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
Uni e si y o he Basque Coun y (UPV/EHU)
Facul y o Enginee ing o Vi o ia-Gas eiz
Depa men o Chemical and En i onmen al Enginee ing
COPPER SOLUBILIZATION STRATEGIES FOR MOLD
BIOMACHINING AND BIOLEACHING FROM
PRINTED CIRCUIT BOARDS
A a e San aolalla Ramí ez
Vi o ia-Gas eiz, 2021
Supe iso s:
D a. As id Ba ona Fe nández
D a. Naia a Rojo Azace a
(cc) 2021 A a e San aolalla Ramí ez (cc by-nc-nd 4.0)

Acknowledgmen s
ACKNOWLEDGMENTS
Después de dedica los úl imos casi cua o años a es a esis y aunque pa ecía no llega
nunca es e momen o, aquí es oy, esc ibiendo los ag adecimien os pa a iniqui a así
es e capí ulo de mi ida. Po an o, es el momen o de ag adece a odas esas pe sonas
que han hecho de es e iaje una expe iencia de ida en ez de un simple p oyec o de
in es igación y que han caminado jun o a mi du an e es a e apa.
Con el pe miso de mi amilia y amigos, me gus a ía empeza ag adeciendo de o ma
especial a quienes han hecho posible que yo aho a mismo es e edac ando es as
palab as.
Quie o empeza mencionando de o ma especial a mis di ec o as de esis, As id y
Naia a. G acias po con ia en mí y da me la opo unidad de hace es a esis con
oso as. G acias po ues o apoyo, po ues o es ue zo y po ues a dedicación a
pesa de ene o as mil cosas que hace . Es e abajo no hab ía sido posible sin ues a
ayuda.
A Go ka y Junkal, po que, aunque no han sido o icialmen e pa e de la di ección de mi
esis, siemp e han es ado pa a ayuda me y colabo a me en odo lo que he necesi ado
(y po esos dulces que siemp e encon aba en mi aza al llega ). Me habéis dado mucho
a cambio de muy poco.
A Es i, que me enseñó odo lo que debía sabe sob e nues os bichos y u o mucha
paciencia pa a que ap endie a odo bien.
Me sien o muy a o unada de habe podido o ma pa e del depa amen o de
ingenie ía química y del Medio Ambien e en gene al, y de la sección de Vi o ia-Gas eiz
en pa icula . Du an e es os años he conocido gen e espec acula que espe o no dejen
de o ma pa e de mi cí culo. G acias po hace me sen i pa e del depa amen o y po
inclui me y con a conmigo siemp e pa a odos los “ olle es” que iban su giendo. A
Zu iñe, Loli, Aina a, Jon, I a i, Nie es, y demás (lo sien o si me dejo a alguien sois
muchos), po esos ca és solos pe o an acompañados de in e esan es con e saciones a
media mañana. A los que no oman ca é, pe o ambién o man pa e de la sección.
I would like o hank also P . Pie Lens o gi ing me he oppo uni y o be pa o his
esea ch g oup du ing my s ay in Galway. I also wan o hank A indam o guiding me
in my expe imen s du ing my s ay. To whole esea ch g oup o he e y wa m welcome
o he g oup. A Bo ja, po su paciencia y po ayuda me en el labo a o io (y ue a de él).
Aho a, con el pe miso de mis di ec o as y compañe os oy a da las g acias a mi amilia
po habe me acompañado a lo la go de es e camino. Todo lo que engo que ag adece os
es demasiado pa a deja lo esc i o, aunque oy a in en a esumi lo.
Acknowledgmen s
A mi pad e, po que es és donde es és sé que es a ás o gulloso de mí. Te echo de menos.
A mi mad e, po habe me pe mi ido llega a se quien soy hoy. Me has enseñado a
abaja du o pa a consegui lo que quie o y a da siemp e lo mejo de mí. A i e debo
odo lo que soy y odo lo que he conseguido.
A mis he manas mayo es, I zia y Lei e. Po apoya me a lo la go de es e camino. Po que
de oso as he ap endido que nunca es su icien e y que siemp e se puede un poqui o
más. Siemp e habéis sido y se éis un ejemplo pa a mí.
A I a xe, mi gemela, mi o a mi ad, po que siemp e es ás pa a mí. G acias po
aguan a me siemp e odas mis chapas, po acompaña me a apaga el baño a úl imas
ho as del día, pe o sob e odo po apoya me siemp e du an e es e camino. Po que aun
es ando ocupada, siemp e me has pe mi ido es a más ocupada que ú. Si es e dad que
es amos conec adas sab ás lo ag adecida que e es oy.
A Asie , po que aún sin sabe muy bien de lo que le hablaba muchas eces, siemp e me
p egun aba po mis bichos. Po adap a se siemp e a mi disponibilidad (que a eces no
ha sido an amplia como nos hubie a gus ado), pe o sob e odo po apoya me siemp e
y es a a mi lado. Po que en su compañía las cosas malas se con ie en en buenas.
G acias po es a siemp e ahí pa a mí.
A mis amigas, po pe dona me los plan ones y es a ahí pa a los buenos a os. A mis
Ai os, po eza le siemp e a San a Ri a pidiéndole que odo me ue a bien. Al es o de
mi amilia. No puedo nomb a a odos po que somos muchos, pe o ellos ya saben lo
impo an es que son pa a mí.
Sien o que odas las palab as aquí esc i as son insu icien es y espe o algún día pode
de ol e os odo lo que me habéis dado.
Eske ik asko!
Table o con en s
i
SUMMARY ........................................................................................................................ ii
RESUMEN ........................................................................................................................... xi
LABURPENA ...................................................................................................................... x
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. Chemoli hoau o ophic bac e ia ................................................................................. 14
2.2. He e o ophic bac e ia ................................................................................................ 16
2.3. Fungi and yeas s .......................................................................................................... 17
2.4. Mic oo ganism conso ium ......................................................................................... 18
3. Acidi hiobacillus e ooxidans GENUS ................................................................................. 20
4. MECHANISMS OF MICROBIAL METAL MOBILIZATION ........................................................ 22
4.1. Di ec mechanism ....................................................................................................... 22
4.2. Indi ec mechanism ..................................................................................................... 23
4.3. Coope a i e leaching/mechanism .............................................................................. 24
4.4. Thiosul a e and polysul ide mechanism ...................................................................... 24
4.5. Mechanisms acco ding o he ype o eac ion .......................................................... 25
4.5.1. Redoxolysis .......................................................................................................... 25
4.5.2. Acidolysis ............................................................................................................. 26
4.5.3. Complexolysis ...................................................................................................... 26
4.5.4. Bioaccumula ion .................................................................................................. 26
5. FACTORS INFLUENCING MICROORGANISM-ASSISTED METAL MOBILIZATION .................. 27
5.1. Tempe a u e ............................................................................................................... 27
5.2. pH ................................................................................................................................ 28
5.3. Shaking speed .............................................................................................................. 28
5.4. Bac e ial concen a ion and biomass immobiliza ion ................................................. 29
5.5. I on concen a ion....................................................................................................... 30
5.6. P esence o p ocess inhibi o s .................................................................................... 31
5.7. P ecipi a e o ma ion .................................................................................................. 32
6. APPLICATIONS OF MICROORGANISM-ASSISTED MOBILIZATION OF METALS .................... 33
TABLE OF CONTENTS
Table o con en s
ii
6.1. Biomachining ............................................................................................................... 35
6.1.1. Me al wo kpieces p epa a ion ............................................................................ 36
6.1.2. Speci ic me al emo al a e (SMRR) .................................................................... 36
6.1.3. Su ace inish ....................................................................................................... 37
6.2. Elec onic was e bioleaching ....................................................................................... 38
6.2.1. PCB p e- ea men .............................................................................................. 42
6.2.2. T ea men mode: single and mul i-s ages bioleaching ....................................... 44
6.2.3. Pulp densi y ......................................................................................................... 45
7. REFERENCES ........................................................................................................................ 45
MATERIALS AND GENERAL METHODS ................................................................................ 65
1. MATERIALS .......................................................................................................................... 67
1.1. Mic oo ganisms ........................................................................................................... 67
1.2. Coppe wo kpieces ...................................................................................................... 67
2. GENERAL METHODS ............................................................................................................ 68
2.1. Acidi hiobacillus e ooxidans bac e ial g ow h .......................................................... 68
2.2. Coppe mobiliza ion expe imen s ............................................................................... 69
2.3. De e mina ion o he bac e ial concen a ion in he medium ................................... 69
2.4. Me al emo al a e and speci ic me al emo al a e .................................................. 71
2.5. De e mina ion o i on species in solu ion ................................................................... 71
2.6. Me al con en analysis (ICP and AAS) ......................................................................... 73
2.7. Scanning elec on mic oscopy (SEM) .......................................................................... 73
2.8. O he me hods ............................................................................................................ 74
3. REFERENCES ........................................................................................................................ 74
CHAPTER 1. OPERATION WITH SUSPENDED BIOMASS ......................................................... 77
1.1. OBJECTIVE ............................................................................................................................ 79
1.2. MATERIALS AND METHODS................................................................................................. 79
1.2.1. Coppe pieces .......................................................................................................... 79
1.2.2. Mic oo ganisms and cul u e media ........................................................................ 80
1.2.3. Me al mobiliza ion expe imen s ............................................................................. 80
1.2.3.1. E ec o i on con en on bac e ial g ow h and me al mobiliza ion ............... 80
1.2.3.2. SMRR as a unc ion o ime du ing he me al mobiliza ion p ocess ............... 81
1.2.4. Al e na e p ocess: me al mobiliza ion + egene a ion ........................................... 82
1.2.5. Analy ical me hods .................................................................................................. 82
1.3. RESULTS ............................................................................................................................... 82
1.3.1. Me al mobiliza ion expe imen s ............................................................................. 82
Summa y
ix
solu ion, p ecipi a ion and elec odeposi ion, ende ed high me al eco e y a a
easonable cos and equi ed he p elimina y oxida ion and p ecipi a ion o i on, which
ob iously implied he addi ional consump ion o eagen s and longe ope a ion ime.
Al hough he p ecipi a ion me hod was mo e a o dable (al hough ime-consuming), he
inal p oduc ob ained by he elec o eco e y was mo e a ac i e o he s ock ma ke .
Bea ing in mind ha he wo ld demand o coppe has been on he ise du ing he las
h ee mon hs and is expec ed o go u he up, he me al eco e y om he PCBs and
he deple ed solu ions can be an en ep eneu ial oppo uni y in eg a ed in he ci cula
economy.
In summa y, he biomachining o me allic molds and he me al bioleaching om PCBs
we e concluded o be wo a ainable biop ocesses whose u u e implemen a ion on a
la ge scale will make an impo an con ibu ion o sus ainable p oduc ion, e icien
was e managemen and ci cula economy.

Summa y
x
Resumen
xi
RESUMEN
Ac ualmen e la bio ecnología desempeña un papel undamen al en el desa ollo
sos enible. En es e ámbi o, los undamen os de los p ocesos de solubilización de
me ales empleando mic oo ganismos, que adicionalmen e han sido empleados en la
ex acción de me ales de mine ales, es án siendo aplicados a o as á eas, como la
ab icación de mic oes uc u as (biomecanizado) y la ecupe ación de me ales
p esen es en esiduos (biolixi iación).
El impa able aumen o de la demanda mundial de componen es a escala mic omé ica
hace que la ecnología del mic omecanizado es é en cons an e desa ollo con el in de
encon a al e na i as más económicas y sos enibles a los p ocesos ísicos-químicos
adicionales. En es e sen ido, el biomecanizado se con ie e en una p ome edo a
al e na i a que u iliza el po encial de los mic oo ganismos pa a mejo a el p oceso de
g abado de mic oes uc u as, esul ando se sos enible y espe uoso con el medio
ambien e po su bajo consumo ene gé ico y su bajo cos e ope acional.
Po o o lado, el in e és en a anza en la ecupe ación de me ales de esiduos de
apa a os eléc icos y elec ónicos (RAEE) se debe a la posibilidad de de ol e los me ales
a la cadena de alo y a la opo unidad de ges iona de mane a sos enible es os esiduos,
cuya gene ación ha aumen ado exponencialmen e en los úl imos años.
A pesa de que es as aplicaciones son p ome edo as y p esen an en ajas en e a o as
ecnologías, oda ía exis e la necesidad de es udia cie os aspec os ope acionales que
pe mi an diseña e implan a p ocesos mejo ados a escala p oduc i a.
El p incipal obje i o de es a esis es in es iga el p oceso de solubilización de cob e en
un medio bac e iano, con el in de p opone es a egias que pe mi an mejo a el
endimien o de ca a a dos aplicaciones conc e as: el biomecanizado de moldes pa a la
ab icación de disposi i os mic o luídicos y la ecupe ación de me ales a pa i de placas
de ci cui o imp eso de elé onos mó iles en desuso.
La conocida bac e ia ex emó ila Acidi hiobacillus e ooxidans ue seleccionada po su
esis encia en medios ácidos y su ni el de biosegu idad. An es de aplica el p oceso de
mo ilización biológica de me ales a los dos usos desc i os, se es udia on algunos
aspec os elacionados an o con el c ecimien o mic obiano, como con la solubilización
del cob e y la ope a i idad del p oceso p opues o.
Dado que el endimien o de solubilización del cob e depende de la concen ación de
oxidan e, inicialmen e se es udió la in luencia de es a a iable en el medio biológico. La
concen ación de hie o (Fe2+ pa a el c ecimien o mic obiano o Fe3+ pa a la solubilización
del me al) más e icaz esul ó se 9 g L-1 (medio 9K). La elocidad de biooxidación del Fe2+
en el medio 9K ue 2.8 eces supe io que en o os medios y la ac i idad bac e iana
Resumen
xii
con ibuyó a que la can idad de cob e solubilizada ue a 25% más ele ada que en un
medio abió ico.
A con inuación, eniendo en cuen a que la máxima asa especí ica de eliminación (TEE)
se alcanzó du an e la p ime a ho a de a amien o, se diseñó un p oceso que al e naba
una e apa de solubilización del me al de 3 ho as con la pieza me álica sume gida en la
disolución y una e apa de bio egene ación del oxidan e sin la pieza. Es a al e na i a
demos ó se e ec i a pa a educi el iempo necesa io pa a solubiliza una de e minada
can idad de me al, así como pa a ex ende el iempo de ida de la disolución de
a amien o.
Pos e io men e, se lle ó a cabo el es udio de la inmo ilización de la biomasa sob e a ios
ma e iales de sopo e, como una es a egia pa a a o ece la ope a i idad del sis ema.
T as un ensayo con a ias al e na i as, se seleccionó la celulosa bac e iana po sus
adecuadas p opiedades mecánicas, es abilidad química, y es uc u a po osa. Asimismo,
se op imiza on las condiciones de ope ación que pe mi ie on una oxidación más ápida
del Fe2+. Es e sopo e p esen ó las en ajas de inmo iliza de mane a sa is ac o ia la
biomasa, de no in e e i en el c ecimien o bac e iano, de pode se almacenada a 4 °C
en es ado ac i o y de ene capacidad pa a oxida el Fe2+ en p esencia de can idades
ele adas de Cu2+. Asimismo, p esen ó mejo compo amien o que o o ma e ial
ambién es ado, el poli inil alcohol.
En elación a las aplicaciones, se es udió el biomecanizado como al e na i a pa a la
gene ación de moldes me álicos pa a la ab icación de es uc u as mic o luídicas. En
es e p oceso es o almen e necesa io p o ege adecuadamen e la supe icie me álica
que no se desea mecaniza (o g aba ). Pa a ello se seleccionó una combinación de una
laca oja y un adhesi o de PSA que no a ec a on a la ac i idad mic obiana. Un aspec o a
des aca de es a aplicación es la epe i i idad del p oceso empleando an o la disolución
esca como la disolución egene ada ( a ios ciclos). Es o pe mi ió es ablece las
ecuaciones ma emá icas que posibili an p edeci el iempo necesa io pa a ob ene un
molde con una al u a de inida (en dos amos de a amien o: 0-1 h y 1-7 h). Los iempos
de egene ación ue on más co os cuando la biomasa es aba inmo ilizada en
biocelulosa, aunque los endimien os de mecanizado ue on simila es a los de la
suspensión. La c ecien e acumulación de cob e disuel o en el medio u o un e ec o
nega i o en la egene ación de la disolución de biomecanizado, aunque no ue
impo an e pa a concen aciones in e io es a 3 g Cu2+ L-1. El empleo de la biomasa
inmo ilizada en BC p esen ó además la en aja de la acilidad ope a i a a la ho a de
eemplaza el medio bac e iano.
En la aplicación de biolixi iación de cob e a pa i de placas de ci cui o imp eso de
mó iles en desuso, la he e ogeneidad de es os ma e iales en é minos de amaño,
composición o es uc u a obligó a diseña una e apa p e ia de adecuación. Se decidió
Resumen
xiii
elimina la cubie a epoxi y a a las piezas en e as o pa e de ellas, pe o sin i u ación
p e ia. La biolixi iación se lle ó a cabo en dos pasos (biooxidación y biolixi iación po
sepa ado). Es a es a egia con ibuyó al aumen o de la mo ilización de los me ales de
las placas, siendo la e icacia de la lixi iación en medio bac e iano signi ica i amen e más
ele ada en compa ación con la lixi iación química. El empleo de placas sin i u a
pe mi ió abaja con un medio “más limpio” y a o able pa a el c ecimien o de la
biomasa.
Las dos aplicaciones desc i as gene an disoluciones ago adas con al o con enido de
me ales que deben se a adas an es de su e ido. La ecupe ación de cob e de esas
disoluciones de uel e es e me al a la cadena de alo , mi iga el impac o ambien al, y
con ibuye a la iabilidad económica de ambas aplicaciones. En es e es udio se ha
abajado con una disolución sin é ica ago ada y se han aplicado dos sencillos
a amien os: p ecipi ación química accionada y elec ólisis. Ambas p opues as ue on
e icaces en é minos de ecupe ación del cob e de la disolución esidual, aunque el
p oceso elec olí ico da como esul ado un p oduc o más a ac i o (Cu0) desde el pun o
de is a de me cado.
En esumen, el mecanizado de moldes me álicos y la ex acción de me ales de placas de
ci cui os imp esos en medio bac e iano son dos aplicaciones p ome edo as cuya
implan ación u u a a mayo escala supond á una impo an e con ibución a la
p oducción sos enible, la ges ión in eg ada de esiduos y la economía ci cula .
Resumen
xi

