waste Printed Circuit Board (PCB) Recycling Techniques. Topics in Current Chemistry. 375, 43. Nithya, R., Sivasankari, C., Thirunavukkarasu, A., 2020. Electronic waste generation, regulation and metal recovery: a review. Environmental Chemistry Letters. Okoh, M.P., Olobayetan, I.W., Machunga-Mambula, S.S., 2018. Bioleaching, a technology for metal extraction and remediation: mitigating health consequences for metal exposure. International Journal of Sustainable Development. 7, 2103-2118. Ongondo, F.O., Williams, I.D., Cherrett, T.J., 2011. How are WEEE doing? A global review of the management of electrical and electronic wastes. Waste Management. 31, 714-730. Orell, A., Navarro, C.A., Arancibia, R., Mobarec, J.C., Jerez, C.A., 2010. Life in blue: Copper resistance mechanisms of bacteria and Archaea used in industrial biomining of minerals. Biotechnology Advances., 28, 839-848. Literature review 57 Ortuño, N., Moltó, J., Egea, S., Font, R., Conesa, J.A., 2013. Thermogravimetric study of the decomposition of printed circuit boards from mobile phones. Journal of Analytical and Applied Pyrolysis. 103, 189-200. Osman, Y., Gebreil, A., Mowafy, A.M., Anan, T.I., Hamed, S.M., 2019. Characterization of Aspergillus niger siderophore that mediates bioleaching of rare earth elements from phosphorites. World Journal of Microbiology and Biotechnology. 35, 93. Pal, S., Pradhan, D., Das, T., Sukla, L.B., Roy Chaudhury, G., 2010. Bioleaching of low-grade uranium ore using Acidithiobacillus ferrooxidans. Indian Journal of Microbiology. 50, 70-75. Palmieri, R., Bonifazi, G., Serranti, S., 2014. Recycling-oriented characterization of plastic frames and printed circuit boards from mobile phones by electronic and chemical imaging. Waste Management. 34, 2120-2130. Panda, S., Akcil, A., Pradhan, N., Deveci, H., 2015. Current scenario of chalcopyrite bioleaching: a review on the recent advances to its heap-leach technology. Bioresource Technology. 196, 694-706. Pant, D., Joshi, D., Upreti, M.K., Kotnala, R.K., 2012. Chemical and biological extraction of metals present in E waste: a hybrid technology. Waste Management. 32, 979-990. Park, C.-Y., Cheong, K.-H., Kim, B.-J., 2010. The bioleaching of sphalerite by moderately thermophilic bacteria. Economic and Environmental Geology. 43, 573-587. Pathak, A., Morrison, L., Healy, M.G., 2017. Catalytic potential of selected metal ions for bioleaching, and potential techno-economic and environmental issues: A critical review. Bioresource Technology. 229, 211-221. Pathak, A., Kothari, R., Vinoba, M., Habibi, N., Tyagi, V.V., 2021. Fungal bioleaching of metals from refinery spent catalysts: A critical review of current research, challenges, and future directions. Journal of Environmental Management. 280, 111789. Peng, T.J., Ma, L.Y., Feng, X., Tao, J.M., Nan, M.H., Liu, Y.D., Li, J.K., Shen, L., Wu, X.L., Yu, R.L., Liu, X.D., Qiu, G.Z., Zeng, W.M., 2017. Genomic and transcriptomic analyses reveal adaptation mechanisms of an Acidithiobacillus ferrivorans strain YL15 to alpine acid mine drainage. PLoS ONE. 12, 0178008. Pham, V., Ting, Y. P., 2009. Gold bioleaching of electronic waste by cyanogenic bacteria and its enhancement with bio-oxidation. Advanced Materials Research. 71, 661-664. Plumb, J.J., Muddle, R., Franzmann, P.D., 2008. Effect of pH on rates of iron and sulfur oxidation by bioleaching organisms. Minerals Engineering. 21, 76-82. Potysz, A., Lens, P.N.L., van de Vossenberg, J., Rene, E.R., Grybos, M., Guibaud, G., Kierczak, J., van Hullebusch, E.D., 2016. Comparison of Cu, Zn and Fe bioleaching from Cumetallurgical slags in the presence of Pseudomonas fluorescens and Acidithiobacillus thiooxidans. Applied Geochemistry. 68, 39-52. Pourhossein, F., Mousavi, S.M., 2018. Enhancement of copper, nickel, and gallium recovery from led waste by adaptation of Acidithiobacillus ferrooxidans. Waste Management. 79, 98108. Pradhan, N., Nathsarma, K., Rao, K.S., Sukla, L., Mishra, B., 2008. Heap bioleaching of chalcopyrite: a review. Minerals Engineering. 21, 355-365. Literature review 58 Pradhan, J.K., Kumar, S., 2012. Metals bioleaching from electronic waste by Chromobacterium violaceum and Pseudomonads sp. Waste Management and Research. 30, 1151-1159. Priya, A., Hait, S., 2017. Comparative assessment of metallurgical recovery of metals from electronic waste with special emphasis on bioleaching. Environmental Science and Pollution Research. 24, 6989-7008. Priya, A., Hait, S., 2018. Extraction of metals from high grade waste printed circuit board by conventional and hybrid bioleaching using Acidithiobacillus ferrooxidans. Hydrometallurgy. 177, 132-139. Priya, A., Hait, S., 2020. Biometallurgical recovery of metals from waste printed circuit boards using pure and mixed strains of Acidithiobacillus ferrooxidans and Acidiphilium acidophilum. Process Safety and Environmental Protection. 143, 262-272. Pronk, J.T., de Bruyn, J.C., Bos, P., Kuenen, J.G., 1992. Anaerobic growth of Thiobacillus ferrooxidans. Applied and Environmental Microbiology. 58, 2227-2230 Qu, Y., Lian, B., Mo, B., Liu, C., 2013. Bioleaching of heavy metals from red mud using Aspergillus niger. Hydrometallurgy. 136, 71-77. Quatrini, R., Appia-Ayme, C., Denis, Y., Ratouchniak, J., Veloso, F., Valdes, J., Lefimil, C., Silver, S., Roberto, F., Orellana, O., Denizot, F., Jedlicki, E., Holmes, D.S., Bonnefoy, V., 2006. Insights into the iron and sulfur energetic metabolism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling. Hydrometallurgy. 83, 263-272. Quatrini, R., Escudero, L.V., Moya-Beltrán, A., Galleguillos, P.A., Issotta, F., Acosta, M., Cárdenas, J.P., Nuñez, H., Salinas, K., Holmes, D.S., 2017. Draft genome sequence of Acidithiobacillus thiooxidans CLST isolated from the acidic hypersaline Gorbea salt flat in northern Chile. Standards in Genomic Sciences. 12, 1-8. Rasoulnia, P., Mousavi, S.M., 2016. Maximization of organic acids production by Aspergillus niger in a bubble column bioreactor for V and Ni recovery enhancement from power plant residual ash in spent-medium bioleaching experiments. Bioresource Technology. 216, 729736. Ravindra P., Kodli B., Veera Rao V.P.R., 2015. Effect of Adaptation of Acidithiobacillus ferrooxidans on Ferrous Oxidation and Nickel Leaching Efficiency. Advances in Bioprocess Technology. 17-26. Rawlings, D.E., 1997. Mesophilic, autotrophic bioleaching bacteria: description, physiology and role. In: Rawlings DE, ed. Biomining: Theory, Microbes and Industrial Processes. Springer-Verlag, Berlin, 229-245. Rawlings, D.E., 2005. Characteristics and adaptability of ironand sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microbial Cell Factories. 4, 13. Rawlings, D.E., Johnson, D.B., 2007 The microbiology of biomining: development and optimization of mineral-oxidizing microbial consortia. Microbiology. 153, 315-324. Ren, W., Li, P., Geng, Y., Li, X., 2009. Biological leaching of heavy metals from a contaminated soil by Aspergillus niger. Journal of Hazardous Materials. 167, 164-169. Literature review 59 Rezza, I., Salinas, E., Elorza, M., Tosetti, M., Donati, E., 2001. Mechanisms involved in bioleaching of an aluminosilicate by heterotrophic microorganisms. Process Biochemistry. 36, 495-500. Rohwerder, T., Gehrke, T., Kinzler, K., Sand, W., 2003. Bioleaching review part A: Progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Applied Microbiology and Biotechnology. 63, 239-248. Rojas-Chapana, J.A., Bärtels, C.C., Pohlmann, L., Tributsch, H., 1998. Co-operative leaching and chemotaxis of Thiobacillus studied with spherical sulfur/sulfide substrates. Process Biochemistry. 33, 239-248. Roshani, M., Shojaosadati, S.A., Safdari, S.J., Vasheghani-Farahani, E., Mirjalili, K., Manafi, Z., 2017. Bioleaching of molybdenum by two new thermophilic strains isolated and characterized. Iranian Journal of Chemistry and Chemical Engineering. 36, 183-194. Roy, J. J., Madhavi, S., Cao, B., 2021. Metal extraction from spent lithium-ion batteries (LIBs) at high pulp density by environmentally friendly bioleaching process. Journal of Cleaner Production. 280, 124242. Rozas, E. E., Mendes, M. A., Nascimento, C. A. O., Espinosa, D. C.R., Oliveira, R., Oliveira, G., Custodio., M. R., 2017. Bioleaching of electronic waste using bacteria isolated from the marine sponge Hymeniacidon heliophila (Porifera). Journal of Hazardous Materials. 329, 20130. Ruan, J., Zhu, X., Qian, Y., Hu, J., 2014. A new strain for recovering precious metals from waste printed circuit boards. Waste Management. 34, 901-907. Sajjad, W., Zheng, G., Din, G., Ma, X., Rafiq, M., Xu, W., 2019. Metals extraction from sulfide ores with microorganisms: the bioleaching technology and recent developments. Transactions of the Indian Institute of Metals. 72, 559-579. Saleh, D.K., Abdollahi, H., Noaparast, M., Nosratabad, A.F., Tuovinen, O.H., 2019. Dissolution of Al from metakaolin with carboxylic acids produced by Aspergillus niger, Penicillium bilaji, Pseudomonas putida, and Pseudomonas koreensis. Hydrometallurgy. 186, 235-243. Samadzadeh Yazdi, M., Abdollahi, M., Mousavi, S., Khodadadi Darban., 2020. A. Comparison of Copper Dissolution in Chalcopyrite Concentrate Bioleaching with Acidianus Brierleyi in Different Initial pH Values. Journal of Mining and Environment. 11, 753-764. Sand, W., Gehrke, T., Jozsa, P-G., Schippers, A., 2001. (Bio)chemistry of bacterial leaching— direct vs indirect bioleaching. Hydrometallurgy. 59, 159-175 Santaolalla A., Gutierrez, J., Gallastegui G., Barona A., Rojo N., 2021. Immobilization of Acidithiobacillus ferrooxidans in bacterial cellulose for a more sustainable bioleaching process. Journal of Environmental Chemical Engineering. 9, 105283 Santhiya, D., Ting, Y. P., 2005. Bioleaching of spent refinery processing catalyst using Aspergillus niger with high-yield oxalic acid. Journal of Biotechnology. 116, 171-184. Senophiyah-Mary, J., Loganath, R., Meenambal, T., 2018. A novel method for the removal of epoxy coating from waste printed circuit board. Waste Management and Research. 36, 645-652. Shabani, M.A., Irannajad, M., Azadmehr, A.R., Meshkini, M., 2013. Bioleaching of copper oxide ore by Pseudomonas aeruginosa. International Journal of Minerals Metallurgy and Materials. 20, 1130-1133. Literature review 60 Shah, M.B., Tipre, D.R., Purohit, M.S., Dave, S.R., 2015. Development of two-step process for enhanced biorecovery of Cu–Zn–Ni from computer printed circuit boards. Journal of Bioscience and Bioengineering. 120, 167-173. Sheng, P.P., Etsell, T.H., 2007. Recovery of gold from computer circuit board scrap using aqua regia. Waste Management and Research. 25, 380-383. Shenker, M., Hadar, Y., Chen, Y., 1999. Kinetics of iron complexing and metal exchange in solutions by rhizoferrin, a fungal siderophore. Soil Science Society of America Journal. 63, 1681-1687. Shin, D., Jeong, J., Lee, S., Pandey, B., Lee, J-C., 2013. Evaluation of bioleaching factors on gold recovery from ore by cyanide-producing bacteria. Minerals Engineering. 48, 20-24. Shin, D., Park, J., Jeong, J., Kim, B., 2015. A biological cyanide production and accumulation system and the recovery of platinum-group metals from spent automotive catalysts by biogenic cyanide. Hydrometallurgy. 158, 10-18. Silvas, F.P.C., Correa, M.M.J., Caldas, M.P.K., de Moraes, V.T., Espinosa, D.C.R., Tenório, J.A.S., 2015. Printed circuit board recycling: physical processing and copper extraction by selective leaching. Waste Management. 46, 503-510. Singh, A., Manikandan, A., Ravi Sankar, M., Paksirajan, K., Roy, L., 2018. Experimental investigations and surface morphology of bio-micromachining on copper. Materials Today: Proceedings. 5, 4225-4234. Sodha, A.B., Tipre, D.R., Dave, S.R., 2020. Optimisation of biohydrometallurgical batch reactor process for copper extraction and recovery from non-pulverized waste printed circuit boards. Hydrometallurgy. 191, 105170. Stott, M.B., Sutton, D.C., Watling, H.R., Franzmann, P.D., 2003. Comparitive leaching of chalcopyrite by selected Acidophilic Bacteria and Archaea. Geomicrobiology Journal. 20, 215-230. Štyriaková, I., Štyriak, I., Nandakumar, M. P., Mattiasson, B., 2003. Bacterial destruction of mica during bioleaching of kaolin and quartz sands by Bacillus cereus. World Journal of Microbiology and Biotechnology. 19, 583-590. Tang, J.A.; Valix, M., 2006. Leaching of low grade limonite and nontronite ores by fungi metabolic acids. Minerals Engineering. 19, 1274–1279. Temple, K.L., Colmer, A.R., 1951. The autotrophic oxidation of iron by a new bacterium: Thiobacillus ferrooxidans. Journal of Bacteriology. 62, 605-611. Tesfaye, F., Lindberg, D., Hamuyuni, J., Taskinen, P., Hupa, L., 2017. Improving urban mining practices for optimal recovery of resources from e-waste. Minerals Engineering. 111, 209221. Thangavelu, V., Tang, J., Ryan, D., Valix, M., 2006. Effect of saline stress on fungi metabolism and biological leaching of weathered saprolite ores. Minerals Engineering. 19, 1266-1273. Tributsch, H., 2001. Direct vs indirect bioleaching. Hydrometallurgy. 59, 177-185. Uno, Y., Kaneeda, T., Yokomizo, S., 1993. Fundamental study on biomachining, machining of metals by Thiobacillus ferrooxidans. JSME International Journal. 39, 837-842. Vakilchap, F., Mousavi, S.M., Shojaosadati, S.A., 2016. Role of Aspergillus niger in recovery enhancement of valuable metals from produced red mud in Bayer process. Bioresource Technology. 218, 991-998. Literature review 61 Valdés, J., Pedroso, I., Quatrini, R., Dodson, R.J., Tettelin, H., Blake, R., Eisen, J.A., Holmes, D.S., 2008. Acidithiobacillus ferrooxidans metabolism: from genome sequence to industrial applications. BMC Genomics. 9, 597. Valix, M., 2017. Bioleaching of electronic waste: milestones and challenges. In: Current Developments in Biotechnology and Bioengineering: Solid Waste Management. Ed: Elsevier. 407-440. Van Yken, J., Cheng, K.Y., Boxall, N.J., Nikoloski, A.N., Moheimani, N., Valix, M., Sahajwalla, V., Kaksonen A.H., 2020. Potential of metals leaching from printed circuit boards with biological and chemical lixiviants. Hydrometallurgy. 196, 105433. Velásquez, L., Dussan, J., 2009. Biosorption and bioaccumulation of heavy metals on dead and living biomass of Bacillus sphaericus. Journal of Hazardous Materials. 167, 713-716. Verma P., Sodhi A.K., Bhanot N., 2019. Application of Aspergillus Niger for Biomachining of Aluminium Alloy 4004. In: Agnihotri A., Reddy K., Bansal A. (eds) Sustainable Engineering. Lecture Notes in Civil Engineering, vol 30. Springer, Singapore. Vermeulen, F., Nicolay, X., 2017. Sequential bioleaching of copper from brake pads residues using encapsulated bacteria. Minerals Engineering. 