Labu pena
x
LABURPENA
Gau egun bio eknologia en aplikazioek un sezko ga an zia du e ga apen jasanga ia
lo zeko. A lo ho e an, mik oo ganismoak e abil zen di uen solubilizazio p ozesua
aspaldi ik e abili izan da mea za i zan, mine ale a ik me alak e auz eko. Biop ozesu
ho en oina iak bes e alo ba zue an e e aplika u di a azken u eo an, hala nola,
mik oegi u en ab ikazioan (biomekaniza uan) e a hondakine an dauden me alen
be esku apenean (biolixibiazioan).
Mik oegi u en ab ikazioa i dagokionez, eskala mik ome ikoko osagaien eskae a asko
igo da mundu mailan azken u eo an. Eskae a ho ek, mik omekanizazio eknologia en
ga apen e engabea bul za u du, ohiko p ozesu isiko-kimikoak baino al e na iba
ekonomikoagoak e a jasanga iagoak au ki zea p emiazkoa delako. Auke en a ean,
biomekanizazioa de i zon eknika aipa dai eke be eziki, hau da, mik oo ganismoek
lagundu a, mik oegi u ak g aba zeko eknika jasanga ia. Ene gia-kon sumo baxua e a
ope azio-kos u mode a ua di a be a en aldeko ezauga ie ako ba zuk.
Bes alde ik, T esna elek iko e a elek onikoen hondakinak (TEEH)ge o e a kan i a e
handiago an pila zen di a munduan, e a ho ien kudeake a a azoa oso la ia da gau
egun. Hondakin ho ien kudeake a e aginko a en e a jasanga ia en helbu u nagusia,
me alak balo iza u e a balio-ka e a (lehengaien me ka u a) buel a zea da, me alen i u i
na u alen ago pena saihes eko. Balo izazio ho i, mik oo ganismoek lagundu ako
biolixibiazioa en bidez bu u u dai eke.
Aipa u ako p ozesu biek (biomekanizazioak e a biolixibiazioak) aban aila naba menak
di uz e be idanik e abili di en eknologiekin alde a u a. Hala e e, zenbai aspek u
ope a ibo sakon az e u beha da hobe u ako p ozedu ak diseina u e a ho iek ekoizpen
eskalan (eskala handian) inplemen a u ahal iza eko.
Tesi honen helbu u nagusia mik oo ganismoek lagundu iko kob ea en solubilizazio
p ozesua ike zea izan da, e a ho e a ako adie azi ako aplikazio bien e endimendua
hobe zeko es a egiak bila u e a az e u zi en. Aplikazio ho iek ondokoak izan zi en:
gailu mik o luidikoak ekoiz eko molde me alikoen biomekanizazioa, e a sakeleko
ele onoen zi kui u inp ima ue an dauden me alen be esku apena.
Acidi hiobacillus e ooxidans bak e ia ex emo ilo ezaguna auke a u zen ike ke an,
ingu une azidoe an haz eko duen gai asun aipaga iaga ik e a maneia zeko duen
biosegu asun maila al uaga ik. Me alen mobilizazio biologikoa adie azi ako bi
e abile a a a aplika u baino lehen, mik oo ganismoen hazkun zan, kob ea en
solubilizazioan e a p oposa u ako p ozesua en e aginko asunean e agina du en
hainba ak o e az e u zi en.
Lehenik, oxida zailea en kon zen azioa en e agina az e u zen, kob ea en
solubilizazioa en e ekina ho en menpekoa bai a. Kob e kon zen azio e aginko ena
Labu pena
x i
(Fe2+ hazkun za- asean e a Fe3+ me ala en solubilizazioan) 9 g L-1 izan zen (9K hazkun za-
medioa). Bu din(II) espeziea en biooxidazio-abiadu a 9K hazkun za-medioan, bes e
kul u a an baino 2.8 aldiz handiagoa izan zen, e a bak e ioen ak ibi a ea i eske
solubiliza u ako kob e kan i a ea ingu une abio ikoan baino % 25 al uagoa izan zen.
Ope a ibi a ea i dagokionez, me ala en solubilizazio-p ozesua e a oxida zailea en
bi so ze-p ozesua xandaka (bana u a) aplika zeko es a egia diseina u zen, ezaba ze-
asa espezi iko maximoa lehenengo o duan lo u zela kon uan ha u a. Es a egia hau
oso egokia izan zen, me al kan i a e jakina solubiliza zeko beha den denbo a
mu iz eko e a disoluzioa en e abilga i asuna luza zeko.
Biomasa en immobilizazioa en e aginko asuna az e zeko, hainba ma e ial hau a u
zi en lehendabizi. Guz ien a ean, zelulosa bak e ianoa auke a u zen os eko
espe imen ue an e abil zeko, ezauga i mekaniko bikainak, egonko asun kimiko al ua,
e a egi u a po o su egokia zi uelako. Gaine a, Fe2+-a en oxidazio azka agoa
ahalbide zen zuela e a ez zuela hazkun za mik obia a kal e zen ondo ioz a u zen.
Mik oo ganismoak i sa si a zi uen zelulosa bak e ianoak (euska i ak iboak) hainba
onu a zi uen suspen sio zelula a ekin alde a u a, hala nola: biomasa e ek iboki
immobiliza zen zuen, 4 °Can bil egian go de zeko egokia izan zen, e a Cu2+ kon zen azio
handien p esen zian Fe2+-a oxida zeko ahalmena man endu zuen.
Lehenengo aplikazioa i dagokionez, biomekanizazioa ike u zen. P ozesu ho e an,
mekaniza u (g aba u) nahi ez den azale a egoki babes u beha da de igo ez e a,
ike ke a hone an, mik oo ganismoen ak ibi a ean kal e ik e agi en ez zu en laka e a PSA
pega ina auke a u zi en helbu u ho e a ako. Naba men zekoa da p ozesua
e epikako a izan zela, bai disoluzio oxida zaile p es a u be ia e a bai a bi so u ako
disoluzioa e abili zi enean. Ho i eske , e a da u espe imen ale an oina i u a, al ue a
jakina duen moldea so zeko beha den denbo a au esa eko ekuazio ma ema iko bi
p oposa u zi en bi a amendu- a e bi an: 0-1 h e a 1-7 h a ee an.
Biomekanizazioa en e ekina an zekoa izan zen biomasa zelulosa bak e ianoan
immobiliza u zenean e a bak e iak esekidu an (suspen sioan) e abili zi enean. Ai zi ik,
disoluzio oxida zailea en bi so zea azka agoa izan zen euska i ak iboa e abili zenean.
Disolba u ako kob e kon zen azioa en igoe a p og esiboak e agin nega iboa izan zuen
disoluzio oxida zailea en bi so zean. Hala e e, 3 g Cu2+ L-1 baino kon zen azio
baxuago an e agina ez zen la ia izan. Zelulosa bak e iano ak iboa e abil zea en bes e
aban aila ba medio biologikoa e azago e a azka ago o dezka zeko auke a ema en
zuela zen.
Sakeleko ele onoen zi kui u inp ima uek du en kob ea en be esku apena i
dagokionez, lehenbizi, laginen egoki ze e apa diseina u egin beha izan zen, zi kui u
hauek oso he e ogenoak zi elako. Epoxi es alkia ken zea e a piezak oso ik (edo ho ien
pa ea) a a zea e abaki zen, bi inke a ik gabe. Biolixibazioa bi pausue an bu u u zen:
Labu pena
x ii
biooxidazioa lehenengo e a os ean biolixibiazioa, zi kui ua medio ik a e a a. Es a egia
ho ek me alen mobilizazioan e agin posi iboa izan zuen e a, ondo ioz, lixibiazioa en
e ekina handiagoa izan zen medio bak e ianoan ingu une kimikoan baino. Zi kui uak
bi indu gabe e abil zea i eske , biomasa en hazkun za ako egokiagoa izan zen medio
“ga biagoa” e abili zen.
Desk iba u ako bi aplikazioek, me al kon zen azio al uko disoluzio ago uak so u
zi uz en e a, ondo ioz, hondakin likido ho i kudea zeko modu egokia au ki u beha izan
zen. Disolba u ik zegoen kob ea be esku a zea i enbide e aka ga ia izan zen, me al
p ezia u ho i balio-ka e a (me ka u a) buel a zeko negozio-auke a zelako, biop ozesuen
ingu umen-inpak ua mu iz en zelako e a bi aplikazioen bide aga i asun ekonomikoan
inpak u posi iboa izan zuelako. Ike ke a-lan hone an disoluzio ago u sin e ikoa e abili
zen e a bi a amendu aplika u zi zaizkion: hauspea ze za ika ua e a elek olisia. Bi
p ozesuak e aginko ak izan zi en e a kob ea en be esku apen-maila al ua lo u zen,
nahiz e a p ozesu elek oli ikoa en bidez salmen a ako p oduk u e aka ga iagoa lo u.
Labu bilduz, ike ke a hone an az e u di en bi biop ozesuak (me alezko moldeen
biomekanizazioa e a zi kui oen inp ima u ik me alen bioe auzke a edo biolixibiazioa)
e aginko ak iza eaz gain, ekoizpen jasanga ia en, hondakinen kudeake a
in eg a ua en e a ekonomia zi kula ean aldeko bul zada ema eko e o kizun handiko
esnak di a.
Labu pena
x iii
OBJECTIVES

Objec i es
9
OBJECTIVES
The main objec i e o his hesis is o imp o e he e iciency o he mic oo ganism-
assis ed solubiliza ion o coppe by p oposing s a egies ha can be applied in he
ollowing p ocesses: he biomachining o coppe pieces o eng a ing mic os uc u es
and he eco e y o ha me al om disused mobile elephones. Bo h applica ions ha e
been s udied using he same bac e ium (A. e ooxidans) as i is a e y e sa ile and
esis an mic oo ganism wi h a low biosa e y isk.
The seconda y objec i es a e:
o To con ibu e o he p oposal o a semi-con inuous me al mobiliza ion p ocess
wi h suspended biomass ha allows he main enance o he maximum emo al
a e o coppe while minimizing he amoun o deple ed solu ion. The con inuous
egene a ion o he oxidan will allow he p ocess o wo k on as long as he
biomass is ac i e.
o To explo e he po en ial o using a suppo ma e ial o immobilizing he biomass.
Tha ma e ial should be easily biosyn hesized in he labo a o y, a o dable and
sus ainable.
o To assess he bene i s o using immobilized biomass o imp o ing he e iciency
o he me al solubiliza ion p ocess, in compa ison o suspended biomass.
o To design a p ocess o eng a ing mic os uc u es on coppe pieces wi h he inal
objec i e o manu ac u ing molds o mic o luidic de ices. This new applica ion o
he biomachining has no been explo ed so a , and i is an inno a i e use o ha
biop ocess wi h manu ac u ing pe spec i es.
o To s udy he me al ex ac ion e iciency when he p in ed ci cui boa ds om
obsole e mobile elephones a e bioleached. The imely p e ea men s o he
he e ogenous PCBs be o e me al ex ac ion is a challenge o be aced.
o To assess he echnical and economic iabili y o wo al e na i es o ea ing he
was e solu ions ob ained in he coppe biomachining: chemical p ecipi a ion and
elec ochemical me hod.
Objec i es
10
LITERATURE
REVIEW

Li e a u e e iew
13
1. MICROORGANISM-ASSISTED METAL MOBILIZATION
The hyd ome allu gical ex ac ion o me als om mine als and he subsequen
p ecipi a ion and eco e y is an ancien echnology ha was i s used in China as ea ly
as 100-200 B.C. (Eh lich, 2001). In he pas , he mobiliza ion o he me als in mine als
was a ibu ed o an abio ic p ocess and i was only in he 1950s ha he i s acidophilic
i on and sul u oxidizing bac e ia we e isola ed and iden i ied om acid mining d ains,
and he on-going esea ch cla i ied he basic mechanisms o he biosolubiliza ion o he
me als (Colme e al., 1950; Mish a e al., 2005).
Coppe ex ac ion om i s o es was in oduced in Spain by he A abs in he mines o Rio
Tin o a ound he 18 h cen u y. P e iously, du ing he 17 h cen u y, mo e han 2 million
ons o coppe we e ob ained by bioleaching in he deposi s o he Falun mine (Sweden)
(Eh lich,2001). I was no un il he middle o he 20 h cen u y when his p ocess began
o be scien i ically s udied and he p esence o he in ol ed bac e ia was disco e ed
(Bosecke e al., 1997). Bac e ia A. e ooxidans ( o me ly Thiobacillus e ooxidans) and
Acidi hiobacillus hiooxidans (A. hiooxidans) we e epo ed o be esponsible o he
bioleaching p ocess, being he mos s udied ones o da e.
Nowadays, biohyd ome allu gy (o bioleaching) is conside ed an en i onmen al iendly
echnology ha uses he ac i i y o mic oo ganisms o he eco e y me als om
mine als, concen a es and ecycled o esidual ma e ials (Mish a e al., 2005; Rawlings
and Johnson, 2007; Gumulya e al., 2018; Kaksonen e al., 2018; Habibi e al., 2020). The
mic obial ole is he con inuous bio- egene a ion o he oxidizing agen (Fe3+)
esponsible o he chemical dissolu ion o he me al. Theo e ically, he oxidan is ne e
deple ed and he ex ac ion con inues as long as he mic obial pe o mance is
main ained. Consequen ly, he oxidizing agen does no ha e o be con inuously
supplied by chemical addi ion, wi h he consequen economic and en i onmen al
bene i s (Ba ona e al., 2018)
The mos impo an applica ion o bioleaching in he las cen u y has been mine al
ex ac ion, bu o he bio echnological al e na i es based on he same p inciple ha e
also been s udied in ecen yea s. Two o hese applica ions a e he biomachining o
me allic pieces ( he machining o me al pieces by biological me hods) and he eco e y
o me als om was e elec ical and elec onic equipmen (WEEE). Bo h o hem will be
de ailed in he las sec ions o his li e a u e e iew.
2. MICROORGANISMS IN MICROORGANISM-ASSISTED METAL MOBILIZATION
PROCESSES
The mos dis inc i e cha ac e is ic o any mic oo ganism-assis ed me al mobiliza ion
p ocess is he use o mic oo ganisms. The main mic obial g oups in ol ed in he p ocess
Li e a u e e iew
14
a e chemoli hoau o ophic p oka yo es, he e o ophic bac e ia and ungi. In addi ion,
mic obial conso iums ha e also been s udied.
2.1. Chemoli hoau o ophic bac e ia
The g oup o au o ophic chemoli ho ophic bac e ia a e he mos s udied
mic oo ganisms bo h in he biomachining o me allic pieces and in he bioleaching o
PCBs. This ype o o ganisms p esen s a high ole ance o hea y me als, being a c ucial
cha ac e is ic ha makes hem sui able o hese applica ions (O ell e al., 2010).
chemoli ho ophic bac e ia ob ain he ene gy equi ed o g owing om he oxida ion
o some ino ganic compounds such us as sul ides, elemen al sul u (S0), e ous ions,
and, some o hem, e en om hyd ogen ions (Hed ich and Johnson, 2013). In
biohyd ome allu gy, he mos employed chemoli ho ophic bac e ia a e acidophilic
bac e ia ha g ow p e e ably a pH alues om 1.5 o 4 unde ae obic condi ions.
Among he acidophilic ones, he mos s udied bac e ium is A. e ooxidans, due o i s
abili y o oxidize bo h soluble and non-soluble ino ganic subs a es (Uno e al., 1993;
Wang e al., 2009; Liang e al., 2010; Hocheng e al., 2012b; Hocheng e al., 2012c; Díaz-
Tena e al., 2014; Xeno on os e al., 2015; Nie e al., 2015a; Muhammad e al., 2015;
Singh e al., 2018). Howe e , he sui abili y o o he mic oo ganisms has also been
e alua ed bo h o hei applica ion in biomachining and in WEEE bioleaching. Figu e 1
shows wo common chemoli ho ophic mic oo ganisms used in bioleaching p ocesses.
Figu e 1. SEM mic og aph: A. hiooxidans (published by Qua ini e al., 2017) (a) and
Lep ospi illum e ooxidans (published by V doljak and Spille , 2005) (b).
Table 1 summa ies some chemoli ho ophic mic oo ganisms epo ed in bibliog aphy.
Li e a u e e iew
15
Table 1. Some chemoli ho ophic mic oo ganisms used in mic oo ganism-assis ed me al
mobiliza ion p ocesses epo ed in li e a u e.
Name
Type
pH
T
(°C)
Re e ence
Acidi hiobacillus
e ooxidans
M.
1.5-4.0
28-35
Wang e al., 2009; Liang e al., 2010;
Hocheng e al., 2012c; Díaz-Tena e al.,
2014; Xeno on os e al., 2015;
Muhammad e al., 2015; Nie e al.,
2015a; Singh e al., 2018; Benzal e al.,
2020
Acidi hiobacillus
hiooxidans
M.
2.0-3.5
28-30
B andl e al., 2001; Chang e al., 2008;
Wang e al., 2009; Liang e al., 2013;
Isilda e al., 2016; Ma a e al., 2018;
Nase i e al., 2019; Lee e al, 2020
Acidi hiobacillus e i o ans
M.
1.9-3.4
27-32
Isilda e al., 2016; Peng e al., 2019
Sul obacillus
he mosul idooxidans
M.T.
1.9-2.4
40-60
De eci e al., 2004; Ilyas e al., 2007;
Díaz-Tena e al., 2018
Acidi hiobacillus caldus
M.T.
2.0-2.5
42-45
Zhou e al., 2007; Fu e al., 2008; Wang
e al., 2012
Sul obacillus sibi icus
M.T.
2.0
50
Zhang e al., 2015
Lep ospi illum e iphilum
M.T.
1.5-1.8
45–50
Fu e al., 2008; Gu e al., 2013; Zhao e
al., 2015
Acidianus manzaensis
E.T.
1.0-5.0
60–90
He e al., 2009; Zhu e al., 2011; Liu e
al., 2016
Sul olobus me allicus
E.T.
1.3-1.7
65-80
Vilcáez e al., 2008; Plumb e al., 2008
Acidianus b ie leyi
E.T.
2.0
65
Konishi e al., 1998; Bha adwaj and
Ting, 2013; Samadzadeh e al., 2020
Acidianus copahuensis
E.T.
3.0
75
Cas o and Dona i, 2016
Me allosphae a sedula
E.T.
2.0-4.5
65-80
Mikkelsen e al., 2007; Yu e al., 2019
Sul olobus sol a a icus
E.T.
2.0-4.0
80
Roshani e al., 2017
Acidi hiobacillus albe ensis
M.
2.0-4.5
28-50
Xia e al., 2007
Acidianus ambi alens
E.T.
2.0-5.0
80
Roshani e al., 2017
Me allosphae a hakonensis
E.T.
3.0
70
K ok e al., 2013
Sul olobus acidocalda ius
E.T.
1.0-6.0
55-85
Linds öm e al., 1993
Me allosphae a p unae
E.T.
2.0-3.0
75
S o e al., 2003
Acidianus in e nus
1.0-5.5
65-96
Mikkelsen e al., 2006
Thiobacillus p ospe us
M.
1.0-4.5
23-41
Hube and S e e , 1989
Fe oplasma cup icumulans
M.T.
1.0-1.2
22-63
Hawkes e al., 2006
M.: Mesophilic; M.T.: Mode a e he mophilic; E.T.: ex eme he mophilic.
Li e a u e e iew
16
The he mophiles g oup can be classi ied in o mode a ely he mophilic bac e ia and
ex emely he mophilic bac e ia acco ding o hei op imal g owing empe a u e ange.
Mode a ely he mophilic bac e ia ha e an op imal g ow h empe a u e be ween 40-60
°C and comp ise s ains such as Sul obacillus he mosul idooxidans (De ici e al., 2004;
Ilyas e al., 2007), Lep ospi illum e iphilum (Gu e al., 2013, Zhao e al., 2015) o
Acidi hiobacillus caldus (Zhou e al., 2007; Wang e al., 2012). The e is an inc easing
in e es in s udying he mophilic bac e ia, especially o applica ion in he chalcopy i e
bioleaching, due o hei unique me abolic cha ac e is ics and hei ole ance o high
empe a u es, which can enhance he bioleaching kine ics (De eci e al., 2004; Vilcáez
e al., 2008; Zhao e al., 2019). Su p isingly, his g oup o bac e ia appa en ly a e mo e
sensi i e o high pulp densi ies and exhibi a lowe ole ance o me al concen a ion
(Panda e al., 2015). The ex emely he mophilic bac e ia ha a e able o g ow a highe
empe a u es (be ween 60 and 90 °C) a e sca ce in li e a u e. As an example, Acidianus
manzaensis (He e al., 2009; Zhu e al., 2011) o Sul olobus me allicus (Vilcáez e al.,
2008; Plumb e al., 2008) s ains can be men ioned.
2.2. He e o ophic bac e ia
He e o ophic bac e ia ob ain he ene gy om he oxida ion o o ganic compounds such
as lipids, alcohols, suga s o hyd oca bons and, unde app op ia e condi ions, hey
p oduce ce ain o ganic amino acids and o he me aboli es which a e esponsible o
he me al dissolu ion in he p ocesses (indi ec mechanism) (Bosecke , 1997; B andl e
al., 2008; Shabani e al., 2013; Ba ne e al., 2018). The abili y o me al bioleaching om
WEEE by he e o ophic mic oo ganisms has also been epo ed in li e a u e (Hassanien
e al., 2014; Shin e al., 2015; Ba ne e al., 2018). The genus Bacillus and Pseudomonas
ha e been desc ibed as he mos e ec i e he e o ophic bac e ia o me al
solubiliza ion (Bosecke e al., 1987; G oude , 1987). Figu e 2 shows wo examples o
hese genus, pa icula ly Pseudomonas ae uginosa and Pseudomonas pu ida.
Figu e 2. SEM mic og aph: Pseudomonas ae uginosa (published by Kasuga e al., 2011)
(a) and Pseudomonas pu ida (published by Me um e al., 2017) (b).
Li e a u e e iew
23
Al hough he exac mechanism used by A. e ooxidans o colonize su aces has no been
cla i y ye , hese bac e ia can co e su aces wi h a dense bio ilm and a co ela ion
be ween pilus exp ession and s ong a achmen has been ound (Li e al., 2010). The
EPSs p oduced by his mic oo ganism a e composed o neu al suga s, a y acids and
u onic acids. They media e a achmen o he su ace and concen a e Fe2+ ions by
complexa ion h ough u onic acids and o he me aboli es (Li e al., 2010)
The ollowing di ec o con ac mechanism o i on leaching has been p oposed ( o A.
e ooxidans bac e ia) (Sand e 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)
O he gene al global equa ion shown below can be used o summa ize he di ec
leaching mechanism om me al sul ides, whe e i can be no ed ha he inal p oduc is
he me al sulpha e (Bosecke e al., 1997):
(Eq. 3)
4.2. Indi ec mechanism
The indi ec bioleaching p ocess akes place wi hou physical con ac be ween he
bac e ia and he solid (Sand e al., 2001; Kuma and Yaashikaa, 2020). The e o e, i is
also called non-con ac mechanism (T ibu sch, 2001). The me al is oxidized by he ac ion
o he e ic ion (oxidizing agen ). Thus, he e ic i on is capable o oxidizing me als while
being educed o e ous i on. The la e is mic obially oxidized again in o Fe3+, leading
o a cyclic oxida ion p ocess (Hamidian, 2011; Mish a and Rhee, 2014) (Figu e 4b).
Indi ec bioleaching mechanism o he oxida ion om me al sul ides can be desc ibed
as ollows ( o A. e ooxidans bac e ia) (Sand e 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)
Fo he speci ic case o pu e me al leaching he equa ions a e as ollows:
Chemical p ocess o me al dissolu ion
M0 (s) + 2 Fe3+ (ac) → M2+ (ac) + 2Fe2+ (ac) (Eq. 7)
MeS + 2 O
2
MeSO
4
Bac e ia