106, 39-45. Vestola, E.A., Kuusenaho, M.K., Närhi, H.M., Tuovinen, O.H., Puhakka, J.A., Plumb, J.J., Kaksonen, A.H., 2010. Acid bioleaching of solid waste materials from copper, steel and recycling industries. Hydrometallurgy. 103, 74-79. Vilcáez, J., Suto, K., Inoue, C., 2008. Bioleaching of chalcopyrite with thermophiles: Temperature-pH-ORP dependence. International Journal of Mineral Processing. 88, 37-44. Vrdoljak, G.A., S.C. Spiller., 2005. Characterization of an Acid Mine Biofilm by Cryo Scanning Electron Microscopy. Microscopy and Microanalysis. 11, 292-293. Wang, J.W., Bai, J.F., Xu, J.Q., Liang, B., 2009. Bioleaching of metals from printed wire boards by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans and their mixture. Journal of Hazardous Materials. 172, 1100-1105. Wang, Y.G., Su, L.J., Zhang, L.J., Zeng, W.M., Wu, J.Z., Wan, L.L., Qiu, G.Z., Chen, X.H., Zhou, H.B., 2012. Bioleaching of chalcopyrite by defined mixed moderately thermophilic consortium including a marine acidophilic halotolerant bacterium. Bioresource Technology. 21, 348-354. Wang, S., Zheng, W., Chen, L., Mahadevan, G. D., Zhao, F., 2016. Enhanced bioleaching efficiency of metals from e-wastes driven by biochar. Journal of Hazardous Materials. 320, 393-400. Wang, S., Chen, L., Zhou, X., Yan, W., Ding, R., Chen, B., Wang, C. T., Zhao, F., 2018. Enhanced bioleaching efficiency of copper from printed circuit boards without iron loss. Hydrometarllurgy. 180, 65-71. Watling, H.R., 2006. The bioleaching of sulphide minerals with emphasis on copper sulphidesA review. Hydrometallurgy. 84, 81-108. Whulanza, Y., Nadhif, H., Istiyanto, J., Supriadi, S., Bachtiar, B., 2016. PDMS surface modification using biomachining method for biomedical application. Journal of Biomimetics Biomaterials and Biomedical Engineering. 26, 66-72. Widmer, R., Oswald-Krapf, H., Sinha-Khetriwal, D., Schnellmann, M., Böni, H., 2005. Global perspectives on e-waste. Environmental Impact Assessment Review. 25, 436-458. Literature review 62 Wu, H.Y., Ting, Y.P., 2006. Metal extraction from municipal solid waste (MSW) incinerator fly ash—Chemical leaching and fungal bioleaching. Enzyme and Microbial Technology. 38, 839-847. Wu, X., Hu, Q., Hou, D., Miao, B., Liu, X., 2010. Differential gene expression in response to copper in Acidithiobacillus ferrooxidans strains possessing dissimilar copper resistance. Journal of General and Applied Microbiology. 56, 491-498. Wu, W., Liu, X., Zhang, X., Zhu, M., Tan, W., 2018. Bioleaching of copper from waste printed circuit boards by bacteria-free cultural supernatant of iron–sulfur-oxidizing bacteria. Bioresources and Bioprocessing. 5, 10. Xenofontos, E., Feidiou, A., Constantinou, M., Constantinides, G., Vyrides, I., 2015. Copper biomachining mechanisms using the newly isolated Acidithiobacillus ferrooxidans B1. Corrosion Science. 100, 642-650. Xia, J-l., Peng, A-a., He, H., Yang, Y., Liu, X-d., Qiu, G-z., 2007. A new strain Acidithiobacillus albertensis BY-05 for bioleaching of metal sulfides ores. The Transactions of Nonferrous Metals Society of China. 17, 168. Xia, M., Bao, P., Liu, A., Wang, M., Shen, L., Yu, R., Liu, Y., Chen, M., Li, J., Wu, X., Qiu, G., Zeng, W., 2018. Bioleaching of low-grade waste printed circuit boards by mixed fungal culture and its community structure analysis. Resources, Conservation and Recycling. 136, 267-275. Xiang, Y., Wu, P., Zhu, N., Zhang, T., Liu, W., Wu, J., Ping, L., 2010. Bioleaching of copper from waste printed circuit boards by bacterial consortium enriched from acid mine drainage. Journal of Hazardous Materials. 184, 812-818. Xin, B., Jiang, W., Li, X., Zhang, K., Liu, C., 2012. Analysis of reasons for decline of bioleaching efficiency of spent Zn–Mn batteries at high pulp densities and exploration measure for improving performance. Bioresource Technology. 112, 186-192. Yamane, L.H., de Moraes, V.T., Espinosa, D.C., Tenório, J.A., 2011. Recycling of WEEE: Characterization of spent printed circuit boards from mobile phones and computers. Waste Management. 31, 2553-2558. Yang, T., Xu, Z., Wen, J., Yang, L., 2009a. Factors influencing bioleaching copper from waste printed circuit boards by Acidithiobacillus ferrooxidans. Hydrometallurgy. 97, 29-32. Yang, Y., Wang, X., Liu, Y., Wang, S., Wen, W., 2009b. Techniques for micromachining using Thiobacillus ferrooxidans based on different culture medium. Applied Mechanics and Materials. 16-19, 1053-105. Liu, J., Yang, J., 2011. Leaching copper from shredded particles of waste printed circuit boards. Journal of Hazardous Materials. 187, 393-400. Yang, Y., Chen, S., Li, S., Chen, M., Chen, H., Liu, B., 2014. Bioleaching waste printed circuit boards by Acidithiobacillus ferrooxidans and its kinetics aspect. Journal of Biotechnology. 173, 24-30. Yang, C., Zhu, N., Shen, W., Zhang, T., Wu, P., 2017. Bioleaching of copper from metal concentrates of waste printed circuit boards by a newly isolated Acidithiobacillus ferrooxidans strain Z1. Journal of Material Cycles and Waste Management. 19, 247-255. Yin, N.H., Sivry, Y., Avril, C., Borensztajn, S., Labanowski, J., Malavergne, V., Lens, P.N.L., Rossano, S., van Hullebusch, E.D., 2014. Bioweathering of lead blast furnace metallurgical Literature review 63 slags by Pseudomonas aeruginosa. International Biodeterioration and Biodegradation. 86, 372-381. Yu, R., Liu, Z., Yu, Z., Wu, X., Shen, L., Liu, Y., Zeng, W., 2019. Relationship among the secretion of extracellular polymeric substances, heat resistance, and bioleaching ability of Metallosphaera sedula. International Journal of Minerals, Metallurgy and Materials. 26, 1504-1511. Zhan, Y., Yang, M., Zhang, S., Zhao, D., Duan, J., Wang, W., Yan, L., 2019. Iron and sulfur oxidation pathways of Acidithiobacillus ferrooxidans. World Journal of Microbiology and Biotechnology. 35, 60. Zhang, Y., Yang, Y., Liu, J., Qiu, G., 2013. Isolation and characterization of Acidithiobacillus ferrooxidans strain QXS-1 capable of unusual ferrous iron and. sulfur utilization. Hydrometallurgy. 136, 51-57. Zhang, G., Chao, X., Guo, P., Cao, J., Yang, C., 2015. Catalytic effect of Ag+ on arsenic bioleaching from orpiment (As2S3) in batch tests with Acidithiobacillus ferrooxidans and Sulfobacillus sibiricus. Journal of Hazardous Materials. 283, 117-22. Zhang, X.Q., Li, Y.S., 2017. Changes in shale oil composition and yield after bioleaching by bacillus mucilaginosus and thiobacillus ferrooxidans. Oil Shale. 34, 146. Zhang, S., Yan, L., Xing, W., Chen, P., Zhang, Y., Wang, W., 2018. Acidithiobacillus ferrooxidans and its potential application. Extremophiles 22, 563-579. Zhao, H., Hu, M., Li, Y., Zhu, S., Qin, W., Qiu, G., 2015. Wang, J. Comparison of electrochemical dissolution of chalcopyrite and bornite in acid culture medium. Transactions of Nonferrous Metals Society of China. 25, 303-313. Zhao, H., Zhang, Y., Zhang, X., Qian, L., Sun, M., Yang, Y., Zhang, Y., Wang, J., Kim, H., Qiu, G., 2019. The dissolution and passivation mechanism of chalcopyrite in bioleaching: An overview. Minerals Engineering. 136, 140-54. Zheng, G., Zhou, L., Wang, S., 2009. An acid-tolerant heterotrophic microorganism role in improving tannery sludge bioleaching conducted in successive multibatch reaction systems. Environmental Science and Technology. 43, 4151-4156. Zhou, H.B., Liu, X., Fu, B., Qiu, G., Huo, Q., Zeng, W.M., Liu, J.S.H., Chen, X.H., 2007. Isolation and characterization of Acidithiobacillus caldus from several typical environments in China. Journal of Central South University of Technology. 14, 163-169 Zhou, X., Guo, J., Lin, K., Huang, K., Deng, J., 2013. Leaching characteristics of heavy metals and brominated flame-retardants from waste printed circuit boards. Journal of Hazardous Materials. 246-247, 96-102. Zhu, W., Xia, J., Yang, Y., Nie, Z.Y., Zheng, L., Ma, C.Y., Zhang, R.Y., Peng, A., Tang, L., Qiu, G., 2011. Sulfur oxidation activities of pure and mixed thermophiles and sulfur speciation in bioleaching of chalcopyrite. Bioresource Technology. 102, 3877-3882. Zhu, N., Shi, C., Shang, R., Yang, C., Xu, Z., Wu, P., 2017. Immobilization of Acidithiobacillus ferrooxidans on cotton gauze for biological oxidation of ferrous ions in a batch bioreactor. Biotechnology and Applied Biochemistry. 64, 27-734. Zotti, M., Di Piazza, S., Roccotiello, E., Lucchetti, G., Mariotti, M. G., Marescotti, P., 2014. Microfungi in highly copper-contaminated soils from an abandoned Fe–Cu sulphide mine: Growth responses, tolerance and bioaccumulation. Chemosphere. 117, 471-476. Literature review 64 Materials and general methods 71 2.4. Metal removal rate and specific metal removal rate The metal removal rate (MRR) and the specific metal removal rate (SMRR) were determined by applying the procedures followed by Jadhav et al. (2013), Muhammad et al. (2015), Diaz-Tena et al. (2016) and Ma et al. (2020). During the copper solubilization tests, each workpiece was regularly taken out from the medium, washed with distilled water and ethanol (96 %), dried and weighed in an analytical balance (Denver instruments, SI-234 230 g/0.1 mg. The MRR and SMRR were calculated as shown in the equations below (Equation 16 and 17): MRR (mg h‐1) = Amount of metal removed (mg) Time (h) (Eq. 15) SMRR (mg h‐1 cm‐2) = MRR Area (cm2) (Eq. 16) 2.5. Determination of iron species in solution The ferrous (Fe2+) and total iron concentration were determined using the 2,2’-dipyridyl molecular absorption spectrophotometry method (adapted from the ‘3500-Fe B’ colorimetric procedure of the Standard Methods for the Examination of Water and Wastewater (Eaton et al., 1998; Diaz-Tena et al., 2016). The details about the reagents used in this analytical method are shown in Table 11. Table 11. Reagents used in the colorimetric method for ferrous and total iron determination. Solution Composition Preparation method Objective Iron standard solution (0.05 mg L-1) Standard iron solution 1 g Fe L-1 in 2 % HNO3 Dilute 5 mL of the standard solution in 100 mL of deionized water Obtaining the calibration curve Ammonium acetate / Acetic acid Buffer Ammonium acetate 98 % Acetic acid glacial 99.8 % Dissolve 280 g of ammonium acetate in 1 L of deionized water. Add glacial acetic acid until pH 5.5 is reached Keep the pH stable around 5.5 Hydroxylamine hydrochloride 10 % (w:v) Hydroxylamine hydrochloride Dissolve 5.0 g of the salt in 50 mL of deionized water Reduce Fe3+ to Fe2+ Solution 2.2 dipyridyl 0.5 % (w:v) 2,2’-dipyridyl Ethanol 96 % Dissolve 0.5 g of the salt in 100 mL of 96 % ethanol (v:v) React with Fe2+ to give a color complex Materials and general methods 72 It is essential to determine the concentration of both ferric and ferrous iron throughout the process, since both values are indicative of the process progress. Nevertheless, only the ferrous Fe2+ ion can be measured by the colorimetric method. Consequently, if the Fe total amount is to be determined, the Fe3+ has to be reduced to Fe2+ and the original Fe2+ and the reduced Fe2+ will be quantified simultaneously, rendering the total iron amount. Obviously, the Fe3+ concentration will be determined by subtraction. Prior to the measurement, it was necessary to obtain the calibration curve. The calibration standards of 0, 1, 2, 5 and 10 mg Fe3+ L-1 were prepared from a certified solution of 0.05 g Fe3+ L-1 by adding the following solutions to each 50 mL volumetric flask (Figure 13): athe corresponding volume of the 0.05 mg Fe3+ L-1 solution for each standard b5 mL of buffer solution (ammonium acetate/acetic acid) to ensure the pH stability during the process c2 mL of the hydroxylamine hydrochloride 10 % solution to reduce the ferric iron to ferrous iron. Shaking vigorously for 5 min was recommended to ensure the complete reduction of ferric iron. This reagent will only be added if the total Fe amount is to be determined. Obviously, it will not be added for Fe2+ determination. The Fe3+ concentration will be calculated by subtracting the Fe2+ amount to the total concentration. d2 mL of the dipyridyl solution. All the ferrous iron containing samples developed a red color. All the solutions were stirred for 5 minutes to ensure the complete reaction. eDeionized water to make up to 50 mL. Figure 13. Iron standards for calibration curve. The absorbance of all the samples were measured with a visible spectrophotometer Jenway 6305 at a wavelength of 520 nm, using 1 cm and 4.5 mL PMMA cuvettes. Materials and general methods 73 Taking into account the dilution factor, the value of total iron and ferrous iron in the original solution was obtained by Equation 17, where Y is the absorbance value, b the cut-off point between the calibration curve and the vertical axis and a the calibration curve slope. Fe (g L‐1) = Y ‐ b a · 50 1 · 100 1 · 1 g 1000 mg (Eq. 17) 2.6. Metal content analysis (ICP and AAS) The metal concentration in the leachated solutions was measured by ICP-OES plasma spectrometry (Figure 14a). Inductively coupled plasma (ICP) is the ionization source that together with an optical emission spectrophotometer (OES) constitutes the ICP-OES equipment. Specifically, a Perkin Elmer OPTIMA 2000DV equipment with a CETAC U 5000AT + ultrasonic nebulizer was used. Additionally, Atomic absorption spectroscopy (Figure 14b) is also commonly used in metal mobilization process to analyze metal elements in liquid samples. In this thesis copper and iron content was also quantified in a Perkin Elmer AAnalyst 100 AAS equipment, depending on the experiment. Figure 14. ICP-OES plasma spectrometry (a) and Atomic absorption spectroscopy (b) equipment. All samples were filtered by a 0.45 μm filter before analysis and the calibration standards were prepared in a matrix as similar as possible to the samples. 2.7. Scanning electron microscopy (SEM) Regarding the scanning electron microscopy (SEM) imaging, samples were fixed in 2 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), washed in iso-osmolar cacodylate/sucrose buffer, and postfixed in 1 % osmium tetroxide in cacodylate buffer. Samples were then dehydrated through an ethanol series and washed in Materials and general methods 74 hexamethyldisilazane prior to air-drying. Finally, samples were mounted onto stubs and gold-coated using a JEOL fine-coat ion sputter JFC-1100. Samples were visualized and micrographed using a SEM (Hitachi S-4800) at 15 kV accelerating voltage. Those SEM imagines were obtained by the Sgiker service (Advanced Research Facilities) of the Universiy of the Basque Country (UPV/EHU). 