Li e a u e e iew
24
Biochemical egene a ion o he oxidan Fe3+
Fe2+ (ac) + O2 (g) + 4H+ (ac) → Fe3+ (ac) + 2H2O (ac) (Eq. 8)
4.3. Coope a i e leaching/mechanism
I has been epo ed ha coope a i e leaching be ween a ached and plank onic cells is
possible (Rojas-Chapana e al., 1998). In his case, he bac e ia a ached o mine al
su aces supply Fe2+ and S o he plank onic cells as hei ene gy sou ce. Then he e ous
ion is oxidized by ee cells, supplying he oxidan which is u he used in indi ec
leaching (T ibu sch, 2001; Li e al., 2013). Consequen ly, i is he e o e a combina ion
be ween he di ec and indi ec mechanism, o con ac and non-con ac mechanisms
(Zhang e al., 2018) (Figu e 4c).
4.4. Thiosul a e and polysul ide mechanism
Thiosul a e mechanism is based on he oxida ion o acid-insoluble sul ides FeS2, MoS2,
and WS2 (py i e, molybdeni e, and wol ami e) by he Fe3+ ions wi h he hiosul a e as
he main in e media e and sul a e as he end-p oduc (Hamidian, 2011).
In he polysul ide mechanism, mine al solubiliza ion such as sphale i e, galena, o
chalcopy i e (ZnS, PbS, CuFeS2, espec i ely) occu s h ough a combined a ack o e ic
i on and p o ons. In his case, he main in e media es a e polysul ides and elemen al
sul u (Figu e 5). The gene a ed elemen al sul u can be oxidized o sul a e in he
p esence o sul u -oxidizing mic obes as shown in Equa ion 13.
The wo mechanisms a e summa ized by he ollowing equa ions (Sand e al., 2001):
Thiosul a e 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)
Polysul ide 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+
Bac e ia
0.125 S8 + 1.5 O2 + H2O SO4- + 2 H+
Bac e ia
Li e a u e e iew
25
Figu e 5. Schema ic diag am o hiosul a e (a) and polysul ide (b) mechanisms in
bioleaching (adap ed om Rohwe de e al., 2003).
4.5. Mechanisms acco ding o he ype o eac ion
Mic obial me al mobiliza ion can also be desc ibed acco ding o he ype o eac ion
in ol ed. Thus, i can ake place by acidolysis ( o ma ion o o ganic o ino ganic acids),
edoxolysis (oxida ion- educ ion p ocess), complexolysis ( o ma ion o complexes and
chela es) and, o a lesse ex en , bioaccumula ion (Bossha d e al., 1996; Wu and Ting,
2006; Okoh e al., 2018).
4.5.1. Redoxolysis
Redoxolysis mechanism is di ided in o di ec and indi ec mechanism (Wa ling, 2006).
Thus, me als can be dissol ed bo h di ec ly by he di ec con ac wi h bac e ial cells o
wi h hei ex acellula compounds (di ec mechanism o “con ac ” mechanism) o
indi ec ly by he me abolic p oduc s gene a ed by he biomass (indi ec o non-con ac
mechanism) (Isilda e al., 2016; Co nu e al., 2017). Mic oo ganisms playing a ole in
edoxolysis p ocesses ob ain ene gy om solid mine als and he i on oxidizing A.
e ooxidans o sul u oxidizing A. hiooxidans bac e ia can be pa icula ly highligh ed.
Fe ic i on (Fe3+) ion is one o he mos common edoxolysis agen s in leaching sys ems
as i is a s ong oxidan wi h a s anda d educ ion po en ial o +0.770 V(SHE) (Diaz-Tena
e al., 2016; Isilda e al., 2016). Redoxolysis p ocess is based on he p e iously desc ibed
non-con ac mechanism whe e a cyclic oxida ion p ocess akes place. In addi ion, some
mic obial s ains a e capable o oxidizing S0 unde anae obic condi ions, educing Fe3+
o Fe2+ (Rawlings, 2005).
Li e a u e e iew
26
In he edoxolysis mechanism, he edox po en ial is an essen ial pa ame e con olling
he p ocess since i is ela ed o he [Fe3+]/[Fe2+] concen a ion a io. This alue inc eases
as long as he e ous i on is being oxidized in o e ic i on and i is ela ed o he
o ma ion o sul u ic acid. Thus, he edox po en ial is a quick and con enien pa ame e
o de e mine he biomachining/bioleaching p og ess (Rohwe de e al., 2003; Diaz-Tena
e al., 2016).
4.5.2. Acidolysis
In his case, he me al solubiliza ion is achie ed by he ac ion o o ganic (malic, oxalic,
gluconic, ace ic, ci ic) and ino ganic (H2SO4) acids p oduced by mic oo ganisms. The
p o ons becoming om he acids syn hesized by hese mic oo ganisms, a e capable o
weakening he bond o me al ions ende ing he dissolu ion o he me al (Bu gs alle e
al., 1992; B andl and Fa ama zi, 2006). The oxygen a oms ha co e he me al su ace
a e quickly p o ona ed. Then he p o ons and he oxygen combine wi h wa e and,
he e o e, he me al is de ached om he su ace (Am i i, 2012; Bahaloo-Ho eh e al.,
2018; Xia e al., 2018).
Al hough his mechanism can be pe o med by au o ophic sul u oxidize
mic oo ganisms (A. hiooxidans), acidolysis is he p incipal mechanism in me al
mobiliza ion p ocesses in ol ing he e o ophic ungal s ains (Anjum, 2009, Vakilchap
e al., 2016).
The ollowing equa ion ep esen s he mechanism o acidolysis,
MeO + 2H+ → Me2+ + H2O (Eq. 14)
(MeO, being he me al oxide)
4.5.3. Complexolysis
Unlike acidolysis, he o ganic acids p oduced by ce ain mic oo ganisms (Aspe gillus
nige , Penicillium spp., o Rhizopus spp., among o he s) ac as chela ing agen s which a e
capable o leaching me als h ough he o ma ion o complexes (Mish a and Rhee, 2014;
Ho eh e al., 2016). Complexolysis p ocess is slowe han acidolysis, al hough bo h
mechanisms can occu join ly (Okoh e al., 2018). Apa om o ganic acids, o he
me aboli es, such as side opho es can o m complex and solubilize me als such as he
e ic i on (Shenke e al., 1999, Gadd, 2004; Osman e al., 2019).
4.5.4. Bioaccumula ion
Bioaccumula ion consis s o he accumula ion o o ganic o ino ganic pollu an s inside
li ing mic oo ganisms (Ba on e al., 1995; Cha e jee e al., 2020). Howe e , he e is a
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27
con o e sy abou he “bioaccumula ion” concep when he pollu an accumula ion
occu s in dead cells (bioso p ion) (Chojnacka, 2007; Velásquez and Dussan, 2009).
Se e al mic oo ganisms such as bac e ia, ungi, yeas , and some algae a e able o
accumula e hea y me als by bioaccumula ion and by bioso p ion (Aksu and Ka abayı ,
2008; Pa k e al., 2010). Fo ins ance, Aspe gillus nige , Aspe gillus oe idus, Aspe gillus
nomius, T ichode ma ha zianum, Aspe gillus len ulus and Ya owia lipoly ica a e
epo ed o be me al bioaccumula o s (Du sun e al., 2003; Ge e al., 2011; Mish a Malik,
2012; Zo i e al., 2014; Cha e jee e al., 2019).
The main d awback o his p ocess is he oxici y o high concen a ions o he me al in
he medium which could cause he dea h o bioaccumula o s ains (Chojnacka, 2007;
Ge e al., 2011).
5. FACTORS INFLUENCING MICROORGANISM-ASSISTED METAL MOBILIZATION
As e e y biological sys em, his p ocess is a ec ed by se e al ac o s such as bac e ial
concen a ion, empe a u e, pH, shaking speed o he p esence o inhibi o s, among
o he s (Díaz-Tena e al., 2016). In his ype o p ocesses, he i on concen a ion (oxidan )
in he medium is also a ele an a iable.
The s udies ca ied ou o da e ha e concluded ha he op imal ope a ing condi ions
(op imal alues o empe a u e, pH, Fe2+ concen a ion, and o he s) a e dependen on
he s ain used in each speci ic case. The e o e, he in luence o hese ac o s is common
o many bio echnological applica ions, al hough speci ic ea u es ha e o be conside ed
in each pa icula case.
The main ac o s ha a ec he mic oo ganism-assis ed me al mobiliza ion p ocess a e
desc ibed below.
5.1. Tempe a u e
In o de o main ain op imal bac e ial g ow h condi ions, i is necessa y o es ablish a
sui able empe a u e ange du ing he bioleaching p ocess. Each mic oo ganism
equi es di e en op imal empe a u e condi ions. Thus, mesophilic mic oo ganisms
( he mos ypical ones in bioleaching/biomachining p ocesses) g ow a empe a u es
be ween 25 and 40 °C (Rawlings, 1997; Leahy e al., 2007; Kuma and Yaashikaa, 2020),
while he mophiles g ow a empe a u es abo e 40 °C (Plumb e al., 2008; Dopson and
Johnson, 2012, Dona i e al., 2016). Fo example, A. e ooxidans, he mos common
mic obe associa ed wi h bioleaching/biomachining p ocesses, has i s op imum g owing
empe a u es a 30 °C, and i loses i s ac i i y a empe a u es below 10 °C o o abo e
40 °C (Modak e al., 1996, Mousa i e al., 2007, Ka imi e al., 2010).
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28
Tempe a u e is also a de e mining pa ame e o inc easing oxida ion eac ion a es, as
deduced om he A henius equa ion (Le enspiel, 2005; F anzmann e al., 2005; Leahy
e al., 2007).
5.2. pH
In addi ion o being ela ed o he mic obial g ow h, he pH alue is closely ela ed o
he me al dissolu ion e iciency and o he o ma ion o p ecipi a es in he cul u e
medium. The pH in he 2.0 and 3.0 ange is he op imal alue o bac e ia o oxidize
e ous i on (Kuma and Yaashikaa, 2020).
The me al solubiliza ion p ocess in ol es acid consuming eac ions, which causes he pH
inc ease ho ough he p ocess (Vilcaez e al., 2008) (Equa ion 8). I he pH exceeds he
alue o 2.2, he p ecipi a ion o me als and he o ma ion o ja osi e a e clea
incon enien s (Xeno on os e al., 2015). The e o e, wo king a pH alues as low as
possible (depending on he bac e ial s ain employed) can inc ease he e iciency o he
me al mobiliza ion since mos o he me als a e usually solubilized unde low pH alues
(Ilyas e al., 2007; Xiang e al., 2010).
5.3. Shaking speed
An op imal shaking speed p o ides he app op ia e condi ions o he mic obial g ow h,
as well as an e icien con ac be ween he ma e ial o be ea ed and he cul u e
medium. In addi ion, shaking speed is a pa ame e ha a ec s he biop ocesses such as
biomachining and bioleaching o PCBs.
As a as he biomachining o me allic wo kpieces is conce ned, some au ho s ha e
concluded ha he speci ic me al emo al a e (SMRR) when using A. e ooxidans
s ains keeps o a minimum i agi a ion is no p o ided, and ha i conside ably inc eases
when inc easing he shaking speed (Jadha e al., 2013; Xeno on os e al., 2015; Díaz-
Tena e al., 2016). Howe e , his inc ease is no linea a high shaking speeds
(Xeno on os e al., 2015). Jadha e al. (2013) concluded ha he op imal shaking speed
is 150 pm unde he es ed condi ions ( ea men o coppe pieces a 30 °C using he
supe na an om a cul u e o A. e ooxidans (13823) s ain wi h an ini ial i on
concen a ion o app oxima ely 3.6 g Fe2+ L-1). These au ho s ob ained he highes SMRR
alue (15.9 ± 1.3 mg Cu h-1 cm-2) a 150 pm, in compa ison o he esul s ob ained when
shaking mo e igo ously.
In he case o bioleaching ea men o elec onic was e, many expe imen s ha e been
epo edly ca ied ou a shaking speeds o 150 pm (B andl e al., 2001; Shah e al.,
2015; Isilda e al., 2016; Ma a e al., 2018; Wang e al., 2018), al hough, in a lesse

Li e a u e e iew
29
ex en , lowe speeds (120 pm) (Xiang e al., 2010) and highe ones (up o 200 pm) (Ilyas
e al., 2007; Liang e al., 2010) ha e also been s udied.
5.4. Bac e ial concen a ion and biomass immobiliza ion
The bac e ial densi y plays an impo an ole in he p ocess e iciency, since i
de e mines he ime equi ed by bac e ia o e-oxidize all he Fe2+ o Fe3+ (Zhu e al.,
2017). In gene al, a cons an cell concen a ion is ecommended du ing he p ocess in
o de o a oid he educ ion in he dissolu ion a e o me als (Díaz-Tena e al., 2016).
One o he al e na i es p oposed by some au ho s o inc ease he cell densi y ho ough
he p ocess is he immobiliza ion o he mic oo ganisms on sui able suppo ma e ials
ha allows g ow h on hei su ace. Some o he ma e ials p oposed by di e en au ho s
o he immobiliza ion o he A. e ooxidans bac e ium a e shown in Table 5.
Table 5. Di e en suppo ma e ials epo ed in bibliog aphy o A. e ooxidans
immobiliza ion.
Suppo ma e ial
Ope a ing condi ionsa
Re e ence
Nickel alloy ib e
pH 1.8, 10 % inoc., 200 pm, 30 °C, 600 mL
Gomez e al., 2000
Ce amic beads
pH 1.6, 10 % inoc., ai , 30 °C, 500 mL
Jun eng e al., 2007
Hemp ib es
pH 1.6, 10 % inoc., 180 pm, 30 °C, 50 mL
Akhlaghi, 2019
Monoli hic
pa icles
pH 1.6, 30 °C, ai
Kah izi e al., 2008
Chi osan beads
pH 1.8, ai , 30 °C, 100 mL
Gia eno e al., 2008
Co on gauze
Ac i a ed ca bon
Zeoli e
pH 2.0, 10 % inoc., ai , 30 °C, 500 mL
Zhu e al., 2017
Co on gauze
pH 2.0, 10 % inoc., ai , 30 °C, 500 mL
Nie e al., 2015b
Foam ma e ial
pH 1.5-2.0, 10 % inoc., 240 pm, 30 °C
Jaisanka and Modak,
2009
Sul SDVB-GAC
Sul SDVB
PUF
pH 1.8, 10 % inoc., 200 pm, 35 °C,
200 mL
Koseoglu-Ime and
Keskinle , 2013
Biocellulose
pH 1.8, 5 % inoc., 170 pm, 31 °C, 100 mL
San aolalla e al., 2021
aP ocesses ha do no speci y shaking speed is because a e shaken by ai injec ion, (indica ed
as ai ).
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30
Mos o he s udies ha e been ca ied ou wi h he A. e ooxidans bac e ium, due o i s
na u al endency o immobiliza ion (Nema i e al., 1998; Gia eno e al., 2008; Jaisanka
e al., 2009). These s udies ha e ocused on in es iga ing he immobiliza ion p ocess
and he capaci y o he immobilized biomass o oxidize Fe2+. On he con a y, li le da a
is a ailable abou he ac i i y o he immobilized biomass du ing he me al leaching
s age o he in luence o he inc easing me al concen a ion h oughou he p ocess.
The encapsula ion o he biomass in a ma ix is ano he al e na i e o biomass
immobiliza ion. In addi ion o ensu ing adequa e cell densi y, his op ion has a
p o ec i e e ec agains shaking u bulences (Ve meulen and Nikolay, 2017). Likewise,
Gia eno e al. (2008) ha e concluded ha a packed eac o wi h chi osan beads in which
A. e ooxidans had p e iously been g own could ope a e wi h a medium low a e up
o eigh imes highe han a eac o wi h ee cells cul u e. Ano he ad an age o
biomass encapsula ion is he g ea e ole ance o inc easing coppe concen a ions, in
compa ison o he cell suspension mode (Ve meulen and Nikolay, 2017).
5.5. I on concen a ion
I on is he main ac o in he bioleaching and biomachining p ocess when he A.
e ooxidans bac e ium is employed, since i con ibu es o he mic obial g ow h and
he me al dissolu ion p ocess. As a as he g ow h s age o A. e ooxidans bac e ia is
conce ned, i is a ec ed by he concen a ion o i on in he medium in di e en ways.
Fi s , he concen a ion o Fe2+ has an impac on he ime equi ed by he cul u e o
oxidize all Fe2+ o Fe3+, wi h longe imes being necessa y as he ini ial Fe2+ concen a ion
inc eases (Jadha e al., 2013). Second, he cell concen a ion also seems o be a ec ed
by he ini ial concen a ion o Fe2+ in he cul u e medium. In a s udy ca ied ou by
Hocheng e al. (2012c) using A. e ooxidans (BCRC 13820) s ain, hese au ho s epo ed
ha he cell concen a ion p ac ically doubled when he concen a ion o Fe2+ in he
medium inc eased om 11 o 22 g Fe2+ L-1 while he inc ease was less no iceable when
he concen a ion o Fe2+ was 3 and 4 imes highe .
Kawabe e al. (2003) concluded ha excessi ely high concen a ions o Fe3+ can inhibi
he oxida ion o Fe2+ by A. e ooxidans bac e ium and ha he deg ee o inhibi ion is a
unc ion o he s ain used. Thus, he T23-3 s ain is capable o oxidizing Fe2+ wi h
concen a ions o 26 g Fe3+ L-1 in he medium, while he ac i i y o he ATCC19859 s ain
is comple ely inhibi ed a 16 g Fe3+ L-1.
In he biomachining p ocess, he inc easing concen a ions o Fe3+ en o ces he
dissolu ion o he me al (coppe in mos s udies) and allows a highe speci ic me al
emo al a e (SMRR) o be achie ed. Howe e , oo high concen a ions o his ion a e
no ecommended o indus ial applica ion, since he inal su ace quali y o he
biomachined piece would be e y poo , he sul u ic acid consump ion would inc ease
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31
and uncon olled ja osi e p ecipi a ion would ake place (Díaz-Tena e al., 2016). Jadha
e al. (2013) ha e obse ed a linea endency in he inc emen o SMRR wi h i on
concen a ion (in he ange 3.6-14.8 g L-1, app oxima ely), al hough he su ace
de e io a ion was conside ably g ea e when he highes concen a ions we e es ed (a
30 °C using he supe na an o A. e ooxidans (13823) cul u e).
In he WEEE bioleaching p ocess, Yang e al. (2009a) ha e concluded ha he highe he
concen a ion o Fe3+ in he medium, he as e he coppe in he PCBs dissol ed. Thus,
he ime equi ed o solubilize all he coppe p esen in he PCB was educed om 96 o
36 h when he concen a ion was inc eased om 2.4 o 6.7 g Fe3+ L-1 (A. e ooxidans
cul u e a 30 °C and 165 pm). In a p ocess in which he g ow h o mic oo ganisms and
he solubiliza ion o me als ook place simul aneously, Xiang e al. (2010) ha e ound
ha he a e o me al leaching inc eased wi h he concen a ion o Fe2+ up o 9 g Fe2+ L-
1, bu i dec eased wi h he p esence o 12 and 15 g Fe2+ L-1, which was a ibu ed o he
ja osi e p ecipi a ion and he consequen passi a ion o he su ace o be ea ed. Xiang
e al. (2010) and Kha i e al. (2018) ha e join ly concluded ha an ini ial concen a ion
o 9 g Fe2+ L-1 is he adequa e one ha allows ob aining a highe solubiliza ion o me als
when using wo conso iums o mic oo ganisms wi h di e en o igins.
5.6. P esence o p ocess inhibi o s
The p esence o a a ie y o me als in he leaching medium can exe a oxicological
e ec on mic obial g ow h. As an example, coppe is essen ial o he me abolic ac i i y
o A. e ooxidans, since i se es as an elec on dono du ing he mic obial g ow h
p ocess and i has been ound in he s uc u e o Rus icyanin, a p o ein ha ac s as an
elec on anspo e (Lilo a e al., 2007; Myky czuk e al., 2011; Co nu e al., 2017).
Con e sely, high concen a ions can induce he dena u a ion o p o eins and nucleic
acids, causing he biomass dea h (Valix, 2017). The e o e, he p esence o ce ain
concen a ions o me als in solu ion, which a e con inuously p oduced du ing he
p ocess, can inhibi bac e ial ac i i y (Lilo a e al., 2007). Ne e heless, some au ho s
ha e sugges ed ha he ole ance o mic oo ganisms o di e en me als can be
inc eased i hey a e adap ed o he me al p esence du ing hei ac i a ion (Ilyas e al.,
2007; Liang e al., 2013; Ra ind a e al., 2015; Pou hossein and Mousa i, 2018).
In he pa icula case o he WEEEs bioleaching, oxic elemen s including a la ge numbe
o me als (Ag, Al, As, Cd, C , Cu, Fe, Hg, Mn, Ni, Pb and Zn) can be dissol ed, as well as
o he o ganic componen s and epoxy esins (Ongondo e al., 2011; Isilda e al., 2016).
In o de o inc ease he ole ance o he mic oo ganisms o he oxic p esence, h ee
main s a egies ha e been s udied: a) he p e-acclima ion o he mic oo ganisms o
hea y me als´ p esence be o e ca ying ou he leaching s age (Ilyas e al., 2007; Liang
e al., 2010), b) he use o conso ia o mic oo ganisms (Ilyas e al., 2007; Ilyas e al.,
Li e a u e e iew
32
2013b) and c) cellula adap a ion by ea ing he was es in se e al s ages (Xiang e al.,
2010).
The me al concen a ion alues wi h oxicological e ec s epo ed in bibliog aphy a y
depending on he bac e ial s ain and he ope a ing condi ions. As an example, in he
case o he cul u e o bac e ia A. e ooxidans isola ed om Río Tin o (Huel a, Spain),
Cab e a e al. (2005) concluded ha his s ain was able o ole a e he ollowing me al
concen a ions: 0.4 g C 3+ L-1, 10 g Cu2+ L-1, 10 g Cd2+ L-1, 30 g Zn2+ L-1 and 30 g Ni2+ L-1.
These limi alues a ied when a conso ium o he e o ophic Acidiphilium bac e ia was
added o he cul u e, ob aining in his case limi alues o 0.4 g C 3+ L-1, 4 g Cu2+ L-1, 10 g
Cd2+ L-1, 40 g Zn2+ L-1 and 15 g Ni2+ L-1. Das e al. (1998) used an A. e ooxidans s ain
isola ed om he Malanjkhand coppe mine (India) and i was ound o su i e in he
p esence o 20 g Cu2+ L-1 in a single cul u e s age.
Tole ance was signi ican ly inc eased in a s udy by Ve meulen and Nikolay (2017), in
which he encapsula ion o A. e ooxidans (DSM 11477) in poly inyl alcohol allowed o
ob ain an oxida ion a e o Fe2+ o Fe3+ in he p esence o 40 g Cu2+ L-1 2-3 imes highe
han ha ob ained by he ee cells.
5.7. P ecipi a e o ma ion
When Fe3+ accumula es in he leaching medium and he pH o he solu ion inc eases,
Fe3+ may p ecipi a e as ja osi e, which can lead o a loss o 77 % o he i on a ailable in
he solu ion (Xiang e al., 2010; Wang e al., 2018). Likewise, p ecipi a es can co e he
su ace o be ea ed, hus p e en ing he dissolu ion o he me als (P adhan e al.,
2008; Xiang e al., 2010). The e o e, he o ma ion o p ecipi a es has an in luence bo h
on he p ocess e iciency and cos .
In he case o a bioleaching/biomachining solu ion con aining Fe3+, he main p ecipi a es
a e i on hyd oxides and ja osi e (MFe3 (SO4)2 (OH)6, whe e M = K+, Na+, NH4+, Ag+ o
H3O+). The o ma ion o hese p ecipi a es is ela ed o pH, empe a u e, ca ion
a ailabili y o SO42− concen a ion (Wang e al., 2018).
In addi ion, he join p esence o a high a ie y o me als (such as in PCB bioleaching
solu ions) can esul in a o ma ion o mo e complex p ecipi a es. Ilyas e al. (2013b)
analyzed he p ecipi a e ob ained in a PCB bioleaching p ocess using a bac e ial
conso ium including Sul obacillus he mosul idooxidans and The moplasma
acidophilum he mophilic bac e ia. The p ecipi a e was de e mined o be composed o
PbSO4, Ag2SO4, SnO2, AgFe3(SO4)2 (OH)6 and H2SnO3, in he p esence o 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 %.
Li e a u e e iew
39
Thus, he p ope managemen o disca ded elec ical and elec onic equipmen (EEE)
has become one o he main en i onmen al conce ns in de eloped coun ies due o
se ious e ec s hei componen s gene a e bo h o human heal h and o he
en i onmen (O uño e al., 2013; Jadhao e al., 2016; Ho lge sson e al., 2017).
Rega ding he en i onmen al impac o WEEE, he Eu opean Union (EU) a emp ed o
limi he use o haza dous subs ances in he manu ac u ing o hese equipmen h ough
he enac men o he RoHS Di ec i e 2002/95/EC (Kuma e al., 2017).
As a as he Di ec i e 2012/19/EU o he Eu opean Pa liamen on WEEE is conce ned,
hese esidues a e classi ied in he ollowing ca ego ies: 1. Tempe a u e exchange
equipmen ; 2. Sc eens, moni o s, and equipmen con aining sc eens ha ing a su ace
g ea e han 100 cm2; 3. Lamps; 4. La ge equipmen (any ex e nal dimension mo e han
50 cm); 5. Small equipmen (no ex e nal dimension mo e han 50 cm); 6. Small IT and
elecommunica ion equipmen (no ex e nal dimension mo e han 50 cm). Among hese
ca ego ies, he o al amoun o WEEE gene a ed in 2019 was basically made up o small
equipmen (17.4 M ), la ge equipmen (13.1 M ), empe a u e exchange equipmen
(10.8 M ) and sc eens (6.7 M ). Lamps and small elecommunica ion equipmen , wi h
0.9 M and 4.7 M espec i ely, ep esen ed smalle ac ions (Figu e 8) (Ni hya e al.,
2020). I has been p edic ed ha he gene a ion o was es om empe a u e exchange
de ices and small and la ge de ices will egis e he highes g ow h a es among all he
ca ego ies. Con e sely, sc een was e is expec ed o dec ease in he coming yea s due o
he eplacemen o hea y ca hode- ay ube (CRT) sc eens by la al e na i es (Baldé e
al., 2017).
Figu e 8. To al WEEE gene a ion in 2019 pe ca ego y (adap ed om Ni hya e al., 2020).
Despi e egula ions in o ce, he e ec i e managemen o mos WEEE is s ill sca ce. In
Eu ope, app oxima ely only 35 % o ha was e is ecycled, while he es is dumped,
ans e ed o de eloping coun ies, o “simply los ” (Isilda e al., 2019). Howe e ,
WEEE is an impo an seconda y sou ce o high- alue me als, so i s sus ainable eco e y
0 5 10 15 20
Lamps
Small IT
Sc eens and moni o s
Hea exchanging de ices
La ge equipmen
Small equipmen
WEEE gene a ion in 2019 (M )

Li e a u e e iew
40
is manda o y o a oid he deple ion o na u al sou ces, mi iga e he pollu ion ha
causes i s disuse and achie e i s e icien ecycling wi hin he applica ion o he ci cula
economy p inciples. Due o i s high con en o aluable ma e ials, se e al s udies on
WEEE ecycling ha e been ca ied ou as an a emp o eco e hose aluable ma e ials
(Mize o e al., 2018; P iya and Hai , 2020; Roy e al., 2021).
In addi ion o legal egula ions, he “was e p inciple” was included in he Was e
F amewo k Di ec i e, Di ec i e 2008/98/EC o he Eu opean Pa liamen and Council, in
2008. Thus, he commonly known was e hie a chy py amid (Figu e 9) is composed o
i e measu es: p e en ion, p epa ing o euse, ecycling, eco e y (including ene gy
eco e y), and disposal. This was e hie a chy py amid gi es he highes p io i y o he
p e en ion and educ ion o was e gene a ion, and, i gene a ed, i gi es p io i y o
di ec euse and ecycling me hods (Di ec i e 2008/98 / CE).
Figu e 9. Was e hie a chy acco ding o di ec i e 2008/98/CE.
Among he di e en WEEE ypes, mobile phones (included in ca ego y 6) signi ican ly
con ibu e o he was e amoun o be managed (Hi a e al., 2017). In 2016, abou 435
K o disused mobile phones we e gene a ed wo ldwide (Baldé e al., 2017). The a e age
weigh composi ion o a s anda d mobile phone is app oxima ely 50 % plas ics, 15 %
glass and ce amic, 15 % me als, and he es o he ma e ials (Mol ó e al., 2011; Tes aye
e al., 2017). I is used as a suppo o elec onic componen s and as a basis o
connec ing hose using conduc i e pa hways (Hadi e al., 2015). The main en i onmen al
impac o his ype o was e is a ibu ed o he p in ed ci cui boa ds (PCBs), which ha e
a high me al con en . Figu e 10 shows he a e age weigh composi ion o a s anda d
mobile phone PCB (Yamane e al., 2011; Palmie i e al., 2014).
Li e a u e e iew
41
Figu e 10. A e age weigh composi ion o a s anda d mobile phone PCB.
A mobile phone can con ain up o 40 di e en elemen s, such as commonly used me als
(coppe and in), p ecious me als (gold and sil e ), and o he s such as pla inum and
palladium (Kaya, 2016). Be ween 65 and 80 % o he ma e ials in a mobile phone a e
ecyclable (Mol ó e al., 2011) bu , ecycling PCBs can be pa icula ly di icul due o he
wide a ie y o componen s.
The ex ac ion o he me als con ained in he PCBs (so called u ban mining) is an
undoub ed economic oppo uni y and a p io i y measu e o p o ec he en i onmen
and public heal h. I is es ima ed ha he alue o he aw ma e ials con ained in
disca ded mobile phones in 2016 was € 9,400 million (Baldé e al., 2017).
The adi ional echniques used o he ecycling o PCBs a e g ouped in o wo main
ypes: hyd ome allu gical p ocesses (based on he ex ac ion o me als using aqueous
and o ganic liquid solu ions) and py ome allu gical p ocesses (based on hea ing). These
ea men s a e esponsible o se e al en i onmen al p oblems such as he o ma ion
o b omina ed and chlo ina ed di-benzo u ans, he gene a ion o dioxins o he oxici y
o he employed eagen s (Ning e al., 2017; Kha i e al., 2018; Liu e al., 2020).
Con e sely, he bioleaching p ocess is an en i onmen ally iendly ecycling al e na i e,
as i has ad an ages o e cu en echnologies: i has a low ea men cos , and i s
en i onmen al impac is mode a e. Howe e , some echnical issues equi e u he
esea ch o imp o ing p ocess pe o mance and subsequen high scale implemen a ion
(Isilda e al., 2019; A ya and Kuma , 2020).
As a as he echno-economic assessmen o hyd ome allu gy and biohyd ome allu gy
is conce ned, Isilda (2018) and Baniasadi e al. (2019) calcula ed he ea men cos pe
kg PCB and he con ibu ion o each me hod o he clima e change. They concluded ha
he mos a o dable and en i onmen ally iendly p ocess is he biological al e na i e
(Table 8, ep oduced om Sodha e al., 2020).
Li e a u e e iew
42
Table 8. Economical and en i onmen al assessmen compa ison o hyd ome allu gy and
biohyd ome allu gy me hods o PCB ea men (Sodha e al., 2020).
Technique
Cos (€ kg-1 PCB)
Clima e change con ibu ion
In es men
Ope a ional
To al cos
kg CO2 kg-1 PCB
Biohyd ome allu gy
0.457
0.159
0.616
8.26
Hyd ome allu gy
0.446
0.224
0.670
14.6
The main d awbacks ha mus be o e come o he indus ial implemen a ion o his
echnology a e: he complex na u e o some PCB ma e ials (B andl e al., 2001; Xiang e
al., 2010), he possible oxici y o he non-me allic ac ion o PCBs (Ilyas e al., 2007;
Shah e al., 2015; Isilda e al., 2019), he inhibi ion o bac e ia by he p esence o high
me al concen a ions in he solu ion (Ilyas e al., 2013b; A shadi and Mousa i, 2015a),
and he lack o a s anda dized p ocess o he eco e y o he me als p esen in he
deple ed solu ions.
The p e ious disman ling o emo al o componen s om PCBs is an addi ional p oblem
ha can be o e come by a no el applica ion o bioleaching: biodisman ling. Monne on-
Enaud e al. (2020) p esen ed he biodisman ling (disman ling using bioleaching) as a
no el easible applica ion o emo ing he PCB componen s. I can be implemen ed as
a new uni ope a ion in he ecycling p ocess.
The speci ic key aspec s o he success ul bioleaching o mobile phone PCB such as he
p e- ea men o he PCBs (en i e pieces o c ushed PCB), he ea men o he ma e ial
in mul i-s ages o he amoun o ma e ial o be ea ed pe olume o medium
cul i a ion (pulp densi y) a e desc ibed below.
6.2.1. PCB p e- ea men
The ma e ials used in he manu ac u ing o mobile phone PCBs a e di ided in wo main
ac ions: non-me allic and me allic componen s. The non-me allic componen s a e
ibe glass (ino ganic), epoxy esin (o ganic) and b omina ed lame e a dan s. The o he
ac ion, made up o me allic elemen s, con ains a single laye o mul ilaye o Cu, lead
solde , in solde , and o he me als such as nickel, i on, lead, cobal , aluminium, gold,
indium, o an imony (A shadi and Mousa i, 2015b). The ecycling o he non-me allic
ac ion has no ecei ed much a en ion in li e a u e because i is no p o i able o da e
(Ilyas e al., 2007; Adhapu e e al., 2014). Two ypes o PCB p e- ea men s ha e been
p oposed: emo al o he non-me allic ac ion and size educ ion.
Li e a u e e iew
43
The non-me allic ac ion (epoxy esin) co e ing he boa ds is oxic o mic oo ganisms
(Isilda e al., 2019) and i hinde s he in e ac ion be ween he me allic su ace and he
bioleaching solu ion. Fo his eason, some au ho s sepa a e he me allic and non-
me allic ac ion be o e imme sing he was e in o he bioleaching medium (Jujun e al.,
2015; Wu e al., 2018).
The emo al o he non-me allic ma e ials by washing he PCB dus samples wi h a
sa u a ed solu ion o NaCl has p o ed o imp o e he p ocess (Ilyas e al., 2007; Shah e
al., 2015). Senophiyah-Ma y e al. (2018) ha e s udied he e iciency o o he di e en
p e- ea men me hods o epoxy coa ing emo al om PCBs. These au ho s ha e
es ed di e en solu ions con aining NaOH and sol en s like e hanol, ace one, ween-
80, ca binol, benzyl alcohol, and acids like sul u ic acid, ni ic acid and hyd ochlo ic acid.
No epoxy emo al has ob ained wi h sol en s while comple e epoxy co e emo al was
achie ed wi h sodium hyd oxide solu ions and wi h sul u ic and ni ic acids. Howe e ,
hey selec ed he NaOH as he bes p e- ea men me hod since he acid a ack
dissol ed many componen s simul aneously. Moyo e al. (2020), ha e also epo ed he
bes esul s when employing NaOH o epoxy co e emo al, and Adhapu e e al. (2014)
concluded ha imme sing he PCBs in 10 M NaOH o e nigh was he op imal p e-
ea men o elimina ing he epoxy co e ing. Once he epoxy esin is emo ed, he
en i e PCB (wi hou c ushing) can also be ea ed. This las al e na i e o en i e pieces is
e y in e es ing o he indus ial scale applica ion, since i simpli ies he p e- ea men
(no c ushing is equi ed) and, acili a es he ex ac ion o he deple ed piece om he
solu ion.
Rega ding size educ ion, pa icle size ob iously de e mines he con ac su ace a ea
be ween he medium and he ma e ial o be ea ed so ha , when he pa icle size
dec eases, he mass ans e inc eases acco dingly. On he con a y, when pa icle size
is high, he collisions be ween he mic oo ganisms and he PCB pa icles can damage
he bac e ial cells (A shadi and Mousa i, 2015a). Mos o he PCB bioleaching s udies
ha e been ca ied ou using pul e ized samples (Liang e al., 2010; A shadi and Mousa i,
2015a; A shadi e al., 2016; Isilda e al., 2016; Wang e al., 2018; Kha i e al., 2018).
The usual equipmen o c ushing he boa ds is a me al c ushe , hamme o ball mill.
Ini ially, he PCB g inding s ep can be ca ied ou in a hamme mill o g ind he coa se
pa icles and subsequen ly a ball mill o g ind he ine ones (Kaspe e al., 2011).
The main di icul ies o hese p e- ea men s a e he loss o ma e ial (which can be up
o 40 %), he high ene gy consump ion and he o ma ion o a ine mix u e o me allic
o non-me allic pa icles ha can be dange ous o heal h and di icul o sepa a e
(Kuma e al., 2017). Once he ma e ial is c ushed, sie es a e used o classi y he pa icles
acco ding o pa icle size.
Li e a u e e iew
44
Mos au ho s employ pa icles smalle han 200 μm, al hough he e a e s udies ha use
la ge pa icle sizes (Table 9).
Table 9. Pulp densi ies (g L-1) and pa icle sizes (µm) epo ed in li e a u e.
Mig oo ganism
Pa icle
size
Pulp densi y
Me alsa
Re e ence
A. e ooxidans
37-150
1-20
(op imal: 8.5)
Cu, Ni
A shadi and Mousa i.,
2015b
Bacillus mega e ium
37-150
1-20
(op imal: 8.1)
Cu, Au
A shadi e al., 2016
S. he mosul idooxidans
50-150
10
Cu, Ni, Al, Zn
Ilyas e al., 2007
A. e ooxidans and
A. hiooxidans
100-
200
4-12
Cu, Zn, Ni
Liang e al., 2010
A. e i o ans and
A. hiooxidans
≤ 500
5-50
(op imal: 10)
Cu, Ag
Isilda e al., 2016
P. pu ida and
P. luo escens
≤ 500
5-50
(op imal: 10)
Cu, Ag
Isilda e al., 2016
En iched mixed cul u e
(o igin: ac i e sludge)
≤ 180
50
Cu
Wang e al., 2018
Mic oo ganism
conso ium domina ed
by L. e iphilum
≤ 200-
250
10-100
(op imal: 10)
Cu, Fe, Zn,
Ni, Pb, Cd,
Au, Ag, Co
Kha i e al., 2018
A. hiooxidans
<75
10-50
(op imal: 30)
Li, Co, Mn
Nase i e al., 2019
A. e ooxidans and
A. acidophilum
75-
1000
7.5−15
(op imal: 7.5)
Cu, Zn. Ni,
Pb, Ag, Au,
Sc, Ce, La, Nd
P iya and Hai , 2020
A. e ooxidans
< 100
5-100
Co, Li
Roy e al., 2021
aanalyzed dissol ed me als
6.2.2. T ea men mode: single and mul i-s ages bioleaching
The bioleaching expe imen s wi h PCBs can be ca ied ou in one (A shadi e al., 2016;
Kha i e al., 2018) o wo s ages (B andl e al., 2001; Shah e al., 2015; Isilda e al.,
2016; Kha i e al., 2018; Ma a e al., 2018). In he i s case, he ex ac ion p ocess
akes place in he p esence o he mic oo ganisms (bio ic medium), while in he wo-
s age p ocess he me al ex ac ion is ca ied ou by imme sing he PCB in he il e ed
supe na an (abio ic medium) ha is ob ained in a p e ious s age whe e he mic obial
oxida ion o Fe2+ o Fe3+ occu s. The wo-s age ope a ion allows a be e con ol o he