2.8. Other methods The pH was measured with a Crison Basic 20 pH-meter equipped with a sensION+ 5010T pH electrode. The redox potential was recorded with a Thermo-Orion 920+ instrument equipped with an Orion 9778BNWPO Sur-Flow® electrode. 3. REFERENCES Almarcegui, R.J., Navarro, C.A., Paradela, A., Albar, J.P., von Bernath, D., Jerez, C.A., 2014. New copper resistance determinants in the extremophile Acidithiobacillus ferrooxidans: a quantitative proteomic analysis. Journal of Proteome Research. 13, 946-960. Meechan, P.J., Potts, J., 2020. Biosafety in microbiological and biomedical laboratories: United States Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institutes of Health. David, D.J., Pradhan, D., Das, T., 2008. Evaluation of iron oxidation rate of Acidithiobacillus ferrooxidans in presence of heavy metal ions. Mineral Processing and Extractive Metallurgy. 117, 56-61. Díaz-Tena, E., Gallastegui. G., Hipperdinger, M., Donati, E. R., Ramírez, M., Rodríguez, A., López de Lacalle, L. N., Elías, A., 2016. New advances in copper biomachining by ironoxidizing bacteria. Corrosion Science. 112, 385-392. Eaton, A.D., Clesceri L.S., Greenberg, A.E., Franson, M.A.H., 1998. Standard Methods forthe Examination of Water and Wastewater, American Public HealthAssociation, American Water Works Association, Water EnvironmentFederation, Washington DC. Istiyanto, J., Ko, T. J., Yoon, I.-C., 2010. A study on copper micromachining using microorganisms. International Journal of Precision Engineering and Manufacturing. 11, 659664, Jadhav, U., Hocheng, H., Weng, W.H., 2013. Innovative use of biologically produced ferric sulfate for machining of copper metal and study of specific metal removal rate and surface roughness during the process. Journal of Materials Processing Technology. 213, 1509-1515. Johnson, D., Warner, R. and Shih, A. J., 2007. Surface roughness and material removal rate in machining using microorganisms. Journal of Manufacturing Science and Engineering. 129, 223-227. Ma F., Huang H., Cui C., Mater J., 2020. Biomachining properties of various metals by microorganisms. Process Technology. 278, 116512. Muhammad, I., Sana Ullah, S. M., Sup Han, D., Jo Ko, T., 2015. Selection of optimum process parameters of biomachining for maximum metal removal rate. International Journal of Precision Engineering and Manufacturing-Green Technology. 2, 307-313. Materials and general methods 75 Nguyen, V.T., Flanagan, B., Gidley, M.J., Dykes, G.A., 2008. Characterization of cellulose production by a Gluconacetobacter xylinus strain from Kombucha. Current Microbiology. 57, 449-53. Silverman M. P., Lundgren D. G., 1959. Studies on the chemoautotrophic iron bacterium Ferroobacillus ferrooxidans: I. An improved medium and a harvesting procedure for securing high cell yields. Journal of Bacteriology. 77, 642-647. Song, Y.X., Zhang, H.P., Chon, C.H., Chen, S., Pan X., Li, D.Q., 2010. Counting bacteria on a microfluidic chip. Analytica Chimica Acta. 681, 82-86. Vazquez, A., Foresti, M.L., Cerrutti, P., Galvagno, M., 2013. Bacterial cellulose from simple and low cost production media by Gluconacetobacter xylinus. Journal of Polymers and the Environment. 21, 545-554. Xenofontos, E., Feidiou, A., Constantinou, M., Constantinides, G., Vyrides, I., 2015. Copper biomachining mechanisms using the newly isolated Acidithiobacillus ferrooxidans B1. Corrosion Science. 100, 642-650. Materials and general methods 76 CHAPTER 1. OPERATION WITH SUSPENDED BIOMASS Chapter 1. Operation with suspended biomass 79 1.1. OBJECTIVE As far as biomachining and bioleaching processes is concerned, most of the studies in these fields are still carried out in a small scale. Therefore, the larger industrial scale of these bioprocesses is still limited (Diaz-tena et al., 2017; Erust et al., 2021). One of the main drawbacks for continuous operation at high scale, even under optimum operating conditions, is the decrease in the amount of mobilized metal as a consequence of the loss of bio-oxidative activity which can be attributed to the increasing toxicity of the medium (Díaz-Tena et al., 2016; Liang et al., 2018). Another feature to be considered is that the final depleted solution requires adequate (and sometimes complex) treatment before discharge, increasing process cost. The objective here was to contribute to the design of a bioprocess with suspended biomass that allows the maintenance of the metal removal rate at high values while minimizing the amount of depleted solution. Copper was selected as a representative metal to assess the influence of iron concentration on both suspended A. ferrooxidans growth and metal mobilization efficiency, and to determine the contribution of bacteria to the specific metal removal rate (SMRR). Bearing in mind the future industrial applications in bioleaching of metals from electronic waste and biomachining, an alternate process composed of two stages (metal removal + regeneration stage) was searched with the aim of maintaining high SMRR and reusing the solution in semicontinuous operation. Figure 1.1 shows the outline of the experimental section of this chapter. Figure 1.1 . Outline of the experimental section of this chapter. 1.2. MATERIALS AND METHODS 1.2.1. Copper pieces Copper workpieces (purity 99.9 %) measuring approximately 2 x 10 x 15 mm were cut employing a Remet LS1 metallographic cutting machine with K-type cutting discs. A 2mm diameter hole was drilled in each workpiece for holding it during immersion into Chapter 1. Operation with suspended biomass 80 cell suspension. Before further treatment, they were cleaned and prepared as described in Section 1.2 Copper workpieces (materials and general methods). 1.2.2. Microorganisms and culture media The A. ferrooxidans bacterium (ATCC 23270) used for this study was cultured in a Silverman and Lundgren medium (Silverman and Lundgren, 1959) as described in Section 1.1 Microorganisms (materials and general methods). 1.2.3. Metal mobilization experiments Metal mobilization experiments were performed to evaluate the influence of Fe2+ concentration on microbial growth (experiment G1), and to record the variation in metal removal over time as function of Fe3+ concentration (experiment BM1). In addition, the biomass’s contribution to the process was studied by comparing biotic and abiotic experiments using the optimum iron concentration selected in BM1 as the initial energy source for bacterial growth (experiment BM2). The experiments were carried out in two-stages following the procedure described in Section 2.2. Copper mobilization experiments (materials and general methods). First, the medium containing varying concentration of ferrous iron (1.5, 6 and 9K) was inoculated with a 2 % v:v of an A. ferrooxidans culture in an exponential growth phase, and each sample was cultured until the complete Fe2+ oxidation (step 1). Thus, the treatment solutions were obtained. Once Fe2+ was oxidized to Fe3+, a previously weighted copper workpiece was immersed in the treatment solution (step 2). The workpieces were removed from the oxidizing medium on an hourly basis, rinsed with deionized water and ethanol (96 % v:v), dried, and then weighed as described in Section 1.2 Copper workpieces (materials and general methods). Thereafter, they were immersed once again in the corresponding culture until the end of each experiment. Additionally, several abiotic tests were used for comparison purposes (control tests without bacteria). In these assays, the solution containing Fe3+ was prepared by filtering the treatment solution obtained as previously described in step 1 (a 0.45 μm polyvinylidene fluoride filter was used). All the experiments were carried out at 31 °C, at a shaking speed of 130 rpm. A pH threshold value of 1.7-1.8 was maintained in both steps by the addition of sulfuric acid (25 % v:v). Each metal mobilization experiment was performed in triplicate. The specifics of each experiment are described below. 1.2.3.1. Effect of iron content on bacterial growth and metal mobilization The effect of the energy source (iron concentration) on A. ferrooxidans growth in step 1 was studied by culturing the bacteria in media with different initial concentrations of Chapter 1. Operation with suspended biomass 87 biotic BM2 experiment) and in the total absence of iron (experiment B2; < 0.5 % of the SMRR obtained in the biotic BM1 experiment). Fe3+ was therefore concluded to be the main oxidant responsible for copper leaching, in agreement with other studies (Xenofontos et al., 2015; Lambert et al., 2015). The presence of microorganisms improved the process efficiency, as the total Cu amount solubilized in the 6K and 9K biotic systems was 10 % and 25 % higher, respectively, than the amount dissolved in the absence of microorganisms (Table 1.2). By contrast, Cu removal after three hours in the 1.5K solution was similar in both cases, which is indicative of the poor oxidation activity attributed to the lower biomass concentration at low iron concentrations (78.1±4.8 and 75.5±1.8 mg Cu for the biotic and abiotic 1.5K experiments, respectively). Table 1.2. Total copper amount removed in the biotic (BM1) and abiotic (SN1) experiments after the three-hour immersion of a copper piece. Initial Fe3+ concentration (g Fe3+ L-1) Experiment BM1 (biotic medium) (mg Cu) Experiment SN1 (abiotic medium) (mg Cu) 1.5 78.1±4.8 75.5±1.8 6 254.5±8.2 231.0±16.3 9 358.4±25.9 287.0±19.7 The total amount of copper solubilized per square centimeter in the 6K and 9K biotic solutions was 12-13 % higher than in the absence of microorganisms (69.2±1.0 vs. 61.6±1.0 mg cm-2 in the 6K; 102.3±5.9 vs. 90.1±3.2 mg cm-2 in the 9K solution), with the bacteria’s effect being greater than that described by other authors. For example, Xenofontos et al. (2015) have reported that the bacterial contribution only accounted for an increase of 5.7 % in the removed copper (from 15.9 mg cm-2 to 16.8 mg cm-2) after 6 h with an initial concentration of 6.5 g Fe3+ L-1. Likewise, Lambert et al. (2015) have reported that this difference never exceeded 7-8 % during the first 10 h in assays performed with initial iron concentrations up to 7 g Fe3+ L-1. Regarding SMRR, the most significant difference between the biotic (BM1) and abiotic (SN1) assays was recorded for the 9 g Fe3+ L-1 initial concentration during the first two hours of operation (Figure 1.6). The high SMRR value in the biotic medium could be attributed to the presence of sufficient amounts of extracellular polymeric substances (EPSs) with attached Fe3+ ions, enhancing the catalytic oxidation of Fe2+, and thus the recovery of the oxidant and the amount of metal mobilization. Sand and Gehrke (2006) Chapter 1. Operation with suspended biomass 88 have concluded that strains of A. ferrooxidans with a high amount of Fe3+ ions in their EPSs recorded greater Fe2+ oxidation activity than those with a lower Fe3+ concentration. In the particular case of metal sulfides, some authors have observed that EPSs, composed mainly of neutral sugars and lipids, provide a controlled reaction layer that concentrates Fe3+ ions by complexation through uronic acids or other metabolites on the compound surface, increasing effective Fe3+ concentration in the attachment points and recording a 20-100 times enhancement over chemical leaching (Kinzler et al., 2003; Silva et al., 2015). Figure 1.6 shows that the maximum SMRR (SMRRmax) values were recorded within the first hour, and then the rate decreased over time. The SMRRmax obtained here is consistent with those described in the literature for experiments using either A. ferrooxidans cultures or a cell-free culture supernatant (Table 1.3). It is noteworthy that the SMRRmax recorded in the biotic 9K experiment (40 mg h-1 cm-2) was slightly higher than that reported by Jadhav et al. (2013) with the strain BCRC 13823 and an initial concentration of 40 g FeSO4 L-1. Consequently, the 9K medium was selected for subsequent experiments in this study. Figure 1.6. Variation of the SMRR during the three-hour experiment for the three initial Fe3+ concentrations (9, 6 and 1.5 g Fe3+ L-1). Regardless of the metal removal rate recorded in the experiments, the Fe3+/Fe2+ ratio followed a similar evolution in all cases and, after the three-hour immersion of a copper piece in the oxidant medium, the Fe3+ content decreased to 55±4 % of its initial value in both biotic and abiotic assays (BM1 and SN1 experiments respectively). Chapter 1. Operation with suspended biomass 89 Table 1.3. Maximum SMRR values obtained in the literature and in this study when using different initial concentrations of Fe3+ at T= 30-31 °C). SMRRmax (mg Cu h-1 cm-2) A. ferrooxidans strain [Fe3+]t=0 (g L-1) pH Reference Biotic experiments 2 ATCC 23270 0.6 1.8 Díaz-Tena et al., 2016 10 ATCC 23270 1.5 1.7 This study 10 Isolated from acidic pit water 5.0 1.8 Ma et al., 2020 22 ATCC 23270 6.0 1.8 Díaz-Tena et al., 2016 30 ATCC 23270 6.0 1.7 This study 40 ATCC 23270 9.0 1.7 This study 36 BCRC 13823 14.7 2.5a Jadhav et al., 2013 Abiotic experiments 10 ATCC 23270 1.5 1.7 This study 16 BCRC 13823 3.7 2.5 a Jadhav et al., 2013 5.4 BCRC 13820 4.0 2.5 a Hocheng et al., 2012b 28 ATCC 23270 6.0 1.7 This study 28 ATCC 23270 9.0 1.7 This study 40 BCRC 13823 14.7 2.5 a Jadhav et al., 2013 aThis value corresponds solely to the initial pH. 1.3.1.3. SMRR as a function of time during metal mobilization process Several authors have studied the variation in the SMRR over time using different A. ferrooxidans strains and operating conditions (Istiyanto et al., 2010; Xenofontos et al., 2015; Díaz-Tena et al., 2018). Nevertheless, the literature on the effect of microbial presence on metal leaching by comparing biotic and abiotic experiments over prolonged periods of time is scarce. In this study, after 20 h of operation the total copper mobilization was 55 % higher in the biotic system (1043±14 mg) than in the abiotic one (672±12 mg) (Figure 1.7a). Assuming that all the dissolved copper was in the Cu2+ ionic form, a final concentration of 7.6 g Cu2+ L-1 and 4.9 g Cu2+ L-1 was reached in the reactor with and without microorganisms, respectively. The evolution of the Fe3+/Fe2+ ratio over time is shown in Figure 1.7a, as this ratio plays a key role in overall metal removal kinetics by influencing the rate of the chemical process (metal dissolution) and the biologically catalyzed process (oxidant bio-regeneration). During the first four hours of operation, the ratio Chapter 1. Operation with suspended biomass 90 quickly decreased due to Fe3+ consumption in the copper oxidation process, with this ion being the acceptor that simultaneously received electrons from the metal. Figure 1.7. Evolution of dissolved copper and [Fe3+]/[Fe2+] ratio (a), and SMRR (b) throughout time. Nevertheless, from hour 8 onwards, the ratio remained almost constant for the biotic and abiotic systems, which can be explained by the limited rate of electron transfer, as suggested by Vargas et al. (2014). As far as the biotic system is concerned, when the rate of electron transfer from copper metal to Fe3+ (rate of Fe2+ produced in the chemical process) equaled the rate of electron transfer from Fe2+ to O2 (rate of Fe2+ consumption in the biological process) a pseudo steady-state was reached, and the amount of Fe3+/Fe2+ was therefore expected to remain constant. However, the abiotic system recorded a similar trend with even slightly higher Fe3+/Fe2+ ratio values during the final hours of the experiment, which means that Fe3+ consumption was lower, resulting in a lower amount of copper dissolved. As shown in Figure 1.7b, during the first four hours the SMRR decreased in both reactors more significantly, in agreement with the results recorded in the previous three-hour Chapter 1. Operation with suspended biomass 91 experiment. Afterwards, from 5 to 8 h the metal solubilization process slowed down in the two assays, and the SMRR value fell to 8 mg h-1 cm-2. Finally, the SMRR value became almost constant at an average value of 6.5±0.7 mg h-1 cm-2 from hour 9 onwards for the biotic system (BM2), whereas this value was 1.8±0.2 mg h-1 cm-2 from 15 h onwards for the abiotic assay (SN2). Thus, the difference between the SMRR in both assays became constant beyond 15 h, with the SMRR in the biological reactor being almost four times higher than in the abiotic assay. These results are consistent with those reported by Istiyanto et al. (2007), who have concluded that the reduction in the MRR (mg h-1) is inversely proportional to machining time, and not simply linear. These authors have observed that the decrease in the MRR in a reactor containing A. ferrooxidans ATCC 21834 is almost negligible from hour 12 onwards. A slight variation in the SMRR was observed in experiment B3, where copper extraction from the workpiece was induced by the spontaneous oxidation of Fe2+ to Fe3+ in the presence of the oxygen dissolved in the acidic medium. In this experiment, the SMRR increased with operation time to an average of 1.8±0.3 mg h-1 cm-2 for the 10-20-h period. This value is almost identical to that recorded in experiment SN2 from 15 h onwards, which means the leaching process in the absence of bacteria (SN2) seems to be limited by the re-oxidation of Fe2+ to Fe3+ assisted by the oxygen dissolved in the medium. According to these results, after 15 h of treatment, 65 % of the SMRR recorded in the microorganism-containing reactor (BM2) was attributed to the presence of bacteria, whose catalytic action greatly enhanced the electron transfer process. 1.3.2. Alternate process: metal mobilization + regeneration As concluded in the previous section, microorganism-assisted metal mobilization performed better than abiotic process. Nevertheless, the SMRR decreased significantly after three-hours of treatment, which would be a technical drawback when longer operation time is required, for example, to engrave specific geometries on a copper piece in biomachining application. Thus, the alternative proposed here was to treat the metal piece in consecutive three-hour leaching stages. In this approach, the reuse of the solution (after regeneration) in several metal mobilization stages is essential for the system’s sustainability. Therefore, the efficiency of the regenerated solution in consecutive treatment stages was studied and the criteria for the solution to be considered “exhausted” was defined. Figure 1.8a shows the SMRR during six metal mobilization + regeneration stages. In addition, Figure 1.8b provides a detailed view of the variation in Fe3+ and Cu2+ concentrations in the medium during stages MR1 and MR2 and the first regeneration stage. Chapter 1. Operation with suspended biomass 92 Figure 1.8. SMRR as a function of time during six three-hour metal removal stages (a); variation in SMRR (bars), [Cu2+] (grey dots) and [Fe3+] (white dots) during two successive treatment stages (MR1 and MR2) and the oxidant regeneration step (b). The trend in the SMRR during each three-hour treatment was similar in all the stages (MR1-MR6). It is noteworthy that the SMRRmax remained almost constant (36.0±0.9 mg h-1 cm-2) in MR1MR5 stages, and decreased in MR6 by about 22 % of the initial value. The stages MR1 and MR2 recorded a slightly better performance than the successive ones in terms of total Cu removal, and the amount of copper dissolved (calculated as the average for each 3 h period) remained in the 97-104 mg cm-2 range (Figure 1.9). In MR3 to MR5, this value was almost constant (89.9±2.7 mg cm-2), and in MR6 this value was 33 % lower than that recorded in MR1. These accumulated values were significantly Chapter 1. Operation with suspended biomass 93 higher than those recently reported by Ma et al. (2020) using an A. ferrooxidans strain isolated from acidic pit water taken from an iron mine in China. These authors have reported a removal of copper of approximately 60 mg cm-2 after an eight-hour leaching experiment using 5 g L-1 of iron as energy source (30 °C, 160 rpm, pH 1.8). Figure 1.9. Copper solubilization in consecutive treatment stages (columns, right axis) and accumulated copper concentration in the solution (white dots, left axis). Overall, the proposed system design being comprised of three-hour copper mobilization stages in consecutive operation (with intermediate regeneration stages) significantly improved the average SMRR for copper extraction in comparison to continuous operation (Section 1.3.1.3 SMRR as a function of time during the metal mobilization process). Indeed, the total amount of removed copper in 5 consecutive stages (15 h of treatment) (471.6 mg cm-1) was 52.4 % higher than the metal amount extracted during 15 h in continuous operation (224.6 mg cm-1). This means that the time for extracting a certain amount of copper can be considerably shortened in the consecutive stage mode, with the positive perspective for sustainability and industrial implementation of the process. Regarding the cumulative copper concentration shown in Figure 1.9, this parameter increased linearly with treatment time and, consequently, with stage number ([Cu2+] (g L-1) = 2.03·stage number + 0.466; R2 = 0.9980). In this experiment, the final concentration recorded after six metal mobilization stages was 12.4 g Cu2+ L-1, and the total mass of copper removed from the workpiece throughout the whole experiment was 1.57 g. Figure 1.10 shows the solutions after six metal mobilization + regeneration stages. Chapter 1. Operation with suspended biomass 94 Figure 1.10. Resulting solutions after six metal mobilization + regeneration stages (in duplicate). A low concentration of dissolved copper in the medium is essential for A. ferrooxidans growth, as high concentration of this metal leads to the denaturation of the proteins and nucleic acids necessary for metabolic activities (Valix et al., 2017), inhibiting bacterial activity, and thus halting the whole process. The results recorded here confirmed that the loss of bacterial activity due to the increasing Cu2+ concentration in the medium impaired the activity of A. ferrooxidans strain ATCC 23270. Nevertheless, under these particular operating conditions, it was not until the dissolved Cu2+ concentration was higher than 10.7 g L-1 (recorded at the end of MR5) that biomass activity was more severely affected, slowing the bacterial Fe2+ re-oxidation rate, and thus reducing the SMRR. The inhibitory copper concentration depends both on the bacterial strain and on the operating conditions. However, the maximum dissolved copper concentration before bacterial inhibition detected in this study was slightly higher than that reported by other authors for other A. ferrooxidans strains. Thus, DíazTena et al. (2016) have observed a 63 % reduction in A. ferrooxidans ATCC 23270 activity for oxidizing Fe2+ ions in the presence of 6.1 g Cu2+ L-1 (30 °C, 130 rpm, pH 1.8,), and Cho et al. (2008) have evidenced that the oxidation capacity of Fe2+ by the A. ferrooxidans ATCC 19859 strain was completely annulled by a Cu2+ concentration above 10.8 g L-1 (30 °C, 200 rpm, pH 2.0). Regarding the reduction in the waste solution generation, the results showed that microorganisms effectively re-oxidized the Fe2+ generated in the metal solubilization stage (Figure 1.11), which allowed the solution reuse and reagent consumption saving. Nevertheless, the medium’s increasing toxicity was responsible for the longer time required for the complete bio-regeneration of the oxidant solution in the consecutive regeneration steps (Figure 1.11). Chapter 1. Operation with suspended biomass 95 Figure 1.11. Regeneration time as a function of the copper concentration in the medium. Figure 1.11 shows the linear increase in the time needed for complete Fe2+ re-oxidation or regeneration (tregen (h) = 1.04·[Cu2+] (g L-1) +12.72; R2 = 0.969) until it sharply increased after MR5 ([Cu2+] = 10.7 g L-1), when the time needed was 3.2 times higher than that required in the first regeneration stage ([Cu2+] = 2.3 g L-1). The final cumulative concentration of Cu2+ (12.4 g L-1) was not considered to be the toxic limit for biomass activity, as the bio-regeneration of the oxidant still took place in the solution after MR6. Nevertheless, the bio-regeneration time was longer than 90 h and it was considered too long for an industrial application. The results obtained in this study suggest that the alternate mode could be successfully implemented at industrial scale by installing two (or more) treatment lines in parallel when using suspended biomass. Thus, the metal pieces (or metal containing wastes) could be consecutively immersed in the regenerated solution of each line without waiting for the oxidant recovery. The operation mode proposed in this study is a relevant contribution for the sustainable application of this metal solubilization process to new sectors, as it prolongs the use of the solutions (and consequently reduces liquid waste generation), and improves process performance until the toxicity limit is reached. 1.4. CONCLUSIONS This chapter has focused on studying the aspects that can contribute to the efficient solubilization of copper in microorganism-assisted processes with suspended biomass. An alternate procedure for enhancing the oxidant bioregeneration and maintaining high SMRR was sought. As far as the influence of the iron concentration is concerned, the 9 K medium (containing 9 g L-1) was concluded to be the optimum one for both A. ferrooxidans ATCC Chapter 1. Operation with suspended biomass 96 23270 growth (Fe2+) and copper solubilization (Fe3+). Under the operating conditions, the Fe2+ oxidation rate in the 9K medium during microbial growth (0.101 g Fe2+ L-1 h-1) was 2.8 times higher than that recorded in the presence of 1.5 g Fe2+ L-1 (0.036 g Fe2+ L-1 h-1), rendering a higher concentration of Fe3+ available in the subsequent step for copper solubilization. Similarly, the presence of microorganisms improved the copper solubilization process efficiency in comparison to the biotic experiment, as the total Cu amount leached in the 9K biotic system was 25 % higher than that in absence of microorganisms. The SMRR peaked during the first hour (SMRRmax of 40 mg Cu h-1 cm-2) and decreased significantly after 3-4 hours. After 15 h, 65 % of the SMRR was attributed to the reoxidation of Fe2+ to Fe3+ due to bacterial activity, but the SMRR values recorded after 15 h in both biotic (6.5±0.7 mg Cu h-1 cm-2) and abiotic samples (1.8±0.2 mg Cu h-1 cm-2) were too low for industrial application. The SMRR decrease after the first hours of treatment could be a technical drawback when longer operation time is required. Therefore, an alternate process in stages (metal solubilization during 3 hours followed by oxidant regeneration) has been proposed with the aim of maintaining high SMRR and reusing the solution in semicontinuous operation. The amount of metal mobilized in 5 consecutive stages (471.6 mg cm-1) was 52.4 % higher than the amount obtained in the continuous operation for the same treatment time (15 h) (224.6 mg cm-1). The inclusion of an oxidant regenerating stage between two consecutive metal solubilization stages contributed to prolonging the solution lifespan. Nevertheless, the regeneration time increased threefold compared to the first regeneration stage when the copper concentration in the medium was 0.7 g Cu2+ L-1. This alternate design allows the reduction of both the treatment time and the amount of depleted solution, which enhances the sustainability of the process. 1.5. REFERENCES Blanch H.W., Clark D.S., Biochemical Engineering, Marcel Dekker Inc., New York, 1996. Cho K. S., Ryu H. W., Choi H. M., 2008. Toxicity Evaluation of Complex Metal Mixtures Using Reduced Metal Concentrations: Application to Iron Oxidation by Acidithiobacillus ferrooxidans. Journal of Microbiology and Biotechnology. 18, 1298-1307. Daoud J., Karamanev D., 2006. Formation of jarosite during Fe2+ oxidation by Acidithiobacillus ferrooxidans. Minerals Engineering. 19, 960-967. Díaz-Tena E., Gallastegui G., Hipperdinger M., Donati E. R., Ramírez M., Rodríguez A., López de Lacalle L. N., Elías A., 2016. New advances in copper biomachining by iron-oxidizing bacteria. Corrosion Science. 