Li e a u e e iew
45
p ocess, since he pa ame e s co esponding o he bio ic s age and he abio ic s age
can be op imized sepa a ely.
6.2.3. Pulp densi y
The pulp densi y is he a io be ween he amoun o ma e ial o be ea ed (usually
powde o dus ) and he olume o leaching medium (Xin e al., 2012). This is ano he
ele an pa ame e because high alues o pulp densi y can be de imen al o he
ex ac ion i he samples a e no p e iously p e ea ed. In his case, he amoun o
dissol ed o ganic ma e ial in he medium can be oo high, which can de ini i ely inhibi
mic obial ac i i y (Ilyas e al., 2007; Ves ola e al., 2010; Zhou e al., 2013; Valix, 2017).
Ob iously he highe he pulp densi y, he highe me al ex ac ion, which would quickly
inc ease oxici y o he medium. Likewise, a low ma e ial:medium a io equi es la ge
eac o s and highe in es men cos s (Valix, 2017), as well as a g ea e ene gy
equi emen o hea ing and mixing. Some o he pulp densi y alues es ed by di e en
au ho s a e shown in Table 9.
The op imal concen a ion epo ed by mos au ho s is equal o o lowe han 10 g PCB
L-1. Howe e , Liang e al. (2010) p oposed a p ocedu e based on he addi ion o he PCB
powde in h ee s eps (4 g L-1 a e 48 h o cul u e, 6 g L-1 a e 96 h and 8 g L-1 a e 144
h). A o al concen a ion o 18 g L-1 was ea ed and he leaching esul s we e 93, 89, 91
and 86 % o Cu, Ni, Zn and Pb, espec i ely. Simila ly, Kha i e al. (2018) epo ed ha
i is possible o ob ain a 95 % solubiliza ion o Cu and mo e han 50 % o Ni by using a
PCB pulp densi y o 100 g L-1 (10 %), al hough he alues ob ained we e lowe han hose
egis e ed wi h lowe pulp densi ies.
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Li e a u e e iew
64
Ma e ials and gene al me hods
71
2.4. Me al emo al a e and speci ic me al emo al a e
The me al emo al a e (MRR) and he speci ic me al emo al a e (SMRR) we e
de e mined by applying he p ocedu es ollowed by Jadha e al. (2013), Muhammad e
al. (2015), Diaz-Tena e al. (2016) and Ma e al. (2020). Du ing he coppe solubiliza ion
es s, each wo kpiece was egula ly aken ou om he medium, washed wi h dis illed
wa e and e hanol (96 %), d ied and weighed in an analy ical balance (Den e
ins umen s, SI-234 230 g/0.1 mg. The MRR and SMRR we e calcula ed as shown in he
equa ions below (Equa ion 16 and 17):
MRR (mg h‐1) = Amoun o me al emo ed (mg)
Time (h) (Eq. 15)
SMRR (mg h‐1 cm‐2) = MRR
A ea (cm2) (Eq. 16)
2.5. De e mina ion o i on species in solu ion
The e ous (Fe2+) and o al i on concen a ion we e de e mined using he 2,2’-dipy idyl
molecula abso p ion spec opho ome y me hod (adap ed om he ‘3500-Fe B’
colo ime ic p ocedu e o he S anda d Me hods o he Examina ion o Wa e and
Was ewa e (Ea on e al., 1998; Diaz-Tena e al., 2016).
The de ails abou he eagen s used in his analy ical me hod a e shown in Table 11.
Table 11. Reagen s used in he colo ime ic me hod o e ous and o al i on
de e mina ion.
Solu ion
Composi ion
P epa a ion me hod
Objec i e
I on s anda d
solu ion
(0.05 mg L-1)
S anda d i on
solu ion 1 g Fe L-1 in
2 % HNO3
Dilu e 5 mL o he s anda d
solu ion in 100 mL o
deionized wa e
Ob aining he
calib a ion
cu e
Ammonium
ace a e / Ace ic
acid Bu e
Ammonium ace a e
98 %
Ace ic acid glacial
99.8 %
Dissol e 280 g o ammonium
ace a e in 1 L o deionized
wa e . Add glacial ace ic acid
un il pH 5.5 is eached
Keep he pH
s able a ound
5.5
Hyd oxylamine
hyd ochlo ide 10 %
(w: )
Hyd oxylamine
hyd ochlo ide
Dissol e 5.0 g o he sal in
50 mL o deionized wa e
Reduce Fe3+ o
Fe2+
Solu ion 2.2
dipy idyl 0.5 %
(w: )
2,2’-dipy idyl
E hanol 96 %
Dissol e 0.5 g o he sal in
100 mL o 96 % e hanol ( : )
Reac wi h Fe2+
o gi e a colo
complex

Ma e ials and gene al me hods
72
I is essen ial o de e mine he concen a ion o bo h e ic and e ous i on h oughou
he p ocess, since bo h alues a e indica i e o he p ocess p og ess. Ne e heless, only
he e ous Fe2+ ion can be measu ed by he colo ime ic me hod. Consequen ly, i he
Fe o al amoun is o be de e mined, he Fe3+ has o be educed o Fe2+ and he o iginal
Fe2+ and he educed Fe2+ will be quan i ied simul aneously, ende ing he o al i on
amoun . Ob iously, he Fe3+ concen a ion will be de e mined by sub ac ion.
P io o he measu emen , i was necessa y o ob ain he calib a ion cu e. The
calib a ion s anda ds o 0, 1, 2, 5 and 10 mg Fe3+ L-1 we e p epa ed om a ce i ied
solu ion o 0.05 g Fe3+ L-1 by adding he ollowing solu ions o each 50 mL olume ic
lask (Figu e 13):
a- he co esponding olume o he 0.05 mg Fe3+ L-1 solu ion o each s anda d
b- 5 mL o bu e solu ion (ammonium ace a e/ace ic acid) o ensu e he pH
s abili y du ing he p ocess
c- 2 mL o he hyd oxylamine hyd ochlo ide 10 % solu ion o educe he e ic i on
o e ous i on. Shaking igo ously o 5 min was ecommended o ensu e he
comple e educ ion o e ic i on. This eagen will only be added i he o al Fe
amoun is o be de e mined. Ob iously, i will no be added o Fe2+
de e mina ion. The Fe3+ concen a ion will be calcula ed by sub ac ing he Fe2+
amoun o he o al concen a ion.
d- 2 mL o he dipy idyl solu ion. All he e ous i on con aining samples de eloped
a ed colo . All he solu ions we e s i ed o 5 minu es o ensu e he comple e
eac ion.
e- Deionized wa e o make up o 50 mL.
Figu e 13. I on s anda ds o calib a ion cu e.
The abso bance o all he samples we e measu ed wi h a isible spec opho ome e
Jenway 6305 a a wa eleng h o 520 nm, using 1 cm and 4.5 mL PMMA cu e es.
Ma e ials and gene al me hods
73
Taking in o accoun he dilu ion ac o , he alue o o al i on and e ous i on in he
o iginal solu ion was ob ained by Equa ion 17, whe e Y is he abso bance alue, b he
cu -o poin be ween he calib a ion cu e and he e ical axis and a he calib a ion
cu e slope.
Fe (g L‐1) = Y ‐ b
a · 50
1 · 100
1 · 1 g
1000 mg (Eq. 17)
2.6. Me al con en analysis (ICP and AAS)
The me al concen a ion in he leacha ed solu ions was measu ed by ICP-OES plasma
spec ome y (Figu e 14a). Induc i ely coupled plasma (ICP) is he ioniza ion sou ce ha
oge he wi h an op ical emission spec opho ome e (OES) cons i u es he ICP-OES
equipmen . Speci ically, a Pe kin Elme OPTIMA 2000DV equipmen wi h a CETAC U
5000AT + ul asonic nebulize was used.
Addi ionally, A omic abso p ion spec oscopy (Figu e 14b) is also commonly used in
me al mobiliza ion p ocess o analyze me al elemen s in liquid samples. In his hesis
coppe and i on con en was also quan i ied in a Pe kin Elme AAnalys 100 AAS
equipmen , depending on he expe imen .
Figu e 14. ICP-OES plasma spec ome y (a) and A omic abso p ion spec oscopy (b)
equipmen .
All samples we e il e ed by a 0.45 μm il e be o e analysis and he calib a ion s anda ds
we e p epa ed in a ma ix as simila as possible o he samples.
2.7. Scanning elec on mic oscopy (SEM)
Rega ding he scanning elec on mic oscopy (SEM) imaging, samples we e ixed in 2 %
glu a aldehyde in 0.1 M cacodyla e bu e (pH 7.4), washed in iso-osmola
cacodyla e/suc ose bu e , and pos ixed in 1 % osmium e oxide in cacodyla e bu e .
Samples we e hen dehyd a ed h ough an e hanol se ies and washed in
Ma e ials and gene al me hods
74
hexame hyldisilazane p io o ai -d ying. Finally, samples we e moun ed on o s ubs and
gold-coa ed using a JEOL ine-coa ion spu e JFC-1100. Samples we e isualized and
mic og aphed using a SEM (Hi achi S-4800) a 15 kV accele a ing ol age. Those SEM
imagines we e ob ained by he Sgike se ice (Ad anced Resea ch Facili ies) o he
Uni e siy o he Basque Coun y (UPV/EHU).
2.8. O he me hods
The pH was measu ed wi h a C ison Basic 20 pH-me e equipped wi h a sensION+ 5010T
pH elec ode. The edox po en ial was eco ded wi h a The mo-O ion 920+ ins umen
equipped wi h an O ion 9778BNWPO Su -Flow® elec ode.
3. REFERENCES
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Ma e ials and gene al me hods
76

CHAPTER 1.
OPERATION WITH
SUSPENDED BIOMASS
Chap e 1. Ope a ion wi h suspended biomass
79
1.1. OBJECTIVE
As a as biomachining and bioleaching p ocesses is conce ned, mos o he s udies in
hese ields a e s ill ca ied ou in a small scale. The e o e, he la ge indus ial scale o
hese biop ocesses is s ill limi ed (Diaz- ena e al., 2017; E us e al., 2021). One o he
main d awbacks o con inuous ope a ion a high scale, e en unde op imum ope a ing
condi ions, is he dec ease in he amoun o mobilized me al as a consequence o he
loss o bio-oxida i e ac i i y which can be a ibu ed o he inc easing oxici y o he
medium (Díaz-Tena e al., 2016; Liang e al., 2018). Ano he ea u e o be conside ed is
ha he inal deple ed solu ion equi es adequa e (and some imes complex) ea men
be o e discha ge, inc easing p ocess cos .
The objec i e he e was o con ibu e o he design o a biop ocess wi h suspended
biomass ha allows he main enance o he me al emo al a e a high alues while
minimizing he amoun o deple ed solu ion. Coppe was selec ed as a ep esen a i e
me al o assess he in luence o i on concen a ion on bo h suspended A. e ooxidans
g ow h and me al mobiliza ion e iciency, and o de e mine he con ibu ion o bac e ia
o he speci ic me al emo al a e (SMRR). Bea ing in mind he u u e indus ial
applica ions in bioleaching o me als om elec onic was e and biomachining, an
al e na e p ocess composed o wo s ages (me al emo al + egene a ion s age) was
sea ched wi h he aim o main aining high SMRR and eusing he solu ion in
semicon inuous ope a ion.
Figu e 1.1 shows he ou line o he expe imen al sec ion o his chap e .
Figu e 1.1 . Ou line o he expe imen al sec ion o his chap e .
1.2. MATERIALS AND METHODS
1.2.1. Coppe pieces
Coppe wo kpieces (pu i y 99.9 %) measu ing app oxima ely 2 x 10 x 15 mm we e cu
employing a Reme LS1 me allog aphic cu ing machine wi h K- ype cu ing discs. A 2-
mm diame e hole was d illed in each wo kpiece o holding i du ing imme sion in o
Chap e 1. Ope a ion wi h suspended biomass
80
cell suspension. Be o e u he ea men , hey we e cleaned and p epa ed as desc ibed
in Sec ion 1.2 Coppe wo kpieces (ma e ials and gene al me hods).
1.2.2. Mic oo ganisms and cul u e media
The A. e ooxidans bac e ium (ATCC 23270) used o his s udy was cul u ed in a
Sil e man and Lundg en medium (Sil e man and Lundg en, 1959) as desc ibed in
Sec ion 1.1 Mic oo ganisms (ma e ials and gene al me hods).
1.2.3. Me al mobiliza ion expe imen s
Me al mobiliza ion expe imen s we e pe o med o e alua e he in luence o Fe2+
concen a ion on mic obial g ow h (expe imen G1), and o eco d he a ia ion in me al
emo al o e ime as unc ion o Fe3+ concen a ion (expe imen BM1). In addi ion, he
biomass’s con ibu ion o he p ocess was s udied by compa ing bio ic and abio ic
expe imen s using he op imum i on concen a ion selec ed in BM1 as he ini ial ene gy
sou ce o bac e ial g ow h (expe imen BM2).
The expe imen s we e ca ied ou in wo-s ages ollowing he p ocedu e desc ibed in
Sec ion 2.2. Coppe mobiliza ion expe imen s (ma e ials and gene al me hods). Fi s , he
medium con aining a ying concen a ion o e ous i on (1.5, 6 and 9K) was inocula ed
wi h a 2 % : o an A. e ooxidans cul u e in an exponen ial g ow h phase, and each
sample was cul u ed un il he comple e Fe2+ oxida ion (s ep 1). Thus, he ea men
solu ions we e ob ained. Once Fe2+ was oxidized o Fe3+, a p e iously weigh ed coppe
wo kpiece was imme sed in he ea men solu ion (s ep 2). The wo kpieces we e
emo ed om he oxidizing medium on an hou ly basis, insed wi h deionized wa e and
e hanol (96 % : ), d ied, and hen weighed as desc ibed in Sec ion 1.2 Coppe
wo kpieces (ma e ials and gene al me hods). The ea e , hey we e imme sed once
again in he co esponding cul u e un il he end o each expe imen .
Addi ionally, se e al abio ic es s we e used o compa ison pu poses (con ol es s
wi hou bac e ia). In hese assays, he solu ion con aining Fe3+ was p epa ed by il e ing
he ea men solu ion ob ained as p e iously desc ibed in s ep 1 (a 0.45 μm
poly inylidene luo ide il e was used). All he expe imen s we e ca ied ou a 31 °C,
a a shaking speed o 130 pm. A pH h eshold alue o 1.7-1.8 was main ained in bo h
s eps by he addi ion o sul u ic acid (25 % : ). Each me al mobiliza ion expe imen was
pe o med in iplica e. The speci ics o each expe imen a e desc ibed below.
1.2.3.1. E ec o i on con en on bac e ial g ow h and me al mobiliza ion
The e ec o he ene gy sou ce (i on concen a ion) on A. e ooxidans g ow h in s ep 1
was s udied by cul u ing he bac e ia in media wi h di e en ini ial concen a ions o
Chap e 1. Ope a ion wi h suspended biomass
87
bio ic BM2 expe imen ) and in he o al absence o i on (expe imen B2; < 0.5 % o he
SMRR ob ained in he bio ic BM1 expe imen ). Fe3+ was he e o e concluded o be he
main oxidan esponsible o coppe leaching, in ag eemen wi h o he s udies
(Xeno on os e al., 2015; Lambe e al., 2015).
The p esence o mic oo ganisms imp o ed he p ocess e iciency, as he o al Cu
amoun solubilized in he 6K and 9K bio ic sys ems was 10 % and 25 % highe ,
espec i ely, han he amoun dissol ed in he absence o mic oo ganisms (Table 1.2).
By con as , Cu emo al a e h ee hou s in he 1.5K solu ion was simila in bo h cases,
which is indica i e o he poo oxida ion ac i i y a ibu ed o he lowe biomass
concen a ion a low i on concen a ions (78.1±4.8 and 75.5±1.8 mg Cu o he bio ic
and abio ic 1.5K expe imen s, espec i ely).
Table 1.2. To al coppe amoun emo ed in he bio ic (BM1) and abio ic (SN1)
expe imen s a e he h ee-hou imme sion o a coppe piece.
Ini ial Fe3+ concen a ion
(g Fe3+ L-1)
Expe imen BM1
(bio ic medium)
(mg Cu)
Expe imen SN1
(abio ic 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 o al amoun o coppe solubilized pe squa e cen ime e in he 6K and 9K bio ic
solu ions was 12-13 % highe han in he absence o mic oo ganisms (69.2±1.0 s.
61.6±1.0 mg cm-2 in he 6K; 102.3±5.9 s. 90.1±3.2 mg cm-2 in he 9K solu ion), wi h he
bac e ia’s e ec being g ea e han ha desc ibed by o he au ho s. Fo example,
Xeno on os e al. (2015) ha e epo ed ha he bac e ial con ibu ion only accoun ed
o an inc ease o 5.7 % in he emo ed coppe ( om 15.9 mg cm-2 o 16.8 mg cm-2) a e
6 h wi h an ini ial concen a ion o 6.5 g Fe3+ L-1. Likewise, Lambe e al. (2015) ha e
epo ed ha his di e ence ne e exceeded 7-8 % du ing he i s 10 h in assays
pe o med wi h ini ial i on concen a ions up o 7 g Fe3+ L-1.
Rega ding SMRR, he mos signi ican di e ence be ween he bio ic (BM1) and abio ic
(SN1) assays was eco ded o he 9 g Fe3+ L-1 ini ial concen a ion du ing he i s wo
hou s o ope a ion (Figu e 1.6). The high SMRR alue in he bio ic medium could be
a ibu ed o he p esence o su icien amoun s o ex acellula polyme ic subs ances
(EPSs) wi h a ached Fe3+ ions, enhancing he ca aly ic oxida ion o Fe2+, and hus he
eco e y o he oxidan and he amoun o me al mobiliza ion. Sand and Geh ke (2006)