112, 385-392. Díaz-Tena E., Gallastegui G., Hipperdinger M., Donati E. R., Rojo N., Santaolalla A., Ramirez M., Barona A., Elías A., 2018. Simultaneous Culture and Biomachining of Copper in MAC Chapter 2. Operation with immobilized biomass 103 BC is a tridimensional natural nanofiber network produced by microorganisms with a high specific surface. It exhibits outstanding qualities, such as a highly porous network structure, biocompatibility, and good mechanical and chemical stability (Azeredo et al., 2019; de Oliveira et al., 2021). Being synthesized by bacteria, it can be obtained with the desired size and shape. PVA is a bioplastic derived from the hydrolysis, alcoholysis or aminolysis of polyvinyl acetate. It is a highly biodegradable thermoplastic polymer and easily soluble in water due to its crystalline structure. It stands out for its flexibility and high chemical resistance and for being a barrier for gases and aromas (Hammannavar and Lobo, 2018; Abral et al., 2020). CTA is a chemical compound obtained by treating cellulose where all cellulose hydroxyl groups are replaced by acetyl groups. It is commonly used to elaborate membranes characterized by well-defined pores (Sikorski et al., 2004; Nabili et al., 2017). Two different CTA-based support materials were tested: modified CTA membrane and CTA spheres. CH is a natural biopolymer that is obtained from the partial deacetylation of chitin. This is a very abundant polysaccharide in nature and is obtained from the shell or exoskeleton of crustaceans, fungi and insects, generally as a by-product of fishing industries. It is insoluble in water with a high molecular weight, nontoxic and biodegradable biopolymer (Muxika et al., 2017; Bakshi et al., 2020). 2.2.2.1. Bacterial cellulose Bacterial cellulose hydrogel was biosynthesized in the laboratory by Gluconacetobacter xylinus bacterial strain. It was obtained by adding 1 % inoculum of Gluconacetobacter xylinus bacteria to the medium containing panela and pineapple dissolved in water (13 % w:v). The culture was incubated under static conditions at 28 °C, until the desired thickness of the pellicle was reached after 25 days (the incubation time can be adjusted to obtain the necessary thickness). In this study, BC membranes with a 0.7 cm thickness were used. The pH was adjusted to 3.5 by the addition of acetic acid (CH3COOH) (Retegi et al., 2010). After biosynthesis, BC pellicle was washed with a 2 % w:v solution of sodium hydroxide (NaOH) for 24 h at room temperature and orbital shaking. Finally, it was rinsed with deionized water several times until the complete neutralization of the BC membrane (Gutierrez et al., 2013). After the purification process, a completely white bacterial cellulose membrane was obtained (Figure 2.3). Chapter 2. Operation with immobilized biomass 104 Figure 2.3. Culture medium and synthesized BC membrane. 2.2.2.2. Polyvinyl alcohol PVA hydrogel was prepared using powder PVA (Mw 130000 g mol-1, Sigma-Aldrich Corporation). First, 2.5, 5 and 10 % w:w PVA solutions were prepared by dissolving PVA in deionized water at 95 °C under vigorous stirring for 3 h. The homogenous PVA solution was subsequently placed into a petri dish and cooled at room temperature. Then, two different techniques were used for synthesizing PVA hydrogels: Freeze-Thawing and Freeze-Drying. In the Freeze-Thawing method (Hassan and Peppas, 2000), previously obtained PVA solutions were subjected to 24 h freezing at -21 °C and 3 h of thawing at 25 °C for a total of 3 cycles. The Freeze–Thaw cycling promotes the crystallization of PVA domains, which results in a strong hydrogel formation with high mechanical stability and good properties such as viscoelasticity (Peppas and Scott, 1992; Vrana et al., 2009). During the freezing the PVA domains get closer due to the formation of frozen water, allowing the formation of cross-links (Holloway et al., 2013). After thawing, these PVA domains result in noncovalent bond formation between the polymer chains (Ou et al., 2017). Figure 2.4 schematically shows the Freeze-Thawing process for PVA labeled samples. Figure 2.4. PVA hydrogel preparation process using Freeze-Thawing technique. Chapter 2. Operation with immobilized biomass 105 In the Freeze-Drying method the PVA solutions were frozen for 24 h at -21.5 °C and subsequently placed in a lyophilization equipment (model Alpha 1_4LD (Martin Christ)) at -85 °C and 0.1 mbar. Figure 2.5 shows the scheme of the process. The samples obtained were labeled L-PVA, referring to lyophilized PVA. In this technique only samples of 2.5 and 5 % w:w by mass were prepared, since 10 % w:w concentration was too high for the resulting material to be porous enough for the desired application. Figure 2.5. Lyophilized PVA (L-PVA) preparation process using Freeze-Drying technique. 2.2.2.3. Modified cellulose triacetate membrane A modified CTA membrane was fabricated by an adaptation of the method published by Kaiser et al. (2017). The solution of the triacetate cellulose biopolymer was prepared with acetone as solvent because it is insoluble in water but soluble in organic solvents. CTA (Sigma Aldrich) was dissolved in 300 mL of acetone (99.5 %, Panreac) and kept in a closed container under stirring conditions for 24 h thus obtaining a solution with a 10 % w:v concentration. Afterwards, the solution was transferred to a container with 20.5 g of calcium carbonate (99.5 %, Panreac) and 8.8 g of glycerol (99 %, 98.09 g mol-1, Labkem). It was then vigorously mixed in a blender for 5 min. Finally, the mixture was introduced into a closed container. Afterwards, two water baths containing 0.25 M HCl (99 %, Panreac) and deionized water, respectively, were prepared in order to completely dissolve both calcium carbonate and glycerol. The obtained liquid polymer was spread on a flat glass of 1-2 mm of thickness and it was dried for 5 min approximately. After this time, it was carefully immersed in the HCl bath for 10 min, and bubble formation on the surface was observed, due to the dissolution of calcium carbonate with the consequent CO2 release. The obtained membrane was introduced then into the deionized water bath for another 10 min. Finally, it was extracted and placed on absorbent paper, until a dry porous membrane was obtained. The material obtained was labelled M-CTA. Figure 2.6 shows the scheme of the procedure carried out for the synthesis of the M-CTA membrane. Chapter 2. Operation with immobilized biomass 106 Figure 2.6. Preparation process of modified CTA membrane (M-CTA). 2.2.2.4. Cellulose triacetate spheres The fabrication of the CTA spheres was carried out as follows. Once the abovementioned CTA solution (10 % w:v) was completely dissolved, a certain quantity of the prepared solution was introduced for 10 s into a beaker containing deionized water at 95 °C. The observed bubbling caused by the evaporation of the acetone allowed the formation of the pores. Afterwards, the solution was removed from the beaker, the spheres were manually rounded, and they were placed in an oven (Selecta P model) at 45 °C until the total evaporation of acetone. This support material was labelled B-CTA. Figure 2.7 shows the scheme of the preparation process. Finally, the spheres were weighed and measured for classification according to size. For this study, the spheres with an average surface area of 4.6±0.4 cm2 were selected. Figure 2.7. Preparation process of CTA spheres (B-CTA). 2.2.2.5. Chitosan The chitosan solution was prepared by dissolving 2.45 g of chitosan (Aldrich's, medium molecular weight) in 120 g of a solvent solution containing deionized water and acetic Chapter 2. Operation with immobilized biomass 107 acid at 2 % w:v (99.8 %, Honeywell). The mixture was maintained in a closed container under stirring for 24 h. Then, it was poured into a Petri dish and placed in the freezer at -21.5 °C. After 24 h the samples were extracted from the freezer and two different procedures were carried out. In the first one, the sample was just kept at room temperature. This chitosan material was identified as CH. In the second treatment, the frozen sample was lyophilized obtaining the so-called lyophilized chitosan material (LCH, Figure 2.8). Figure 2.8. Preparation process of lyophilized chitosan L-CH. 2.2.3. Pre-treatment of the support material The four synthesized materials, namely bacterial cellulose (BC), polyvinyl alcohol (PVA), cellulose triacetate (CTA) and chitosan (CH) were tested as immobilization support materials in this study. As far as BC and PVA hydrogels are concerned, they were cut into rectangular parallelepiped pieces, rendering an external surface area (ESA) of 4.8±0.4 cm2. CTA spheres were rounded to a mean diameter of 1.21±0.05 cm which corresponds to an ESA of 4.6±0.4 cm2. CH and modified CTA membrane support materials were prepared trying to maintain the same ESA area. It should be noted that the presence of trace amounts of the reagents used for the synthesis of the materials could hinder microbial growth, thereby making the cleaning of all the support materials mandatory before bacterial immobilization. Therefore, the pieces were rinsed with deionized water at 130 rpm and 31 °C for 1 h. This process was repeated twice and subsequently the same protocol was carried out (twice) replacing the deionized water by fresh nutrient medium (6K or 9K depending on the experiment). Finally, pieces of the different support materials were immersed in fresh nutrient medium for 24 h (130 rpm, 31 °C). Chapter 2. Operation with immobilized biomass 108 2.2.4. The design of a decision-making protocol A decision-making protocol was designed to study the viability of the synthesized materials in bacterial immobilization procedures. Therefore, the main objective of this protocol was to assess the materials´ suitability for microbial immobilization for its application in the metal removal process. Figure 2.9 shows the lay-out of the decisionmaking protocol. Figure 2.9. Decision-making protocol. In the first stage, the possible degradation when being immersed in 9K medium (without bacterial inoculation) was tested by introducing the materials into the aforementioned solution under shaking conditions for 1 day (130 rpm, 31 °C). They were directly discarded when the physical integrity was not maintained and, otherwise, they proceeded to the next stage. The second stage consisted of evaluating whether the materials exerted any adverse influence on microbial growth. Bearing in mind this objective, an immobilization test was carried out by inoculating A. ferrooxidans bacteria (5 % v:v of culture in the Chapter 2. Operation with immobilized biomass 109 exponential growth phase) in the 9K culture medium in the presence of each support (130 rpm, 31 °C). The last stage was focused on assessing the microbial colonization. Once the materials were proved not to be degraded by the medium and not to impair microbial growth, the supports already exposed to biomass in the previous step were cleaned using fresh medium and then cultured in 9K medium without further bacterial inoculation. In case of successful Fe2+ oxidation by the “active material”, the supports were concluded to be favorable for A. ferrooxidans immobilization. If not, they were definitively disregarded for further experimentation. Thus, it was assumed that the microbial film was active if, after the immobilization procedure, the freshly fed Fe2+ was oxidized into Fe3+ without further inoculation. 2.2.5. Selection of the inoculum percentage As this chapter is focused on the immobilization procedure of the A. ferrooxidans bacteria, it was considered relevant to investigate the optimum amount of inoculum that shortened the incubation period while ensuring an adequate bacterial growth. Thus, different percentages (2, 5 and 10 %) of an A. ferrooxidans culture in an exponential growth phase were inoculated in 250 mL Erlenmeyers containing 150 mL of the 6K culture medium (6 g Fe2+ L-1). The experiments were carried out at 31 °C and shaking speed of 130 rpm. A pH threshold value of 1.8 was maintained by the addition of sulfuric acid (25 % v:v). 2.2.6. Suitability of bacterial cellulose as support material for A. ferrooxidans immobilization Among the suitable materials selected according to the decision-making protocol previously described in Figure 2.9., BC has not yet been proposed as support for A. ferrooxidans immobilization. Consequently, it was selected for the experiments focused on selecting the most adequate operating conditions for obtaining and using an Active Bacterial Cellulose (A-BC) capable of transforming the ferrous iron dissolved in the nutrient medium into ferric iron. The particular interest on BC hydrogel lied in its outstanding properties as a viable support material and its novel use for this application. 2.2.6.1. Preparation of the Active Bacterial Cellulose (A-BC) The procedure to obtain the A-BC started with the immersion of the BC pieces into an Erlenmeyer flask containing the nutrient medium (6K and 9K depending on the experiment), and then the A. ferrooxidans bacteria (5 % v:v of a culture in an exponential growth phase) was inoculated and cultured at 31 °C and pH 1.8 (adjusted with sulfuric Chapter 2. Operation with immobilized biomass 110 acid 25 % v:v). This immobilization step was carried out in duplicate under lowdemanding conditions until the complete oxidation of Fe2+ (shaking rate of 130 rpm and a Nutrient medium volume (mL) to external surface area (cm2) (NMV:ESA) ratio of 1:0.2). Thus, biologically active biocellulose (A-BC) was obtained (Figure 2.10). 2.2.6.2. Effect of operating parameters on Fe2+ bio-oxidation The previously prepared A-BC material was used in several experiments with the objective of ascertaining the effect of the shaking mode (orbital vs linear), NMV:ESA ratio (in the 1:0.1-1:0.6 range), initial Fe2+ concentration (6 vs 9 g Fe2+ L-1) and shaking speed (130 vs 170 rpm) on the iron bio-oxidation performance. The experimental conditions are detailed in Table 2.1. During the experiments, the pH was adjusted to 1.8 with sulfuric acid (25 % v:v) and the temperature was maintained at 31 °C. All the BC pieces were incubated until 100 % Fe2+ contained in the nutrient medium was oxidized. Table 2.1. Experimental conditions for the iron bio-oxidation experiments using the ABC pieces at 31 °C and pH 1.8. Operation parameter Experiment Shaking mode NMV:ESA ratio (mL:cm2) Dissolved [Fe2+] (g L-1) Shaking speed (rpm) Shaking mode Orbital Linear 1:0.2 6 130 NMV:ESA ratio Orbital 1:0.1 1:0.2 1:0.3 1:0.6 6 130 Dissolved [Fe2+] Orbital 1:0.6 6 9 130 Shaking speed Orbital 1:0.6 9 130 170 In all the experiments (except for the shaking mode experiment) the same A-BC pieces were used in two consecutive bio-oxidation stages under the same operating conditions (Figure 2.10). This procedure allowed to study the activity of the material in successive stages and to identify the possible problems that could arise from its long-term use. In addition, control tests were conducted using 6K and 9K medium inoculated with 5 % v:v of A. ferrooxidans culture in exponential growth phase but without hydrogel pieces. Figure 2.10 illustrates the scheme of the whole process including A-BC preparation and the subsequent bio-oxidation stages. Chapter 2. Operation with immobilized biomass 111 Figure 2.10. Scheme of bacterial immobilization procedure and subsequent oxidation stages. 2.2.6.3. Influence of dissolved copper concentration on bacterial immobilization and subsequent iron bio-oxidation stages The influence of the presence of dissolved copper (Cu2+) on the preparation of A-BC was tested, as increasingly dissolved metal concentration originated throughout the processes is known to affect bacterial activity and survival (Liang et al., 2018). Thus, the protocol described in Section 2.2.6.1 Preparation of the Active Bacterial Cellulose (A-BC) was repeated using 9K medium, 1:0.6 NMV:ESA ratio, and variable concentrations of dissolved copper (from 0 to 40 g Cu2+ L-1). The BAM pieces were labelled as “A-BCX” being X the concentration of dissolved copper. Several consecutive bio-oxidation stages were carried out for each copper concentration with the active materials pieces, and the time required for complete iron oxidation in each stage was recorded. In addition, the ability of immobilized A. ferrooxidans to adapt to the presence of dissolved copper was tested by immersing the A-BC15 pieces into the medium with increasing Cu2+ concentrations (20, 25 and 30 g Cu2+ L-1) in several consecutive biooxidation assays. Table 2.2 shows the whole procedure particularly applied to the sample A-BC15. Table 2.2. Copper concentration in the medium during A-BC15 preparation and subsequent bio-oxidation stages with the sample A-BC15. Stages [Cu2+] (g L-1) A-BC15 preparation 15 S1-S4 15 S5-S7 20 S8 25 S9 30 Chapter 2. Operation with immobilized biomass 112 The operating conditions in those experiments were 31 °C, pH 1.8 and orbital shaking at 170 rpm. 2.2.6.4. Influence of active BC storage on bacterial activity The proper preservation of the biologically active material (A-BC) would allow its storage for further use, which would contribute to facilitate the future full-scale application. The influence of two storage temperatures (4 and 22 °C) on the recovery of the activity of the A-BC after the storage period was assessed as follows. First, several A-BC pieces underwent two iron bio-oxidation stages using 1:0.1, 1:0.2 and 1:0.3 NMV:ESA ratios (31 °C, pH 1.8, 130 rpm of orbital shaking). The A-BC pieces for each ratio were then divided into two sets and stored in wet conditions (in fresh 6K medium) at 4 °C and at 22 °C, respectively. After 15 days, the A-BC pieces were transferred to an Erlenmeyer flask containing 6K fresh medium (without further inoculation) and incubated in two consecutive bio-oxidation cycles under the same operating conditions used before storage (31 °C, pH 1.8, orbital shaking, 130 rpm). The long-term storage (6 months) at 4 °C was also studied by repeating the abovementioned procedure. In this case, a single iron bio-oxidation stage was carried out after storage. The operating conditions used in the tests before and after storage were those selected from the results obtained in the experiments described in Section 2.2.6.2. Effect of operating parameters on Fe2+ bio-oxidation. 2.2.6.5. Biocellulose cleaning and reuse The strategy of washing the A-BC pieces covered with jarosite for removing the precipitate and re-using the cleaned BC for subsequent bacterial immobilization procedures was tested, in order to expand the life-span of the support material and reduce both A-BC preparation time and reagent consumption. Biocellulose pieces containing precipitated jarosite were introduced in a 50 mL Erlenmeyer flask and covered with 20 mL of 10 % w:v oxalic acid solution for 1 h (W1). Then, the oxalic medium was removed and 20 mL of fresh oxalic acid were added in two consecutive washing stages (W2-W3). The experiment was carried out at 25 °C and 150 rpm to ensure an adequate shaking speed with the objective of cleaning of the material. Once the cleaning operation was concluded, the BC pieces were immersed in fresh 9K medium and inoculated with a 5 % v:v of A. ferrooxidans culture in the exponential growth phase to assess the possibility of reusing the biopolymer again as support material. Once the first bio-oxidation stage was completed, the BC pieces were reimmersed in fresh 9K medium with no further inoculation to check if the attached bacteria were able to successfully oxidize the Fe2+. The immobilization procedure after Chapter 2. Operation with immobilized biomass 119 precipitate was observed on the material´s surface by SEM analysis (Figure 2.16b). This deposit of jarosite has often been reported to precipitate in this medium and to be an effective adsorbent for bacteria, contributing to biofilm formation (Pogliani and Donati, 2000; Junfeng et al., 2007; Chowdhury and Ojumu, 2014). Bearing in mind that several authors have proposed that the bacteria are first wrapped by EPS and then embedded in aggregates of deposited jarosite, the visualization of individual cells by SEM photograph could be hindered by the deposit (Harneit et al., 2006, Lei et al., 2009; Africa et al., 2010, Mulopo and Schaefer, 2015). Pogliani and Donati (2000) observed that bacterial populations immobilized by jarosite precipitation were not easily leached, indicating the biofilm’s strong attachment to the deposit. Figure 2.16. Picture and SEM photograph of the pretreated biocellulose (a) and the surface of the biologically active bacterial cellulose (A-BC) obtained after the immobilization protocol (b). Under the low-demanding operating conditions selected for the preparation of A-BC, the bacteria required 79 h and 97 h for the complete bio-oxidation of 6 and 9 g Fe2+ L-1, respectively. A. ferrooxidans immobilization on different solid supports has already been studied by other authors in order to increase the bio-oxidation rates of Fe2+ during the metal removal processes. Although the accurate comparison of the performance of the BC with other support materials reported in the literature is not feasible due to the influence of the bacterial strain and particular operating conditions, Table 2.4 summarizes the results reported for several A. ferrooxidans strains immobilization under similar pH and temperature conditions. Fe2+ oxidation occurred faster during bacterial immobilization on BC than when using non-biobased materials such as nickel alloy fiber (Gomez et al., 2000) and monolithic particles (Kahrizi et al., 2008). For example, the time needed for 95 % conversion in the reactors containing 6 g Fe2+ L-1 was slightly lower than that reported by Kahrizi et al. (2008) for a similar initial Fe2+ concentration (6.7 g Fe2+ L-1) when immobilizing A. ferrooxidans DSM 584 in a monolithic reactor using a twofold higher inoculum percentage (Table 2.4). These authors have reported that it took 72 h to convert 95 % of the initial Fe2+ concentration, which is 7 % longer than that needed Chapter 2. Operation with immobilized biomass 120 in this study for the same iron conversion (62 h). BC also recorded a better performance during immobilization when compared to sulfonated polystyrene-divinylbenzene copolymer (SlufSDVB), sulfonated polystyrene-divinylbenzene copolymer with granular activated carbon (SulfSCVB-GAC), and polyurethane foam (PUF) (Koseoglu-Imer and Keskinler, 2013). Thus, the time needed to fully convert the Fe2+ was three to four times shorter despite this study’s higher iron concentration (6 and 9 vs 5 g Fe2+ L-1), lower inoculum percentage (5 % vs 10 %), and lower temperature (30 °C vs 35 °C) (KoseogluImer and Keskinler, 2013). Table 2.4. Time required for the bio-oxidation of the Fe2+ during the immobilization of A. ferrooxidans on different support materials (pH = 1.6-2.0 and T = 30-31 °C). Support material A. ferrooxidans strain Time (h) [Fe2+]0 (g L-1) Conversiona (%) Reference Nickel alloy fibre Isolated from Rio Tinto minesb 65c 2 100 (Gomez et al., 2000) Hemp fibres PTCC 1646 34 5 80-90 (Akhlaghi, 2019) Monolithic particles DSM 584 72c 6.7 95 (Kahrizi et al., 2008) Chitosan beads DSM 11477 58 9 100 (Giaveno et al., 2008) Cotton gauze Activated carbon Zeolite CCTCC M2013102d 48 56c 56c 9 100 (Zhu et al., 2017) Biocellulose DSM 14882 79 97 6 9 100 This study aPercentage of initial Fe2+ converted to Fe3+ when the experiment was considered to be completed. bThe strain has the same properties and characteristics as NCIMB 9490. cGraphically inferred. dIsolated from acid mine drainage collected from a local pyrite mine in Guangdong, People´s Republic of China. After the bacterial immobilization procedure, and despite the acidic pH and orbital shaking, the material maintained its physical integrity, which was attributed to the outstanding mechanical properties conferred by its 3D network structure and morphology (Tercjak et al., 2015; de Oliveira et al., 2021). Therefore, BC proved to be a suitable material for the immobilization of A. ferrooxidans. Chapter 2. Operation with immobilized biomass 121 2.3.3.2. Effect of operating parameters on Fe2+ bio-oxidation 2.3.3.2.1. Shaking mode The shaking of the bacterial suspended cultures has a positive impact on growth rate, as it provides better aeration and higher availability of oxygen and nutrients (Juergensmeyer et al., 2007). Two shaking systems can be used: linear reciprocal movement or orbital movement, being the latter one more widely used (Klöckner and Büchs, 2012). In this study, both shaking modes were compared and, as a result, the biooxidation time needed for the complete oxidation of 6 Fe2+ L-1 by A-BC in a NMV:ESA 1:0.2 ratio was found to decrease a 40 % when the reactors were shaken using the orbital mode in contrast to the horizontal shaking (43.5±0.7 h vs. 73.0±4.2 h). Therefore, the orbital shaking mode was selected for further studies. 2.3.3.2.2. Nutrient medium volume (mL) to external surface area (cm2) ratio Bacterial density plays a crucial role in the bioleaching and biomachining processes since it determines ferric iron productivity and, hence, bio-oxidation time (Jaisankar and Modak, 2009; Zhu et al., 2017). Therefore, increasing the amount of active material inside the reactor will obviously reduce the time necessary for the complete Fe2+oxidation. Nevertheless, according to Jaisankar and Modak (2009), different NMV:ESA ratios could lead to different patterns of material-media contact, which could affect ferric iron productivity (Fe3+ produced per medium volume and time). Therefore, the influence of the amount of A-BC on Fe2+ bio-oxidation was analyzed by increasing the NMV:ESA (mL:cm2) ratio. Figure 2.17 (left axis) shows the time required by the A-BC to oxidize 6 g Fe2+ L-1 in two consecutive bio-oxidation stages (S1 and S2) using different NMV:ESA (mL:cm2) ratios. The time recorded when using the cell suspension culture (control) and the average Fe3+ productivity (right axis) for each ratio are also shown. When comparing the time recorded for the complete Fe2+ bio-oxidation in A-BC preparation step and A-BC bio-oxidation stages (S1-S2) for the same NMV:ESA ratio (1:0.2 mL:cm2), it was concluded that the process was significantly shorter in the latter case, as it was reduced almost to a half (45 % reduction). Junfeng et al. (2007) observed the same reduction percentage between bacterial immobilization on ceramic beads and the first bio-oxidation stage using the active material. The behavior is also in agreement with the results obtained when using A. ferrooxidans (DSM 584) immobilized on a monolithic reactor by Kahrizi et al. (2008), who reported a 33 % reduction of the biooxidation time in the second and third batch cultures compared to the time needed during the immobilization step. Similarly, Akhlaghi (2019) reported a 38 % reduction of this parameter when using hemp fibers as immobilization material. Chapter 2. Operation with immobilized biomass 122 Figure 2.17. Average time required for the complete Fe2+ bio-oxidation in two consecutive stages (left axis) and average Fe3+ productivity (right axis) for different NMV:ESA (mL: cm2) ratios when using A-BC pieces. When comparing the time recorded for the complete Fe2+ bio-oxidation in A-BC preparation step and A-BC bio-oxidation stages (S1-S2) for the same NMV:ESA ratio (1:0.2 mL:cm2), it was concluded that the process was significantly shorter in the latter case, as it was reduced almost to a half (45 % reduction). Junfeng et al. (2007) observed the same reduction percentage between bacterial immobilization on ceramic beads and the first bio-oxidation stage using the active material. The behavior is also in agreement with the results obtained when using A. ferrooxidans (DSM 584) immobilized on a monolithic reactor by Kahrizi et al. (2008), who reported a 33 % reduction of the biooxidation time in the second and third batch cultures compared to the time needed during the immobilization step. Similarly, Akhlaghi (2019) reported a 38 % reduction of this parameter when using hemp fibers as immobilization material. It is noteworthy that there were not significant differences between the bio-oxidizing time for S1 and S2 at each NMV:ESA ratio (individual data not shown), and thus only average results are plotted in Figure 2.17. A similar finding was observed by other authors, who reported constant bio-oxidation times after the first immobilization batch on support materials such as cotton gauze (Nie et al., 2015) or nickel allow fiber (Gomez et al., 2000). Conversely, the NMV:ESA ratio influenced the Fe2+ bio-oxidation time. In this study, the time required for full Fe2+ conversion at a 1:0.1 NMV:ESA ratio was similar to that achieved when cells were grown in suspension (5 % v:v of inoculum). By contrast, this Chapter 2. Operation with immobilized biomass 123 parameter decreased linearly with the A-BC loading for a selected nutrient medium volume (Figure 2.17), which was attributed to a higher initial cell density. In general terms, there was a 19 % reduction in the time required for the complete Fe2+ biooxidation between the reactors with a 1:0.6 and a 1:0.1 NMV:ESA ratio. This difference between samples was more significant when comparing the percentage of Fe2+ converted during the process. For example, after 33 h, the sample with a 1:0.6 NMV:ESA ratio reached a 90 % Fe2+ conversion while only the 60 % was transformed in the case of the 1:0.1 relationship in the same period. The average Fe3+ productivity increased with the NMV:ESA ratio. These results are in agreement with those presented by Jaisankar and Modak (2009), who also reported that lower (media volume):(support material) ratios require longer times for complete iron oxidation and render lower productivities when using biologically active polyurethane foam pieces (BAPUF). By contrast, Pogliani and Donati (2000) found that average Fe3+ productivity was very similar for cultures with densities of glass beads of 1, 5, 10 and 15 %, which was attributed to low amounts of bacteria attached to the glass beads. The results obtained in this experiment led to the conclusion that the highest NMV:ESA ratio tested (1:0.6) was the most favorable when using A-BC for A. ferrooxidans immobilization, as the time for Fe2+ bio-oxidation was significantly reduced and the average productivity increased compared to samples with lower NMV:ESA ratios and cell suspension cultures (control). Additionally, the suspension of A-BC in the media was uniform and the system was adequately shaken. Higher ratios were discarded because more active material pieces in the medium volume might have adverse effects, such as the difficulty for effective shaking or the excessive loss of iron (precipitated as jarosite) on the material´s surface. Therefore, 1:0.6 NMV:ESA ratio was selected for further experiments. 