Chap e 1. Ope a ion wi h suspended biomass
88
ha e concluded ha s ains o A. e ooxidans wi h a high amoun o Fe3+ ions in hei
EPSs eco ded g ea e Fe2+ oxida ion ac i i y han hose wi h a lowe Fe3+ concen a ion.
In he pa icula case o me al sul ides, some au ho s ha e obse ed ha EPSs,
composed mainly o neu al suga s and lipids, p o ide a con olled eac ion laye ha
concen a es Fe3+ ions by complexa ion h ough u onic acids o o he me aboli es on
he compound su ace, inc easing e ec i e Fe3+ concen a ion in he a achmen poin s
and eco ding a 20-100 imes enhancemen o e chemical leaching (Kinzle e al., 2003;
Sil a e al., 2015).
Figu e 1.6 shows ha he maximum SMRR (SMRRmax) alues we e eco ded wi hin he
i s hou , and hen he a e dec eased o e ime. The SMRRmax ob ained he e is
consis en wi h hose desc ibed in he li e a u e o expe imen s using ei he A.
e ooxidans cul u es o a cell- ee cul u e supe na an (Table 1.3). I is no ewo hy ha
he SMRRmax eco ded in he bio ic 9K expe imen (40 mg h-1 cm-2) was sligh ly highe
han ha epo ed by Jadha e al. (2013) wi h he s ain BCRC 13823 and an ini ial
concen a ion o 40 g FeSO4 L-1. Consequen ly, he 9K medium was selec ed o
subsequen expe imen s in his s udy.
Figu e 1.6. Va ia ion o he SMRR du ing he h ee-hou expe imen o he h ee ini ial
Fe3+ concen a ions (9, 6 and 1.5 g Fe3+ L-1).
Rega dless o he me al emo al a e eco ded in he expe imen s, he Fe3+/Fe2+ a io
ollowed a simila e olu ion in all cases and, a e he h ee-hou imme sion o a coppe
piece in he oxidan medium, he Fe3+ con en dec eased o 55±4 % o i s ini ial alue in
bo h bio ic and abio ic assays (BM1 and SN1 expe imen s espec i ely).
Chap e 1. Ope a ion wi h suspended biomass
89
Table 1.3. Maximum SMRR alues ob ained in he li e a u e and in his s udy when using
di e en ini ial concen a ions o Fe3+ a T= 30-31 °C).
SMRRmax
(mg Cu h-1 cm-2)
A. e ooxidans s ain
[Fe3+] =0
(g L-1)
pH
Re e ence
Bio ic expe imen s
2
ATCC 23270
0.6
1.8
Díaz-Tena e al., 2016
10
ATCC 23270
1.5
1.7
This s udy
10
Isola ed om acidic pi wa e
5.0
1.8
Ma e al., 2020
22
ATCC 23270
6.0
1.8
Díaz-Tena e al., 2016
30
ATCC 23270
6.0
1.7
This s udy
40
ATCC 23270
9.0
1.7
This s udy
36
BCRC 13823
14.7
2.5a
Jadha e al., 2013
Abio ic expe imen s
10
ATCC 23270
1.5
1.7
This s udy
16
BCRC 13823
3.7
2.5 a
Jadha e al., 2013
5.4
BCRC 13820
4.0
2.5 a
Hocheng e al., 2012b
28
ATCC 23270
6.0
1.7
This s udy
28
ATCC 23270
9.0
1.7
This s udy
40
BCRC 13823
14.7
2.5 a
Jadha e al., 2013
aThis alue co esponds solely o he ini ial pH.
1.3.1.3. SMRR as a unc ion o ime du ing me al mobiliza ion p ocess
Se e al au ho s ha e s udied he a ia ion in he SMRR o e ime using di e en A.
e ooxidans s ains and ope a ing condi ions (Is iyan o e al., 2010; Xeno on os e al.,
2015; Díaz-Tena e al., 2018). Ne e heless, he li e a u e on he e ec o mic obial
p esence on me al leaching by compa ing bio ic and abio ic expe imen s o e p olonged
pe iods o ime is sca ce.
In his s udy, a e 20 h o ope a ion he o al coppe mobiliza ion was 55 % highe in
he bio ic sys em (1043±14 mg) han in he abio ic one (672±12 mg) (Figu e 1.7a).
Assuming ha all he dissol ed coppe was in he Cu2+ ionic o m, a inal concen a ion
o 7.6 g Cu2+ L-1 and 4.9 g Cu2+ L-1 was eached in he eac o wi h and wi hou
mic oo ganisms, espec i ely. The e olu ion o he Fe3+/Fe2+ a io o e ime is shown in
Figu e 1.7a, as his a io plays a key ole in o e all me al emo al kine ics by in luencing
he a e o he chemical p ocess (me al dissolu ion) and he biologically ca alyzed
p ocess (oxidan bio- egene a ion). Du ing he i s ou hou s o ope a ion, he a io
Chap e 1. Ope a ion wi h suspended biomass
90
quickly dec eased due o Fe3+ consump ion in he coppe oxida ion p ocess, wi h his
ion being he accep o ha simul aneously ecei ed elec ons om he me al.
Figu e 1.7. E olu ion o dissol ed coppe and [Fe3+]/[Fe2+] a io (a), and SMRR (b)
h oughou ime.
Ne e heless, om hou 8 onwa ds, he a io emained almos cons an o he bio ic
and abio ic sys ems, which can be explained by he limi ed a e o elec on ans e , as
sugges ed by Va gas e al. (2014). As a as he bio ic sys em is conce ned, when he a e
o elec on ans e om coppe me al o Fe3+ ( a e o Fe2+ p oduced in he chemical
p ocess) equaled he a e o elec on ans e om Fe2+ o O2 ( a e o Fe2+ consump ion
in he biological p ocess) a pseudo s eady-s a e was eached, and he amoun o
Fe3+/Fe2+ was he e o e expec ed o emain cons an . Howe e , he abio ic sys em
eco ded a simila end wi h e en sligh ly highe Fe3+/Fe2+ a io alues du ing he inal
hou s o he expe imen , which means ha Fe3+ consump ion was lowe , esul ing in a
lowe amoun o coppe dissol ed.
As shown in Figu e 1.7b, du ing he i s ou hou s he SMRR dec eased in bo h eac o s
mo e signi ican ly, in ag eemen wi h he esul s eco ded in he p e ious h ee-hou
Chap e 1. Ope a ion wi h suspended biomass
91
expe imen . A e wa ds, om 5 o 8 h he me al solubiliza ion p ocess slowed down in
he wo assays, and he SMRR alue ell o 8 mg h-1 cm-2. Finally, he SMRR alue became
almos cons an a an a e age alue o 6.5±0.7 mg h-1 cm-2 om hou 9 onwa ds o he
bio ic sys em (BM2), whe eas his alue was 1.8±0.2 mg h-1 cm-2 om 15 h onwa ds o
he abio ic assay (SN2). Thus, he di e ence be ween he SMRR in bo h assays became
cons an beyond 15 h, wi h he SMRR in he biological eac o being almos ou imes
highe han in he abio ic assay. These esul s a e consis en wi h hose epo ed by
Is iyan o e al. (2007), who ha e concluded ha he educ ion in he MRR (mg h-1) is
in e sely p opo ional o machining ime, and no simply linea . These au ho s ha e
obse ed ha he dec ease in he MRR in a eac o con aining A. e ooxidans ATCC
21834 is almos negligible om hou 12 onwa ds.
A sligh a ia ion in he SMRR was obse ed in expe imen B3, whe e coppe ex ac ion
om he wo kpiece was induced by he spon aneous oxida ion o Fe2+ o Fe3+ in he
p esence o he oxygen dissol ed in he acidic medium. In his expe imen , he SMRR
inc eased wi h ope a ion ime o an a e age o 1.8±0.3 mg h-1 cm-2 o he 10-20-h
pe iod. This alue is almos iden ical o ha eco ded in expe imen SN2 om 15 h
onwa ds, which means he leaching p ocess in he absence o bac e ia (SN2) seems o
be limi ed by he e-oxida ion o Fe2+ o Fe3+ assis ed by he oxygen dissol ed in he
medium. Acco ding o hese esul s, a e 15 h o ea men , 65 % o he SMRR eco ded
in he mic oo ganism-con aining eac o (BM2) was a ibu ed o he p esence o
bac e ia, whose ca aly ic ac ion g ea ly enhanced he elec on ans e p ocess.
1.3.2. Al e na e p ocess: me al mobiliza ion + egene a ion
As concluded in he p e ious sec ion, mic oo ganism-assis ed me al mobiliza ion
pe o med be e han abio ic p ocess. Ne e heless, he SMRR dec eased signi ican ly
a e h ee-hou s o ea men , which would be a echnical d awback when longe
ope a ion ime is equi ed, o example, o eng a e speci ic geome ies on a coppe
piece in biomachining applica ion. Thus, he al e na i e p oposed he e was o ea he
me al piece in consecu i e h ee-hou leaching s ages. In his app oach, he euse o he
solu ion (a e egene a ion) in se e al me al mobiliza ion s ages is essen ial o he
sys em’s sus ainabili y. The e o e, he e iciency o he egene a ed solu ion in
consecu i e ea men s ages was s udied and he c i e ia o he solu ion o be
conside ed “exhaus ed” was de ined.
Figu e 1.8a shows he SMRR du ing six me al mobiliza ion + egene a ion s ages. In
addi ion, Figu e 1.8b p o ides a de ailed iew o he a ia ion in Fe3+ and Cu2+
concen a ions in he medium du ing s ages MR1 and MR2 and he i s egene a ion
s age.
Chap e 1. Ope a ion wi h suspended biomass
92
Figu e 1.8. SMRR as a unc ion o ime du ing six h ee-hou me al emo al s ages (a);
a ia ion in SMRR (ba s), [Cu2+] (g ey do s) and [Fe3+] (whi e do s) du ing wo successi e
ea men s ages (MR1 and MR2) and he oxidan egene a ion s ep (b).
The end in he SMRR du ing each h ee-hou ea men was simila in all he s ages
(MR1-MR6). I is no ewo hy ha he SMRRmax emained almos cons an (36.0±0.9 mg
h-1 cm-2) in MR1- MR5 s ages, and dec eased in MR6 by abou 22 % o he ini ial alue.
The s ages MR1 and MR2 eco ded a sligh ly be e pe o mance han he successi e
ones in e ms o o al Cu emo al, and he amoun o coppe dissol ed (calcula ed as
he a e age o each 3 h pe iod) emained in he 97-104 mg cm-2 ange (Figu e 1.9). In
MR3 o MR5, his alue was almos cons an (89.9±2.7 mg cm-2), and in MR6 his alue
was 33 % lowe han ha eco ded in MR1. These accumula ed alues we e signi ican ly

Chap e 1. Ope a ion wi h suspended biomass
93
highe han hose ecen ly epo ed by Ma e al. (2020) using an A. e ooxidans s ain
isola ed om acidic pi wa e aken om an i on mine in China. These au ho s ha e
epo ed a emo al o coppe o app oxima ely 60 mg cm-2 a e an eigh -hou leaching
expe imen using 5 g L-1 o i on as ene gy sou ce (30 °C, 160 pm, pH 1.8).
Figu e 1.9. Coppe solubiliza ion in consecu i e ea men s ages (columns, igh axis)
and accumula ed coppe concen a ion in he solu ion (whi e do s, le axis).
O e all, he p oposed sys em design being comp ised o h ee-hou coppe mobiliza ion
s ages in consecu i e ope a ion (wi h in e media e egene a ion s ages) signi ican ly
imp o ed he a e age SMRR o coppe ex ac ion in compa ison o con inuous
ope a ion (Sec ion 1.3.1.3 SMRR as a unc ion o ime du ing he me al mobiliza ion
p ocess). Indeed, he o al amoun o emo ed coppe in 5 consecu i e s ages (15 h o
ea men ) (471.6 mg cm-1) was 52.4 % highe han he me al amoun ex ac ed du ing
15 h in con inuous ope a ion (224.6 mg cm-1). This means ha he ime o ex ac ing a
ce ain amoun o coppe can be conside ably sho ened in he consecu i e s age mode,
wi h he posi i e pe spec i e o sus ainabili y and indus ial implemen a ion o he
p ocess.
Rega ding he cumula i e coppe concen a ion shown in Figu e 1.9, his pa ame e
inc eased linea ly wi h ea men ime and, consequen ly, wi h s age numbe ([Cu2+] (g
L-1) = 2.03·s age numbe + 0.466; R2 = 0.9980). In his expe imen , he inal concen a ion
eco ded a e six me al mobiliza ion s ages was 12.4 g Cu2+ L-1, and he o al mass o
coppe emo ed om he wo kpiece h oughou he whole expe imen was 1.57 g.
Figu e 1.10 shows he solu ions a e six me al mobiliza ion + egene a ion s ages.
Chap e 1. Ope a ion wi h suspended biomass
94
Figu e 1.10. Resul ing solu ions a e six me al mobiliza ion + egene a ion s ages (in
duplica e).
A low concen a ion o dissol ed coppe in he medium is essen ial o A. e ooxidans
g ow h, as high concen a ion o his me al leads o he dena u a ion o he p o eins
and nucleic acids necessa y o me abolic ac i i ies (Valix e al., 2017), inhibi ing
bac e ial ac i i y, and hus hal ing he whole p ocess. The esul s eco ded he e
con i med ha he loss o bac e ial ac i i y due o he inc easing Cu2+ concen a ion in
he medium impai ed he ac i i y o A. e ooxidans s ain ATCC 23270. Ne e heless,
unde hese pa icula ope a ing condi ions, i was no un il he dissol ed Cu2+
concen a ion was highe han 10.7 g L-1 ( eco ded a he end o MR5) ha biomass
ac i i y was mo e se e ely a ec ed, slowing he bac e ial Fe2+ e-oxida ion a e, and
hus educing he SMRR. The inhibi o y coppe concen a ion depends bo h on he
bac e ial s ain and on he ope a ing condi ions. Howe e , he maximum dissol ed
coppe concen a ion be o e bac e ial inhibi ion de ec ed in his s udy was sligh ly
highe han ha epo ed by o he au ho s o o he A. e ooxidans s ains. Thus, Díaz-
Tena e al. (2016) ha e obse ed a 63 % educ ion in A. e ooxidans ATCC 23270 ac i i y
o oxidizing Fe2+ ions in he p esence o 6.1 g Cu2+ L-1 (30 °C, 130 pm, pH 1.8,), and Cho
e al. (2008) ha e e idenced ha he oxida ion capaci y o Fe2+ by he A. e ooxidans
ATCC 19859 s ain was comple ely annulled by a Cu2+ concen a ion abo e 10.8 g L-1 (30
°C, 200 pm, pH 2.0).
Rega ding he educ ion in he was e solu ion gene a ion, he esul s showed ha
mic oo ganisms e ec i ely e-oxidized he Fe2+ gene a ed in he me al solubiliza ion
s age (Figu e 1.11), which allowed he solu ion euse and eagen consump ion sa ing.
Ne e heless, he medium’s inc easing oxici y was esponsible o he longe ime
equi ed o he comple e bio- egene a ion o he oxidan solu ion in he consecu i e
egene a ion s eps (Figu e 1.11).
Chap e 1. Ope a ion wi h suspended biomass
95
Figu e 1.11. Regene a ion ime as a unc ion o he coppe concen a ion in he medium.
Figu e 1.11 shows he linea inc ease in he ime needed o comple e Fe2+ e-oxida ion
o egene a ion ( egen (h) = 1.04·[Cu2+] (g L-1) +12.72; R2 = 0.969) un il i sha ply inc eased
a e MR5 ([Cu2+] = 10.7 g L-1), when he ime needed was 3.2 imes highe han ha
equi ed in he i s egene a ion s age ([Cu2+] = 2.3 g L-1). The inal cumula i e
concen a ion o Cu2+ (12.4 g L-1) was no conside ed o be he oxic limi o biomass
ac i i y, as he bio- egene a ion o he oxidan s ill ook place in he solu ion a e MR6.
Ne e heless, he bio- egene a ion ime was longe han 90 h and i was conside ed oo
long o an indus ial applica ion.
The esul s ob ained in his s udy sugges ha he al e na e mode could be success ully
implemen ed a indus ial scale by ins alling wo (o mo e) ea men lines in pa allel
when using suspended biomass. Thus, he me al pieces (o me al con aining was es)
could be consecu i ely imme sed in he egene a ed solu ion o each line wi hou
wai ing o he oxidan eco e y.
The ope a ion mode p oposed in his s udy is a ele an con ibu ion o he sus ainable
applica ion o his me al solubiliza ion p ocess o new sec o s, as i p olongs he use o
he solu ions (and consequen ly educes liquid was e gene a ion), and imp o es p ocess
pe o mance un il he oxici y limi is eached.
1.4. CONCLUSIONS
This chap e has ocused on s udying he aspec s ha can con ibu e o he e icien
solubiliza ion o coppe in mic oo ganism-assis ed p ocesses wi h suspended biomass.
An al e na e p ocedu e o enhancing he oxidan bio egene a ion and main aining high
SMRR was sough .
As a as he in luence o he i on concen a ion is conce ned, he 9 K medium
(con aining 9 g L-1) was concluded o be he op imum one o bo h A. e ooxidans ATCC
Chap e 1. Ope a ion wi h suspended biomass
96
23270 g ow h (Fe2+) and coppe solubiliza ion (Fe3+). Unde he ope a ing condi ions,
he Fe2+ oxida ion a e in he 9K medium du ing mic obial g ow h (0.101 g Fe2+ L-1 h-1)
was 2.8 imes highe han ha eco ded in he p esence o 1.5 g Fe2+ L-1 (0.036 g Fe2+
L-1 h-1), ende ing a highe concen a ion o Fe3+ a ailable in he subsequen s ep o
coppe solubiliza ion. Simila ly, he p esence o mic oo ganisms imp o ed he coppe
solubiliza ion p ocess e iciency in compa ison o he bio ic expe imen , as he o al Cu
amoun leached in he 9K bio ic sys em was 25 % highe han ha in absence o
mic oo ganisms.
The SMRR peaked du ing he i s hou (SMRRmax o 40 mg Cu h-1 cm-2) and dec eased
signi ican ly a e 3-4 hou s. A e 15 h, 65 % o he SMRR was a ibu ed o he e-
oxida ion o Fe2+ o Fe3+ due o bac e ial ac i i y, bu he SMRR alues eco ded a e 15
h in bo h bio ic (6.5±0.7 mg Cu h-1 cm-2) and abio ic samples (1.8±0.2 mg Cu h-1 cm-2)
we e oo low o indus ial applica ion.
The SMRR dec ease a e he i s hou s o ea men could be a echnical d awback
when longe ope a ion ime is equi ed. The e o e, an al e na e p ocess in s ages (me al
solubiliza ion du ing 3 hou s ollowed by oxidan egene a ion) has been p oposed wi h
he aim o main aining high SMRR and eusing he solu ion in semicon inuous ope a ion.
The amoun o me al mobilized in 5 consecu i e s ages (471.6 mg cm-1) was 52.4 %
highe han he amoun ob ained in he con inuous ope a ion o he same ea men
ime (15 h) (224.6 mg cm-1). The inclusion o an oxidan egene a ing s age be ween wo
consecu i e me al solubiliza ion s ages con ibu ed o p olonging he solu ion li espan.
Ne e heless, he egene a ion ime inc eased h ee old compa ed o he i s
egene a ion s age when he coppe concen a ion in he medium was 0.7 g Cu2+ L-1.
This al e na e design allows he educ ion o bo h he ea men ime and he amoun
o deple ed solu ion, which enhances he sus ainabili y o he p ocess.
1.5. REFERENCES
Blanch H.W., Cla k D.S., Biochemical Enginee ing, Ma cel Dekke Inc., New Yo k, 1996.
Cho K. S., Ryu H. W., Choi H. M., 2008. Toxici y E alua ion o Complex Me al Mix u es Using
Reduced Me al Concen a ions: Applica ion o I on Oxida ion by Acidi hiobacillus
e ooxidans. Jou nal o Mic obiology and Bio echnology. 18, 1298-1307.
Daoud J., Ka amane D., 2006. Fo ma ion o ja osi e du ing Fe2+ oxida ion by
Acidi hiobacillus e ooxidans. Mine als Enginee ing. 19, 960-967.
Díaz-Tena E., Gallas egui G., Hippe dinge M., Dona i E. R., Ramí ez M., Rod íguez A., López
de Lacalle L. N., Elías A., 2016. New ad ances in coppe biomachining by i on-oxidizing
bac e ia. Co osion Science. 112, 385-392.
Díaz-Tena E., Gallas egui G., Hippe dinge M., Dona i E. R., Rojo N., San aolalla A., Rami ez
M., Ba ona A., Elías A., 2018. Simul aneous Cul u e and Biomachining o Coppe in MAC
Chap e 2. Ope a ion wi h immobilized biomass
103
BC is a idimensional na u al nano ibe ne wo k p oduced by mic oo ganisms wi h a
high speci ic su ace. I exhibi s ou s anding quali ies, such as a highly po ous ne wo k
s uc u e, biocompa ibili y, and good mechanical and chemical s abili y (Aze edo e al.,
2019; de Oli ei a e al., 2021). Being syn hesized by bac e ia, i can be ob ained wi h he
desi ed size and shape.
PVA is a bioplas ic de i ed om he hyd olysis, alcoholysis o aminolysis o poly inyl
ace a e. I is a highly biodeg adable he moplas ic polyme and easily soluble in wa e
due o i s c ys alline s uc u e. I s ands ou o i s lexibili y and high chemical esis ance
and o being a ba ie o gases and a omas (Hammanna a and Lobo, 2018; Ab al e
al., 2020).
CTA is a chemical compound ob ained by ea ing cellulose whe e all cellulose hyd oxyl
g oups a e eplaced by ace yl g oups. I is commonly used o elabo a e memb anes
cha ac e ized by well-de ined po es (Siko ski e al., 2004; Nabili e al., 2017). Two
di e en CTA-based suppo ma e ials we e es ed: modi ied CTA memb ane and CTA
sphe es.
CH is a na u al biopolyme ha is ob ained om he pa ial deace yla ion o chi in. This
is a e y abundan polysaccha ide in na u e and is ob ained om he shell o
exoskele on o c us aceans, ungi and insec s, gene ally as a by-p oduc o ishing
indus ies. I is insoluble in wa e wi h a high molecula weigh , non oxic and
biodeg adable biopolyme (Muxika e al., 2017; Bakshi e al., 2020).
2.2.2.1. Bac e ial cellulose
Bac e ial cellulose hyd ogel was biosyn hesized in he labo a o y by Gluconace obac e
xylinus bac e ial s ain. I was ob ained by adding 1 % inoculum o Gluconace obac e
xylinus bac e ia o he medium con aining panela and pineapple dissol ed in wa e (13
% w: ). The cul u e was incuba ed unde s a ic condi ions a 28 °C, un il he desi ed
hickness o he pellicle was eached a e 25 days ( he incuba ion ime can be adjus ed
o ob ain he necessa y hickness). In his s udy, BC memb anes wi h a 0.7 cm hickness
we e used. The pH was adjus ed o 3.5 by he addi ion o ace ic acid (CH3COOH) (Re egi
e al., 2010).
A e biosyn hesis, BC pellicle was washed wi h a 2 % w: solu ion o sodium hyd oxide
(NaOH) o 24 h a oom empe a u e and o bi al shaking. Finally, i was insed wi h
deionized wa e se e al imes un il he comple e neu aliza ion o he BC memb ane
(Gu ie ez e al., 2013). A e he pu i ica ion p ocess, a comple ely whi e bac e ial
cellulose memb ane was ob ained (Figu e 2.3).

Chap e 2. Ope a ion wi h immobilized biomass
104
Figu e 2.3. Cul u e medium and syn hesized BC memb ane.
2.2.2.2. Poly inyl alcohol
PVA hyd ogel was p epa ed using powde PVA (Mw 130000 g mol-1, Sigma-Ald ich
Co po a ion). Fi s , 2.5, 5 and 10 % w:w PVA solu ions we e p epa ed by dissol ing PVA
in deionized wa e a 95 °C unde igo ous s i ing o 3 h. The homogenous PVA solu ion
was subsequen ly placed in o a pe i dish and cooled a oom empe a u e. Then, wo
di e en echniques we e used o syn hesizing PVA hyd ogels: F eeze-Thawing and
F eeze-D ying.
In he F eeze-Thawing me hod (Hassan and Peppas, 2000), p e iously ob ained PVA
solu ions we e subjec ed o 24 h eezing a -21 °C and 3 h o hawing a 25 °C o a o al
o 3 cycles. The F eeze–Thaw cycling p omo es he c ys alliza ion o PVA domains, which
esul s in a s ong hyd ogel o ma ion wi h high mechanical s abili y and good p ope ies
such as iscoelas ici y (Peppas and Sco , 1992; V ana e al., 2009). Du ing he eezing
he PVA domains ge close due o he o ma ion o ozen wa e , allowing he o ma ion
o c oss-links (Holloway e al., 2013). A e hawing, hese PVA domains esul in non-
co alen bond o ma ion be ween he polyme chains (Ou e al., 2017).
Figu e 2.4 schema ically shows he F eeze-Thawing p ocess o PVA labeled samples.
Figu e 2.4. PVA hyd ogel p epa a ion p ocess using F eeze-Thawing echnique.
Chap e 2. Ope a ion wi h immobilized biomass
105
In he F eeze-D ying me hod he PVA solu ions we e ozen o 24 h a -21.5 °C and
subsequen ly placed in a lyophiliza ion equipmen (model Alpha 1_4LD (Ma in Ch is ))
a -85 °C and 0.1 mba . Figu e 2.5 shows he scheme o he p ocess. The samples
ob ained we e labeled L-PVA, e e ing o lyophilized PVA. In his echnique only
samples o 2.5 and 5 % w:w by mass we e p epa ed, since 10 % w:w concen a ion was
oo high o he esul ing ma e ial o be po ous enough o he desi ed applica ion.
Figu e 2.5. Lyophilized PVA (L-PVA) p epa a ion p ocess using F eeze-D ying echnique.
2.2.2.3. Modi ied cellulose iace a e memb ane
A modi ied CTA memb ane was ab ica ed by an adap a ion o he me hod published by
Kaise e al. (2017). The solu ion o he iace a e cellulose biopolyme was p epa ed
wi h ace one as sol en because i is insoluble in wa e bu soluble in o ganic sol en s.
CTA (Sigma Ald ich) was dissol ed in 300 mL o ace one (99.5 %, Pan eac) and kep in a
closed con aine unde s i ing condi ions o 24 h hus ob aining a solu ion wi h a 10 %
w: concen a ion. A e wa ds, he solu ion was ans e ed o a con aine wi h 20.5 g
o calcium ca bona e (99.5 %, Pan eac) and 8.8 g o glyce ol (99 %, 98.09 g mol-1,
Labkem). I was hen igo ously mixed in a blende o 5 min. Finally, he mix u e was
in oduced in o a closed con aine .
A e wa ds, wo wa e ba hs con aining 0.25 M HCl (99 %, Pan eac) and deionized wa e ,
espec i ely, we e p epa ed in o de o comple ely dissol e bo h calcium ca bona e and
glyce ol. The ob ained liquid polyme was sp ead on a la glass o 1-2 mm o hickness
and i was d ied o 5 min app oxima ely. A e his ime, i was ca e ully imme sed in
he HCl ba h o 10 min, and bubble o ma ion on he su ace was obse ed, due o he
dissolu ion o calcium ca bona e wi h he consequen CO2 elease. The ob ained
memb ane was in oduced hen in o he deionized wa e ba h o ano he 10 min.
Finally, i was ex ac ed and placed on abso ben pape , un il a d y po ous memb ane
was ob ained. The ma e ial ob ained was labelled M-CTA. Figu e 2.6 shows he scheme
o he p ocedu e ca ied ou o he syn hesis o he M-CTA memb ane.
Chap e 2. Ope a ion wi h immobilized biomass
106
Figu e 2.6. P epa a ion p ocess o modi ied CTA memb ane (M-CTA).
2.2.2.4. Cellulose iace a e sphe es
The ab ica ion o he CTA sphe es was ca ied ou as ollows. Once he abo emen ioned
CTA solu ion (10 % w: ) was comple ely dissol ed, a ce ain quan i y o he p epa ed
solu ion was in oduced o 10 s in o a beake con aining deionized wa e a 95 °C. The
obse ed bubbling caused by he e apo a ion o he ace one allowed he o ma ion o
he po es. A e wa ds, he solu ion was emo ed om he beake , he sphe es we e
manually ounded, and hey we e placed in an o en (Selec a P model) a 45 °C un il he
o al e apo a ion o ace one. This suppo ma e ial was labelled B-CTA. Figu e 2.7 shows
he scheme o he p epa a ion p ocess.
Finally, he sphe es we e weighed and measu ed o classi ica ion acco ding o size. Fo
his s udy, he sphe es wi h an a e age su ace a ea o 4.6±0.4 cm2 we e selec ed.
Figu e 2.7. P epa a ion p ocess o CTA sphe es (B-CTA).
2.2.2.5. Chi osan
The chi osan solu ion was p epa ed by dissol ing 2.45 g o chi osan (Ald ich's, medium
molecula weigh ) in 120 g o a sol en solu ion con aining deionized wa e and ace ic
Chap e 2. Ope a ion wi h immobilized biomass
107
acid a 2 % w: (99.8 %, Honeywell). The mix u e was main ained in a closed con aine
unde s i ing o 24 h. Then, i was pou ed in o a Pe i dish and placed in he eeze a
-21.5 °C. A e 24 h he samples we e ex ac ed om he eeze and wo di e en
p ocedu es we e ca ied ou . In he i s one, he sample was jus kep a oom
empe a u e. This chi osan ma e ial was iden i ied as CH. In he second ea men , he
ozen sample was lyophilized ob aining he so-called lyophilized chi osan ma e ial (L-
CH, Figu e 2.8).
Figu e 2.8. P epa a ion p ocess o lyophilized chi osan L-CH.
2.2.3. P e- ea men o he suppo ma e ial
The ou syn hesized ma e ials, namely bac e ial cellulose (BC), poly inyl alcohol (PVA),
cellulose iace a e (CTA) and chi osan (CH) we e es ed as immobiliza ion suppo
ma e ials in his s udy.
As a as BC and PVA hyd ogels a e conce ned, hey we e cu in o ec angula
pa allelepiped pieces, ende ing an ex e nal su ace a ea (ESA) o 4.8±0.4 cm2. CTA
sphe es we e ounded o a mean diame e o 1.21±0.05 cm which co esponds o an
ESA o 4.6±0.4 cm2. CH and modi ied CTA memb ane suppo ma e ials we e p epa ed
ying o main ain he same ESA a ea.
I should be no ed ha he p esence o ace amoun s o he eagen s used o he
syn hesis o he ma e ials could hinde mic obial g ow h, he eby making he cleaning
o all he suppo ma e ials manda o y be o e bac e ial immobiliza ion. The e o e, he
pieces we e insed wi h deionized wa e a 130 pm and 31 °C o 1 h. This p ocess was
epea ed wice and subsequen ly he same p o ocol was ca ied ou ( wice) eplacing
he deionized wa e by esh nu ien medium (6K o 9K depending on he expe imen ).
Finally, pieces o he di e en suppo ma e ials we e imme sed in esh nu ien
medium o 24 h (130 pm, 31 °C).
Chap e 2. Ope a ion wi h immobilized biomass
108
2.2.4. The design o a decision-making p o ocol
A decision-making p o ocol was designed o s udy he iabili y o he syn hesized
ma e ials in bac e ial immobiliza ion p ocedu es. The e o e, he main objec i e o his
p o ocol was o assess he ma e ials´ sui abili y o mic obial immobiliza ion o i s
applica ion in he me al emo al p ocess. Figu e 2.9 shows he lay-ou o he decision-
making p o ocol.
Figu e 2.9. Decision-making p o ocol.
In he i s s age, he possible deg ada ion when being imme sed in 9K medium (wi hou
bac e ial inocula ion) was es ed by in oducing he ma e ials in o he a o emen ioned
solu ion unde shaking condi ions o 1 day (130 pm, 31 °C). They we e di ec ly
disca ded when he physical in eg i y was no main ained and, o he wise, hey
p oceeded o he nex s age.
The second s age consis ed o e alua ing whe he he ma e ials exe ed any ad e se
in luence on mic obial g ow h. Bea ing in mind his objec i e, an immobiliza ion es
was ca ied ou by inocula ing A. e ooxidans bac e ia (5 % : o cul u e in he