2.3.3.2.3. Fe2+ concentration Besides facilitating biomass handling and reducing iron bio-oxidation time, the use of ABC can enhance the metal removal process performance if higher iron concentrations are bio-oxidized in shorter treatment times (compared to cell suspension cultures). Thus, in this study the influence of Fe2+ concentration (6 vs. 9 g Fe2+ L-1) in the biooxidation time required by A-BC was studied using the selected NMV:ESA ratio (1:0.6 mL:cm2). Higher iron concentrations were not studied as they can exert an inhibitory effect on the activity of immobilized A. ferrooxidans, as concluded by Junfeng et al. (2007) and Jaisankar and Modak (2009). The time required by A-BC for the complete bio-oxidation of Fe2+ remained almost constant in two successive stages in the presence of 6 (36.8±1.6 h) and 9 g Fe2+ L-1 (47.0±1.7 h) (calculated as the average value for two consecutive stages). Conversely, Chapter 2. Operation with immobilized biomass 124 oxidation time decreased 18 % and 28 % for the 6K and 9K media, respectively, in comparison to the cell suspension mode. In addition, the highest Fe3+ average productivity was recorded in the samples with 9K medium (190 mg Fe3+ L-1 h-1), being this value a 16 % and 39 % higher than that obtained in the samples with 6K medium (163 mg Fe3+ L-1 h-1) and the 9K control (136 mg Fe3+ L-1 h-1), respectively. The use of A-BC in a 1:0.6 NMV:ESA ratio was especially recommended for an initial concentration of 9 g Fe2+ L-1, as the bio-oxidation time was reduced and Fe3+ productivity was increased compared to cell suspension growth using the same initial Fe2+ concentration. In addition, Fe3+ productivity was higher than that recorded with A-BC in the 6K medium, which means that a higher amount of the oxidant would readily become available in the medium. It could lead to a higher metal removal rate in any bioleaching and biomachining process, being of great interest for future industrial applications. 2.3.3.2.4. Shaking speed Many authors have demonstrated the influence of shaking speed in bacterial activity during bioleaching and biomachining processes (Ting et al., 2000; Jadhav et al., 2013; Xenofontos et al., 2015; Singh et al., 2018; Chakankar et al., 2019). However, scarce data have been reported about the influence of this parameter on bacterial immobilization and on the time needed by the active material for Fe2+ bio-oxidation in subsequent stages. In this study two shaking speeds were tested at the optimum operating conditions (NMV:ESA ratio = 1:0.6, [Fe2+] = 9 g L-1): 130 and 170 rpm. Higher shaking speed values were not evaluated since excessively vigorous shaking could damage the integrity of the material and, therefore, reduce the lifespan of A-BC. In this study, A-BC preparation time significantly decreased from 63.7±2.3 to 44±0.1 h when the shaking speed was increased from 130 to 170 rpm (Table 2.5). This result was attributed to a more efficient oxygenation and contact between the support material and the culture medium. As far as biobased materials are concerned, Giaveno et al. (2008) have reported a Fe2+ bio-oxidation time of 60 h (graphically inferred) in the first colonization stage of A. ferrooxidans DSM11477 on cross-linked chitosan beads at 180 rpm (9 g Fe2+ L-1, 30 °C, pH = 1.8). When cotton gauze was used, it took 48 h to successfully immobilize A. ferrooxidans CCTCC M2013102 at 165 rpm (9 g Fe2+ L-1, 30 °C, pH = 2.0). Zhu et al. (2017) have concluded that this support material needed a shorter immobilization time than zeolite and activated carbon because the biocompatibility and biosorption capacity of cellulose favored the process (Zhu et al., 2017). Thus, the lower oxidation times shown in Table 2.5 could be attributed to the structural and particular properties of BC in comparison with plant cellulose (de Oliveira et al., 2021). After bacterial immobilization, the Fe2+ bio-oxidation time decreased compared to the A-BC Chapter 2. Operation with immobilized biomass 125 preparation step, probably due to the formation of a denser biofilm during the first biooxidation stage (Table 2.5). Table 2.5. Influence of shaking speed on bio-oxidation time during both A-BC preparation and A-BC use in two consecutive stages. Shaking speed (rpm) Fe2+ bio-oxidation time (h) preparation use 130 63.7±2.3 47.5±2.0 170 44.0±0.1 40.0±0.6 The advantageous results and the fact that material integrity was not affected during the experiments at 170 rpm supported the selection of this shaking speed for further experiments. 2.3.3.3. Influence of dissolved copper concentration on bacterial immobilization and subsequent iron bio-oxidation stages Several studies have reported that the inhibitory effect of dissolved copper in the medium varies between unadapted and adapted bacteria, as well as among A. ferrooxidans strains (Liang et al., 2010; Martinez-Bussenius et al., 2016; Liang et al., 2018). In this study, the influence of dissolved copper on A. ferrooxidans activity was studied during the A-BC preparation and the subsequent bio-oxidation stages (S1-S4) under the previously selected operating conditions (1:0.6 NMV:ESA ratio, 9 g Fe2+ L-1, 170 rpm orbital shaking). The active material was prepared in several bioreactors in the presence of different copper concentrations ranging from 5 to 20 g Cu2+ L-1. The absence of dissolved copper was used as the control sample. The time required for the complete bio-oxidation during the immobilization period increased linearly (R2 = 0.9839) as the dissolved copper concentration increased from 5 to 20 g Cu2+ L-1 (Figure 2.18). Thus, the bio-oxidation time in the presence of 20 g Cu2+ L-1 was 3.1-fold longer than for the control sample during bacterial immobilization stage. Chapter 2. Operation with immobilized biomass 126 Figure 2.18. Fe2+ bio-oxidation time during A-BC preparation and subsequent biooxidation stages at different Cu2+ concentrations (a) and evolution of Fe2+ concentration in the reactor with [Cu2+] = 10 g L-1 (b). It is noteworthy that when the same A-BC pieces were used for four consecutive biooxidation stages (S1-S4) the average process time decreased considerably compared to the A-BC preparation time (Figure 2.18), which was attributed to bacterial adaptation to the metal. Slight variations were recorder among the A-BC5, A-BC10 and A-BC15 samples, and the average value (46.4±4.3 h) was only 14 % higher than that recorded in the copper-free sample (control sample). Another salient result obtained in these samples is that the bio-oxidation time for the four cycles was very similar in all cases, as shown in upper Figure 2.18 for the concentration of 10 g Cu2+ L-1 as a representative example. As far as the results for the A-BC sample with 20 g Cu2+ L-1 are concerned, the time for complete Fe2+ bio-oxidation gradually decreased until 64 % reduction was recorded in stages S2-S4, compared to the A-BC preparation step (Figure 2.18). This response was attributed to the activity inhibition that might be due to the presence of 20 g Cu2+ L-1. Chapter 2. Operation with immobilized biomass 127 Bacterial inactivation was even more severe in the presence of 30 and 40 g Cu2+ L-1, as the amount of oxidized Fe2+ during BAM30 and BAM40 preparation was only 30 % after a prolonged period of 140 h (when the experiment was concluded). Figure 2.19 shows the aspect of the culture medium with A-BC before and after Fe2+ biooxidation in the presence of different concentrations of dissolved copper (0-20 g Cu2+ L-1). Figure 2.19. Reactors with A-BC before (a) and after (b) Fe2+ bio-oxidation in the presence of different concentrations of dissolved copper (0-20 g Cu2+ L-1). Based on the promising results with the A-BC samples, the ability of immobilized A. ferrooxidans to adapt to the presence of even higher concentrations of dissolved copper was explored by exposing the sample A-BC15 to Cu2+ concentrations up to 30 g Cu2+ L-1 in several consecutive bio-oxidation steps. Thus, bacterial resistance and adaptation were verified when the A-BC15 sample was finally able to successfully oxidize all the Fe2+ in the presence of 30 g Cu2+ L-1 in 89.0±1.0 h (Table 2.6). Although the bio-oxidation time increased with the dissolved copper concentration (time = 7.5·[Cu2+] + 66.2, R2 = 0.9941, [Cu2+] range = 20-30 g Cu2+ L-1), the values were significantly lower than those obtained in the A-BC preparation step using unadapted bacteria in the presence of 20 g Cu2+ L-1. These results are consistent with those reported by Liang et al. (2018), who have concluded that adapted A. ferrooxidans has a relatively higher Fe2+ oxidation rate during the process than unadapted bacteria. Chapter 2. Operation with immobilized biomass 128 Table 2.6. Fe2+ bio-oxidation time of sample A-BC15 when exposed to higher dissolved copper concentrations. Stage [Cu2+] (g L-1) Fe2+ bio-oxidation time (h) Immobilization 15 121.0±0.5 S1-S4 15 50.3±2.8 S5-S7 20 74.0±3.6 S8 25 80.5±0.5 S9 30 89.0±1.0 The inhibitory copper concentration achieved in this study for A. ferrooxidans DSM 14882 is higher or similar to those reported in literature for other strains. For example, Mykytczuk et al. (2011) obtained that the copper tolerance for six A. ferrooxidans strains cultivated in suspension ranged from 0.3 to 9.5 g Cu2+ L-1. Similarly, Orellana and Jerez (2011) reported that a 50 % inhibition of growth was registered when unadapted ATCC23270 cells were grown in 0.6 g Cu2+ L-1 and, conversely, an equivalent percentage of inhibition was observed for ATCC53993 at a concentration of 6.5 g Cu2+ L-1. Additionally, Oetiker et al. (2018) reported that strain ATCC 53993 still actively expressed the proteins related to the RND efflux systems at 12.7 g Cu2+ L-1. Higher inhibitory concentrations were achieved by Novo et al. (2000) who reported an oxygen uptake inhibition in the 40-80 % range for eight A. ferrooxidans strains cultivated in the presence of 25.4 g Cu2+ L-1. 2.3.3.4. Material integrity The maintenance of the material´s physical integrity after successive bio-oxidation stages is crucial to ensure process efficiency and avoid issues in its long-term use. Conventionally, bio-based support materials have a shorter lifespan and deteriorate more easily than inorganic ones in biological systems (Lebrero et al., 2014). Nevertheless, they have the advantage of being biodegradable, which contributes to the process sustainability and waste reduction. In this study, the material maintained its physical integrity (corroborated by visual inspection) despite being subjected to extreme acidic conditions (pH 1.8) and vigorous shaking (170 rpm) during several successive stages. During the process, a precipitate appeared on the surface of the A-BC (Figure 2.20a), which was identified as jarosite by FTIR analysis according to the most distinctive bands in the FTIR spectrum (Figure 2.20b) (Giaveno et al., 2008). Zhu et al. (2017) have GENERAL CONCLUSIONS AND FUTURE OVERLOOK General Conclusions and Future Overlook 233 GENERAL CONCLUSIONS This thesis was focused on improving the process of microorganism-assisted mobilization of metals for two applications: the biomachining of copper pieces for engraving microstructures and the bioleaching of copper from printed circuit boards. Both applications have been studied using the same bacterium (A. ferrooxidans). This versatile, resistant and safe microorganism was able to successfully oxidize Fe2+, rendering a bioregenerated oxidant for both applications. The main general conclusions are summarized below. • The 9K solution (containing Fe2+ or Fe3+) was found to be the most suitable medium for both A. ferrooxidans growth and metal oxidation. Copper solubilization was noticeably increased by the presence of microorganisms in comparison to the abiotic system. • An alternate bioprocess with the regeneration and copper solubilization steps in consecutive stages was proposed. The SMRR peaked during the first hour of treatment and the amount of copper mobilized in five consecutive solubilization steps (471.6 mg cm-1) was 52.4 % higher than the amount obtained in the continuous operation for the same treatment time (15 h) (224.6 mg cm-1). This strategy allowed the reduction of both the treatment time and the amount of depleted solution, which enhances the sustainability of the process by prolonging the solution lifespan. • The strategy of immobilizing the biomass on a support material was searched by selecting biocellulose (BC), polyvinyl alcohol (PVA), cellulose triacetate (CTA), and chitosan (CH) to be tested for that purpose. Only BC and PVA hydrogels fulfilled the suitability protocol, and, finally the BC was selected for further experiments based on its novelty and its outstanding properties such as its biodegradability, highly porous network structure and chemical stability. • The best operating conditions for biomass immobilization were established for the BC material. Under these conditions (1:0.6 for the NMV:ESA ratio, 170 rpm for the orbital shaking, and 9 g Fe2+ L-1), the time required for the oxidant regeneration was reduced by 30 % when compared with the cell suspension. The immobilization time was affected by the presence of increasing concentrations of dissolved copper. Nevertheless, when the active material was consecutively exposed to higher concentrations of the metal, it was finally able to successfully oxidize all the Fe2+ in the present of up to 30 g Cu2+ L-1. • The biomachining of copper pieces for engraving structures requires the efficient protection the metal surface not to be exposed to the oxidant solution. The combination of one common lacquer (red light bulb lacquer) and a PSA adhesive General Conclusions and Future Overlook 234 was found to be a feasible and efficient alternative for the selective protection of surfaces without impairing bacterial activity. • The average specific metal removal rate (SMRRav) in the biomachining assays was maximum in the 0-1 h range (20-24 mg h-1 cm-2) and, afterwards, it decreased in a logarithmic trend. The equations for predicting the treatment time required for the selected height to be machined were proposed. • Although similar SMRRav and height values were achieved when suspended or immobilized biomass were used in the biomachining experiments, shorter regeneration times were needed in the presence of the active biocellulose. In addition, other benefit of using immobilized microorganisms would lie in the easily handling (feeding and replacing) of the biomass in large-scale operation. • The repeatability of the process when reusing the biomachining solution is particularly remarkable for this application, as extends its lifespan and increases the sustainability of the process. • As far as microorganism-assisted solubilization of metals from PCBs is concerned, the entire pieces without the epoxy cover were selected for the experiments, because grinding pre-treatment entailed an important cost and generated particulate matter in the environment. In addition, the use of the entire PCBs facilitated the management and separation of the pieces from the leachate, as well as providing a “cleaner” medium for microbial activity. • The alternate (two step) bioleaching experiment with the entire PCBs rendered a total amount of 82 % of copper dissolved by the end of the assay (300 hours), which was attributed to the relevant contribution of the biomass in regenerating the oxidant. • The bioleaching and biomachining processes generated liquid residues with high metal concentrations. Two alternatives were studied for recovering copper from a synthetic solution: precipitation and electrodeposition. The experimental results revealed that both procedures rendered high metal recovery (98 %) at a reasonable cost and required the preliminary oxidation and precipitation of iron, which obviously implied the additional consumption of reagents and longer operation time. • Although the precipitation method was more affordable (although time-consuming) than the electrorecovery, the final product obtained (CuO) is less attractive than Cu0 for the stock market. Bearing in mind that the world demand of copper (and the LME official prices) has been on the rise during the last three months and is expected to go further up, the metal recovery from these depleted solutions can be an entrepreneurial opportunity integrated in the circular economy. General Conclusions and Future Overlook 235 FUTURE OVERLOOK The results of this thesis suggested that further research on several additional aspects would benefit the final productive implementation of both applications. Thus, the future overlook could include: • To search for a consortium of safe microorganisms (instead of one single bacterium type) that could face and mitigate the medium increasing toxicity caused by copper solubilization throughout the process. • To assess the economical and environmental feasibility of the biomachining in detail, in order to disseminate the suitability of that bioprocess for manufacturing molds for other additional productive applications. • To investigate how to apply the proposed alternate bioleaching process to other electronic wastes for metal recovery. In addition, the sustainable and affordable bioextraction of other precious metals (Ag, Au, Pt…) could be an attractive opportunity for entrepreneurs. • To explore the automatization of the mandatory oxidation of iron prior to the copper electrodeposition in waste solutions for operation cost reduction. • To design and install a pilot-scale plant for the bioleaching of entire PCBs in order to obtain technical information and make a decision about the implementation at higher scale. General Conclusions and Future Overlook 236 APPENDIX 1 239 LIST OF PUBLISHED ARTICLES Santaolalla, A., Gutierrez, J., Gallastegui, G., Barona, A., Rojo, N., 2021. Immobilization of Acidithiobacillus ferrooxidans in bacterial cellulose for a more sustainable bioleaching process. Journal of Environmental Chemical Engineering. 9 (4), 105283. Santaolalla, A., García J., Rojo, N., Barona, A., Gallastegui, G., 2020. Viability of two alternatives for treating waste solutions from the biomachining process. Journal of Cleaner Production. 270, 122549. Santaolalla, A., Rojo, N., Gutierrez, J., Barona, A., 2020. Immobilization of Acidithiobacillus Ferrooxidans on Two Hydrogels. Chemical engineering transactions. 79, 7-12. Santaolalla, A., Alvarez-Braña, Y., Benito-Lopez, F., Basabe-Desmonts, L., Barona, A., Gallastegui, G., Rojo, N. Biomachining: an environmentally friendly technique for the fabrication of PDMS microfluidic devices. Sent to Lab on a Chip. LIST OF CONFERENCES Rojo, N., Gallastegui, G., Díaz-Tena, E., Santaolalla, A., Elías, A., Barona., A., 2017. Joint assessment of biomachining and e-waste biorecovery. 10th World Congress of Chemical Engineering. 1-5 October; Barcelona, Spain. Type of presentation: poster. Santaolalla, A., Rojo, N., Crespo, A., Díaz-Tena, E., Gallastegui, G., Barona, A., 2018. Treating waste printed circuit boards from mobile phones: copper leaching in abiotic and biotic media. 23rd International Congress of Chemical and Process Engineering. 25-29 August; Prague, Czech Republic. Poster. Santaolalla, A., Rojo, N., Gallastegui, G., Barona, A., 2018. A sequential process for biomachining copper pieces including oxidant regeneration. 2nd International Conference on Bioresource Technology for Bioenergy, Bioproducts and Environmental Sustainability. 16-19 September; Sitges, Spain. Type of presentation: poster. Santaolalla, A., Rojo, N., Elías, A., Gallastegui, G., Benito-Lopez, F., Basabe-Desmonts, L., Barona, A., 2019. Assessment of the biomachining time for the sustainable engraving of microstructures on metal workpieces. 4th Green and Sustainable Chemistry Conference. 5-8 May; Dresde, Germany. Type of presentation: poster. Santaolalla, A., Rojo, N., Gutierrez, J., Gallastegui, G., Barona, A., 2019. Novel biosupport material for A. ferrooxidans immobilization. 5th European Congress of Applied Biotechnology (ECCE12-ECAB5). 15-19 September; Florence, Italy. Type of presentation: oral communication. Santaolalla, A., Alvarez-Braña, Y., Gallastegui, G., Basabe-Desmonts, L., Rojo, N., BenitoLopez, F., 2019. PDMS microfluidic devices fabrication by a cyclic biomachining process. 240 The 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS 2019). 27-31 October; Basel, Switzerland. Type of presentation: poster. Gallastegui, G., Santaolalla, A., Rojo, N., Urbina, L., García, J., Barona, A.,2019. Options for Recovering Copper from Biomachining Waste Solutions. 3rd International Congress of Chemical Engineering (ANQUE - ICCE -CIBIQ). 19-21 June; Santander, Spain. Type of presentation: poster. Santaolalla, A., Rojo, N., Gutierrez, J., Ureña, I., Gutierrez, I., Barona, A., 2019. An Insight into Novel Materials for A. ferrooxidans Immobilization. 3rd International Congress of Chemical Engineering (ANQUE - ICCE -CIBIQ). 19-21 June; Santander, Spain. Type of presentation: poster. 247 LIST OF TABLES Table 1. Some chemolithotrophic microorganisms used in microorganism-assisted metal mobilization processes reported in literature............................................................................. 15 Table 2. Some heterotrophic microorganisms reported in bibliography for microorganismassisted metal mobilization processes. ....................................................................................... 17 Table 3. Some of the microorganism consortiums employed in microorganism-assisted metal mobilization processes. ............................................................................................................... 19 Table 4. Metal solubilization efficiencies (%) reported in literature employing A. ferrooxidans bacteria. ...................................................................................................................................... 21 Table 5. Different support materials reported in bibliography for A. ferrooxidans immobilization............................................................................................................................. 29 Table 6. Advantages and drawbacks of microorganism-assisted metal mobilization process compared to other metal extraction processes. ......................................................................... 34 Table 7. SMRR (mg h-1 cm-2) values reported in literature for different strains of the A. ferrooxidans bacterium. .............................................................................................................. 37 Table 8. Economical and environmental assessment comparison of hydrometallurgy and biohydrometallurgy methods for PCB treatment (Sodha et al., 2020). ...................................... 42 Table 9. Pulp densities (g L-1) and particle sizes (µm) reported in literature. ............................. 44 Table 10. Composition of culture medium according to final iron concentration. .................... 68 Table 11. Reagents used in the colorimetric method for ferrous and total iron determination. ..................................................................................................................................................... 71 Table 1.1. Experimental conditions for bacterial growth, metal mobilization and abiotic assays. ..................................................................................................................................................... 81 Table 1.2. Total copper amount removed in the biotic (BM1) and abiotic (SN1) experiments after the three-hour immersion of a copper piece. .................................................................... 87 Table 1.3. Maximum SMRR values obtained in the literature and in this study when using different initial concentrations of Fe3+ at T= 30-31 °C). .............................................................. 89 Table 2.1. Experimental conditions for the iron bio-oxidation experiments using the A-BC pieces at 31 °C and pH 1.8. ........................................................................................................ 110 Table 2.2. Copper concentration in the medium during A-BC15 preparation and subsequent bio-oxidation stages with the sample A-BC15. ......................................................................... 111 Table 2.3. Selection of suitable materials for A. ferrooxidans immobilization. ........................ 114 Table 2.4. Time required for the bio-oxidation of the Fe2+ during the immobilization of A. ferrooxidans on different support materials (pH = 1.6-2.0 and T = 30-31 °C). ......................... 120 Table 2.5. Influence of shaking speed on bio-oxidation time during both A-BC preparation and A-BC use in two consecutive stages. ......................................................................................... 125 Table 2.6. Fe2+ bio-oxidation time of sample A-BC15 when exposed to higher dissolved copper concentrations. ......................................................................................................................... 128 Table 2.7. Optimum operating conditions for A-BC preparation and use. ............................... 129 Table 2.8. Fe2+ oxidation time required by A-BC after 15 days of storage (average of two successive bio-oxidation cycles). ............................................................................................... 130 Table 3. 1. Immersion times in the mold-etching experiments. ............................................... 149 Table 3. 2. Experimental conditions in metal removal experiments when using A-BC and cell suspension (control). ................................................................................................................. 151 248 Table 3. 3. SMRR (mg h-1 cm-2), structure´s height (Ht, μm), and removed copper (mCu, mg) variation according to the two treatment time intervals (h) for the circular geometry. ......... 157 Table 3. 4. Height and depths values (µm) reported in bibliography by other authors. .......... 160 Table 3. 5. Linear equations that correlate mold´s height (µm) and removed copper mass (mCu, g) with the number of mold-etching stage (N). .............................................................. 164 Table 3. 6. Relationship between the regeneration time (tregen, h) and the concentration of dissolved copper (Cu, g L-1). ...................................................................................................... 166 Table 3. 7. Linear equations that correlate the height of the structure (Ht, μm) and the amount of copper removed (mCu, g) with the number of mold-etching stages (N). ............................ 169 Table 4. 1. Metal concentration (mg g-1 PCB) of mobile’s PCBs reported in literature and market price according to the London Metal Exchange (LME, € kg-1). ..................................... 188 Table 4. 2. Average metal content in PCBs in this study (mg metal g-1 PCB). ........................... 189 Table 4. 3. Results of the EDXRF analysis of different areas of the PCB powder...................... 189 Table 4. 4. Results of the EDXRF analysis of different areas of the entire PCB. ....................... 190 Table 4. 5. Metal removal values obtained for Cu, Zn, Ni, and Pb. .......................................... 192 Table 4. 6. Leaching efficiencies for several metals reported in the literature when treating PCB pieces. ....................................................................................................................................... 200 Table 5. 1. Parameters measured in the SLR0, SLR1 and SLR2 samples. .................................... 222 Table 5. 2. Cost analysis for copper electrodeposition at 10 V and pH 4.0. ............................. 226