Chap e 2. Ope a ion wi h immobilized biomass
109
exponen ial g ow h phase) in he 9K cul u e medium in he p esence o each suppo
(130 pm, 31 °C).
The las s age was ocused on assessing he mic obial coloniza ion. Once he ma e ials
we e p o ed no o be deg aded by he medium and no o impai mic obial g ow h, he
suppo s al eady exposed o biomass in he p e ious s ep we e cleaned using esh
medium and hen cul u ed in 9K medium wi hou u he bac e ial inocula ion. In case
o success ul Fe2+ oxida ion by he “ac i e ma e ial”, he suppo s we e concluded o be
a o able o A. e ooxidans immobiliza ion. I no , hey we e de ini i ely dis ega ded
o u he expe imen a ion. Thus, i was assumed ha he mic obial ilm was ac i e i ,
a e he immobiliza ion p ocedu e, he eshly ed Fe2+ was oxidized in o Fe3+ wi hou
u he inocula ion.
2.2.5. Selec ion o he inoculum pe cen age
As his chap e is ocused on he immobiliza ion p ocedu e o he A. e ooxidans
bac e ia, i was conside ed ele an o in es iga e he op imum amoun o inoculum
ha sho ened he incuba ion pe iod while ensu ing an adequa e bac e ial g ow h.
Thus, di e en pe cen ages (2, 5 and 10 %) o an A. e ooxidans cul u e in an
exponen ial g ow h phase we e inocula ed in 250 mL E lenmeye s con aining 150 mL o
he 6K cul u e medium (6 g Fe2+ L-1). The expe imen s we e ca ied ou a 31 °C and
shaking speed o 130 pm. A pH h eshold alue o 1.8 was main ained by he addi ion
o sul u ic acid (25 % : ).
2.2.6. Sui abili y o bac e ial cellulose as suppo ma e ial o A. e ooxidans
immobiliza ion
Among he sui able ma e ials selec ed acco ding o he decision-making p o ocol
p e iously desc ibed in Figu e 2.9., BC has no ye been p oposed as suppo o A.
e ooxidans immobiliza ion. Consequen ly, i was selec ed o he expe imen s ocused
on selec ing he mos adequa e ope a ing condi ions o ob aining and using an Ac i e
Bac e ial Cellulose (A-BC) capable o ans o ming he e ous i on dissol ed in he
nu ien medium in o e ic i on. The pa icula in e es on BC hyd ogel lied in i s
ou s anding p ope ies as a iable suppo ma e ial and i s no el use o his applica ion.
2.2.6.1. P epa a ion o he Ac i e Bac e ial Cellulose (A-BC)
The p ocedu e o ob ain he A-BC s a ed wi h he imme sion o he BC pieces in o an
E lenmeye lask con aining he nu ien medium (6K and 9K depending on he
expe imen ), and hen he A. e ooxidans bac e ia (5 % : o a cul u e in an exponen ial
g ow h phase) was inocula ed and cul u ed a 31 °C and pH 1.8 (adjus ed wi h sul u ic
Chap e 2. Ope a ion wi h immobilized biomass
110
acid 25 % : ). This immobiliza ion s ep was ca ied ou in duplica e unde low-
demanding condi ions un il he comple e oxida ion o Fe2+ (shaking a e o 130 pm and
a Nu ien medium olume (mL) o ex e nal su ace a ea (cm2) (NMV:ESA) a io o 1:0.2).
Thus, biologically ac i e biocellulose (A-BC) was ob ained (Figu e 2.10).
2.2.6.2. E ec o ope a ing pa ame e s on Fe2+ bio-oxida ion
The p e iously p epa ed A-BC ma e ial was used in se e al expe imen s wi h he
objec i e o asce aining he e ec o he shaking mode (o bi al s linea ), NMV:ESA
a io (in he 1:0.1-1:0.6 ange), ini ial Fe2+ concen a ion (6 s 9 g Fe2+ L-1) and shaking
speed (130 s 170 pm) on he i on bio-oxida ion pe o mance. The expe imen al
condi ions a e de ailed in Table 2.1. Du ing he expe imen s, he pH was adjus ed o 1.8
wi h sul u ic acid (25 % : ) and he empe a u e was main ained a 31 °C. All he BC
pieces we e incuba ed un il 100 % Fe2+ con ained in he nu ien medium was oxidized.
Table 2.1. Expe imen al condi ions o he i on bio-oxida ion expe imen s using he A-
BC pieces a 31 °C and pH 1.8.
Ope a ion pa ame e
Expe imen
Shaking mode
NMV:ESA a io
(mL:cm2)
Dissol ed [Fe2+]
(g L-1)
Shaking speed
( pm)
Shaking mode
O bi al
Linea
1:0.2
6
130
NMV:ESA a io
O bi al
1:0.1
1:0.2
1:0.3
1:0.6
6
130
Dissol ed [Fe2+]
O bi al
1:0.6
6
9
130
Shaking speed
O bi al
1:0.6
9
130
170
In all he expe imen s (excep o he shaking mode expe imen ) he same A-BC pieces
we e used in wo consecu i e bio-oxida ion s ages unde he same ope a ing condi ions
(Figu e 2.10). This p ocedu e allowed o s udy he ac i i y o he ma e ial in successi e
s ages and o iden i y he possible p oblems ha could a ise om i s long- e m use.
In addi ion, con ol es s we e conduc ed using 6K and 9K medium inocula ed wi h 5 %
: o A. e ooxidans cul u e in exponen ial g ow h phase bu wi hou hyd ogel pieces.
Figu e 2.10 illus a es he scheme o he whole p ocess including A-BC p epa a ion and
he subsequen bio-oxida ion s ages.
Chap e 2. Ope a ion wi h immobilized biomass
111
Figu e 2.10. Scheme o bac e ial immobiliza ion p ocedu e and subsequen oxida ion
s ages.
2.2.6.3. In luence o dissol ed coppe concen a ion on bac e ial
immobiliza ion and subsequen i on bio-oxida ion s ages
The in luence o he p esence o dissol ed coppe (Cu2+) on he p epa a ion o A-BC was
es ed, as inc easingly dissol ed me al concen a ion o igina ed h oughou he
p ocesses is known o a ec bac e ial ac i i y and su i al (Liang e al., 2018). Thus, he
p o ocol desc ibed in Sec ion 2.2.6.1 P epa a ion o he Ac i e Bac e ial Cellulose (A-BC)
was epea ed using 9K medium, 1:0.6 NMV:ESA a io, and a iable concen a ions o
dissol ed coppe ( om 0 o 40 g Cu2+ L-1).
The BAM pieces we e labelled as “A-BCX” being X he concen a ion o dissol ed coppe .
Se e al consecu i e bio-oxida ion s ages we e ca ied ou o each coppe concen a ion
wi h he ac i e ma e ials pieces, and he ime equi ed o comple e i on oxida ion in
each s age was eco ded.
In addi ion, he abili y o immobilized A. e ooxidans o adap o he p esence o
dissol ed coppe was es ed by imme sing he A-BC15 pieces in o he medium wi h
inc easing Cu2+ concen a ions (20, 25 and 30 g Cu2+ L-1) in se e al consecu i e bio-
oxida ion assays. Table 2.2 shows he whole p ocedu e pa icula ly applied o he
sample A-BC15.
Table 2.2. Coppe concen a ion in he medium du ing A-BC15 p epa a ion and
subsequen bio-oxida ion s ages wi h he sample A-BC15.
S ages
[Cu2+]
(g L-1)
A-BC15 p epa a ion
15
S1-S4
15
S5-S7
20
S8
25
S9
30
Chap e 2. Ope a ion wi h immobilized biomass
112
The ope a ing condi ions in hose expe imen s we e 31 °C, pH 1.8 and o bi al shaking a
170 pm.
2.2.6.4. In luence o ac i e BC s o age on bac e ial ac i i y
The p ope p ese a ion o he biologically ac i e ma e ial (A-BC) would allow i s s o age
o u he use, which would con ibu e o acili a e he u u e ull-scale applica ion. The
in luence o wo s o age empe a u es (4 and 22 °C) on he eco e y o he ac i i y o
he A-BC a e he s o age pe iod was assessed as ollows. Fi s , se e al A-BC pieces
unde wen wo i on bio-oxida ion s ages using 1:0.1, 1:0.2 and 1:0.3 NMV:ESA a ios (31
°C, pH 1.8, 130 pm o o bi al shaking). The A-BC pieces o each a io we e hen di ided
in o wo se s and s o ed in we condi ions (in esh 6K medium) a 4 °C and a 22 °C,
espec i ely. A e 15 days, he A-BC pieces we e ans e ed o an E lenmeye lask
con aining 6K esh medium (wi hou u he inocula ion) and incuba ed in wo
consecu i e bio-oxida ion cycles unde he same ope a ing condi ions used be o e
s o age (31 °C, pH 1.8, o bi al shaking, 130 pm).
The long- e m s o age (6 mon hs) a 4 °C was also s udied by epea ing he
abo emen ioned p ocedu e. In his case, a single i on bio-oxida ion s age was ca ied
ou a e s o age. The ope a ing condi ions used in he es s be o e and a e s o age
we e hose selec ed om he esul s ob ained in he expe imen s desc ibed in Sec ion
2.2.6.2. E ec o ope a ing pa ame e s on Fe2+ bio-oxida ion.
2.2.6.5. Biocellulose cleaning and euse
The s a egy o washing he A-BC pieces co e ed wi h ja osi e o emo ing he
p ecipi a e and e-using he cleaned BC o subsequen bac e ial immobiliza ion
p ocedu es was es ed, in o de o expand he li e-span o he suppo ma e ial and
educe bo h A-BC p epa a ion ime and eagen consump ion.
Biocellulose pieces con aining p ecipi a ed ja osi e we e in oduced in a 50 mL
E lenmeye lask and co e ed wi h 20 mL o 10 % w: oxalic acid solu ion o 1 h (W1).
Then, he oxalic medium was emo ed and 20 mL o esh oxalic acid we e added in wo
consecu i e washing s ages (W2-W3). The expe imen was ca ied ou a 25 °C and 150
pm o ensu e an adequa e shaking speed wi h he objec i e o cleaning o he ma e ial.
Once he cleaning ope a ion was concluded, he BC pieces we e imme sed in esh 9K
medium and inocula ed wi h a 5 % : o A. e ooxidans cul u e in he exponen ial
g ow h phase o assess he possibili y o eusing he biopolyme again as suppo
ma e ial. Once he i s bio-oxida ion s age was comple ed, he BC pieces we e e-
imme sed in esh 9K medium wi h no u he inocula ion o check i he a ached
bac e ia we e able o success ully oxidize he Fe2+. The immobiliza ion p ocedu e a e
Chap e 2. Ope a ion wi h immobilized biomass
119
p ecipi a e was obse ed on he ma e ial´s su ace by SEM analysis (Figu e 2.16b). This
deposi o ja osi e has o en been epo ed o p ecipi a e in his medium and o be an
e ec i e adso ben o bac e ia, con ibu ing o bio ilm o ma ion (Pogliani and Dona i,
2000; Jun eng e al., 2007; Chowdhu y and Ojumu, 2014). Bea ing in mind ha se e al
au ho s ha e p oposed ha he bac e ia a e i s w apped by EPS and hen embedded
in agg ega es o deposi ed ja osi e, he isualiza ion o indi idual cells by SEM
pho og aph could be hinde ed by he deposi (Ha nei e al., 2006, Lei e al., 2009; A ica
e al., 2010, Mulopo and Schae e , 2015). Pogliani and Dona i (2000) obse ed ha
bac e ial popula ions immobilized by ja osi e p ecipi a ion we e no easily leached,
indica ing he bio ilm’s s ong a achmen o he deposi .
Figu e 2.16. Pic u e and SEM pho og aph o he p e ea ed biocellulose (a) and he
su ace o he biologically ac i e bac e ial cellulose (A-BC) ob ained a e he
immobiliza ion p o ocol (b).
Unde he low-demanding ope a ing condi ions selec ed o he p epa a ion o A-BC,
he bac e ia equi ed 79 h and 97 h o he comple e bio-oxida ion o 6 and 9 g Fe2+ L-1,
espec i ely. A. e ooxidans immobiliza ion on di e en solid suppo s has al eady been
s udied by o he au ho s in o de o inc ease he bio-oxida ion a es o Fe2+ du ing he
me al emo al p ocesses. Al hough he accu a e compa ison o he pe o mance o he
BC wi h o he suppo ma e ials epo ed in he li e a u e is no easible due o he
in luence o he bac e ial s ain and pa icula ope a ing condi ions, Table 2.4
summa izes he esul s epo ed o se e al A. e ooxidans s ains immobiliza ion unde
simila pH and empe a u e condi ions. Fe2+ oxida ion occu ed as e du ing bac e ial
immobiliza ion on BC han when using non-biobased ma e ials such as nickel alloy ibe
(Gomez e al., 2000) and monoli hic pa icles (Kah izi e al., 2008). Fo example, he ime
needed o 95 % con e sion in he eac o s con aining 6 g Fe2+ L-1 was sligh ly lowe han
ha epo ed by Kah izi e al. (2008) o a simila ini ial Fe2+ concen a ion (6.7 g Fe2+ L-1)
when immobilizing A. e ooxidans DSM 584 in a monoli hic eac o using a wo old
highe inoculum pe cen age (Table 2.4). These au ho s ha e epo ed ha i ook 72 h
o con e 95 % o he ini ial Fe2+ concen a ion, which is 7 % longe han ha needed

Chap e 2. Ope a ion wi h immobilized biomass
120
in his s udy o he same i on con e sion (62 h). BC also eco ded a be e pe o mance
du ing immobiliza ion when compa ed o sul ona ed polys y ene-di inylbenzene
copolyme (Slu SDVB), sul ona ed polys y ene-di inylbenzene copolyme wi h g anula
ac i a ed ca bon (Sul SCVB-GAC), and polyu e hane oam (PUF) (Koseoglu-Ime and
Keskinle , 2013). Thus, he ime needed o ully con e he Fe2+ was h ee o ou imes
sho e despi e his s udy’s highe i on concen a ion (6 and 9 s 5 g Fe2+ L-1), lowe
inoculum pe cen age (5 % s 10 %), and lowe empe a u e (30 °C s 35 °C) (Koseoglu-
Ime and Keskinle , 2013).
Table 2.4. Time equi ed o he bio-oxida ion o he Fe2+ du ing he immobiliza ion o
A. e ooxidans on di e en suppo ma e ials (pH = 1.6-2.0 and T = 30-31 °C).
Suppo ma e ial
A. e ooxidans
s ain
Time
(h)
[Fe2+]0
(g L-1)
Con e siona
(%)
Re e ence
Nickel alloy ib e
Isola ed om
Rio Tin o minesb
65c
2
100
(Gomez e al., 2000)
Hemp ib es
PTCC 1646
34
5
80-90
(Akhlaghi, 2019)
Monoli hic
pa icles
DSM 584
72c
6.7
95
(Kah izi e al., 2008)
Chi osan beads
DSM 11477
58
9
100
(Gia eno e al.,
2008)
Co on gauze
Ac i a ed ca bon
Zeoli e
CCTCC
M2013102d
48
56c
56c
9
100
(Zhu e al., 2017)
Biocellulose
DSM 14882
79
97
6
9
100
This s udy
aPe cen age o ini ial Fe2+ con e ed o Fe3+ when he expe imen was conside ed o be
comple ed.
bThe s ain has he same p ope ies and cha ac e is ics as NCIMB 9490.
cG aphically in e ed.
dIsola ed om acid mine d ainage collec ed om a local py i e mine in Guangdong, People´s
Republic o China.
A e he bac e ial immobiliza ion p ocedu e, and despi e he acidic pH and o bi al
shaking, he ma e ial main ained i s physical in eg i y, which was a ibu ed o he
ou s anding mechanical p ope ies con e ed by i s 3D ne wo k s uc u e and
mo phology (Te cjak e al., 2015; de Oli ei a e al., 2021). The e o e, BC p o ed o be a
sui able ma e ial o he immobiliza ion o A. e ooxidans.
Chap e 2. Ope a ion wi h immobilized biomass
121
2.3.3.2. E ec o ope a ing pa ame e s on Fe2+ bio-oxida ion
2.3.3.2.1. Shaking mode
The shaking o he bac e ial suspended cul u es has a posi i e impac on g ow h a e, as
i p o ides be e ae a ion and highe a ailabili y o oxygen and nu ien s
(Jue gensmeye e al., 2007). Two shaking sys ems can be used: linea ecip ocal
mo emen o o bi al mo emen , being he la e one mo e widely used (Klöckne and
Büchs, 2012). In his s udy, bo h shaking modes we e compa ed and, as a esul , he bio-
oxida ion ime needed o he comple e oxida ion o 6 Fe2+ L-1 by A-BC in a NMV:ESA
1:0.2 a io was ound o dec ease a 40 % when he eac o s we e shaken using he
o bi al mode in con as o he ho izon al shaking (43.5±0.7 h s. 73.0±4.2 h). The e o e,
he o bi al shaking mode was selec ed o u he s udies.
2.3.3.2.2. Nu ien medium olume (mL) o ex e nal su ace a ea (cm2)
a io
Bac e ial densi y plays a c ucial ole in he bioleaching and biomachining p ocesses since
i de e mines e ic i on p oduc i i y and, hence, bio-oxida ion ime (Jaisanka and
Modak, 2009; Zhu e al., 2017). The e o e, inc easing he amoun o ac i e ma e ial
inside he eac o will ob iously educe he ime necessa y o he comple e
Fe2+oxida ion. Ne e heless, acco ding o Jaisanka and Modak (2009), di e en
NMV:ESA a ios could lead o di e en pa e ns o ma e ial-media con ac , which could
a ec e ic i on p oduc i i y (Fe3+ p oduced pe medium olume and ime). The e o e,
he in luence o he amoun o A-BC on Fe2+ bio-oxida ion was analyzed by inc easing
he NMV:ESA (mL:cm2) a io. Figu e 2.17 (le axis) shows he ime equi ed by he A-BC
o oxidize 6 g Fe2+ L-1 in wo consecu i e bio-oxida ion s ages (S1 and S2) using di e en
NMV:ESA (mL:cm2) a ios. The ime eco ded when using he cell suspension cul u e
(con ol) and he a e age Fe3+ p oduc i i y ( igh axis) o each a io a e also shown.
When compa ing he ime eco ded o he comple e Fe2+ bio-oxida ion in A-BC
p epa a ion s ep and A-BC bio-oxida ion s ages (S1-S2) o he same NMV:ESA a io
(1:0.2 mL:cm2), i was concluded ha he p ocess was signi ican ly sho e in he la e
case, as i was educed almos o a hal (45 % educ ion). Jun eng e al. (2007) obse ed
he same educ ion pe cen age be ween bac e ial immobiliza ion on ce amic beads and
he i s bio-oxida ion s age using he ac i e ma e ial. The beha io is also in ag eemen
wi h he esul s ob ained when using A. e ooxidans (DSM 584) immobilized on a
monoli hic eac o by Kah izi e al. (2008), who epo ed a 33 % educ ion o he bio-
oxida ion ime in he second and hi d ba ch cul u es compa ed o he ime needed
du ing he immobiliza ion s ep. Simila ly, Akhlaghi (2019) epo ed a 38 % educ ion o
his pa ame e when using hemp ibe s as immobiliza ion ma e ial.
Chap e 2. Ope a ion wi h immobilized biomass
122
Figu e 2.17. A e age ime equi ed o he comple e Fe2+ bio-oxida ion in wo
consecu i e s ages (le axis) and a e age Fe3+ p oduc i i y ( igh axis) o di e en
NMV:ESA (mL: cm2) a ios when using A-BC pieces.
When compa ing he ime eco ded o he comple e Fe2+ bio-oxida ion in A-BC
p epa a ion s ep and A-BC bio-oxida ion s ages (S1-S2) o he same NMV:ESA a io
(1:0.2 mL:cm2), i was concluded ha he p ocess was signi ican ly sho e in he la e
case, as i was educed almos o a hal (45 % educ ion). Jun eng e al. (2007) obse ed
he same educ ion pe cen age be ween bac e ial immobiliza ion on ce amic beads and
he i s bio-oxida ion s age using he ac i e ma e ial. The beha io is also in ag eemen
wi h he esul s ob ained when using A. e ooxidans (DSM 584) immobilized on a
monoli hic eac o by Kah izi e al. (2008), who epo ed a 33 % educ ion o he bio-
oxida ion ime in he second and hi d ba ch cul u es compa ed o he ime needed
du ing he immobiliza ion s ep. Simila ly, Akhlaghi (2019) epo ed a 38 % educ ion o
his pa ame e when using hemp ibe s as immobiliza ion ma e ial.
I is no ewo hy ha he e we e no signi ican di e ences be ween he bio-oxidizing
ime o S1 and S2 a each NMV:ESA a io (indi idual da a no shown), and hus only
a e age esul s a e plo ed in Figu e 2.17. A simila inding was obse ed by o he
au ho s, who epo ed cons an bio-oxida ion imes a e he i s immobiliza ion ba ch
on suppo ma e ials such as co on gauze (Nie e al., 2015) o nickel allow ibe (Gomez
e al., 2000).
Con e sely, he NMV:ESA a io in luenced he Fe2+ bio-oxida ion ime. In his s udy, he
ime equi ed o ull Fe2+ con e sion a a 1:0.1 NMV:ESA a io was simila o ha
achie ed when cells we e g own in suspension (5 % : o inoculum). By con as , his
Chap e 2. Ope a ion wi h immobilized biomass
123
pa ame e dec eased linea ly wi h he A-BC loading o a selec ed nu ien medium
olume (Figu e 2.17), which was a ibu ed o a highe ini ial cell densi y. In gene al
e ms, he e was a 19 % educ ion in he ime equi ed o he comple e Fe2+ bio-
oxida ion be ween he eac o s wi h a 1:0.6 and a 1:0.1 NMV:ESA a io. This di e ence
be ween samples was mo e signi ican when compa ing he pe cen age o Fe2+
con e ed du ing he p ocess. Fo example, a e 33 h, he sample wi h a 1:0.6 NMV:ESA
a io eached a 90 % Fe2+ con e sion while only he 60 % was ans o med in he case o
he 1:0.1 ela ionship in he same pe iod. The a e age Fe3+ p oduc i i y inc eased wi h
he NMV:ESA a io. These esul s a e in ag eemen wi h hose p esen ed by Jaisanka
and Modak (2009), who also epo ed ha lowe (media olume):(suppo ma e ial)
a ios equi e longe imes o comple e i on oxida ion and ende lowe p oduc i i ies
when using biologically ac i e polyu e hane oam pieces (BAPUF). By con as , Pogliani
and Dona i (2000) ound ha a e age Fe3+ p oduc i i y was e y simila o cul u es wi h
densi ies o glass beads o 1, 5, 10 and 15 %, which was a ibu ed o low amoun s o
bac e ia a ached o he glass beads.
The esul s ob ained in his expe imen led o he conclusion ha he highes NMV:ESA
a io es ed (1:0.6) was he mos a o able when using A-BC o A. e ooxidans
immobiliza ion, as he ime o Fe2+ bio-oxida ion was signi ican ly educed and he
a e age p oduc i i y inc eased compa ed o samples wi h lowe NMV:ESA a ios and
cell suspension cul u es (con ol). Addi ionally, he suspension o A-BC in he media was
uni o m and he sys em was adequa ely shaken. Highe a ios we e disca ded because
mo e ac i e ma e ial pieces in he medium olume migh ha e ad e se e ec s, such as
he di icul y o e ec i e shaking o he excessi e loss o i on (p ecipi a ed as ja osi e)
on he ma e ial´s su ace. The e o e, 1:0.6 NMV:ESA a io was selec ed o u he
expe imen s.
2.3.3.2.3. Fe2+ concen a ion
Besides acili a ing biomass handling and educing i on bio-oxida ion ime, he use o A-
BC can enhance he me al emo al p ocess pe o mance i highe i on concen a ions
a e bio-oxidized in sho e ea men imes (compa ed o cell suspension cul u es).
Thus, in his s udy he in luence o Fe2+ concen a ion (6 s. 9 g Fe2+ L-1) in he bio-
oxida ion ime equi ed by A-BC was s udied using he selec ed NMV:ESA a io (1:0.6
mL:cm2). Highe i on concen a ions we e no s udied as hey can exe an inhibi o y
e ec on he ac i i y o immobilized A. e ooxidans, as concluded by Jun eng e al.
(2007) and Jaisanka and Modak (2009).
The ime equi ed by A-BC o he comple e bio-oxida ion o Fe2+ emained almos
cons an in wo successi e s ages in he p esence o 6 (36.8±1.6 h) and 9 g Fe2+ L-1
(47.0±1.7 h) (calcula ed as he a e age alue o wo consecu i e s ages). Con e sely,
Chap e 2. Ope a ion wi h immobilized biomass
124
oxida ion ime dec eased 18 % and 28 % o he 6K and 9K media, espec i ely, in
compa ison o he cell suspension mode. In addi ion, he highes Fe3+ a e age
p oduc i i y was eco ded in he samples wi h 9K medium (190 mg Fe3+ L-1 h-1), being
his alue a 16 % and 39 % highe han ha ob ained in he samples wi h 6K medium
(163 mg Fe3+ L-1 h-1) and he 9K con ol (136 mg Fe3+ L-1 h-1), espec i ely.
The use o A-BC in a 1:0.6 NMV:ESA a io was especially ecommended o an ini ial
concen a ion o 9 g Fe2+ L-1, as he bio-oxida ion ime was educed and Fe3+ p oduc i i y
was inc eased compa ed o cell suspension g ow h using he same ini ial Fe2+
concen a ion. In addi ion, Fe3+ p oduc i i y was highe han ha eco ded wi h A-BC in
he 6K medium, which means ha a highe amoun o he oxidan would eadily become
a ailable in he medium. I could lead o a highe me al emo al a e in any bioleaching
and biomachining p ocess, being o g ea in e es o u u e indus ial applica ions.
2.3.3.2.4. Shaking speed
Many au ho s ha e demons a ed he in luence o shaking speed in bac e ial ac i i y
du ing bioleaching and biomachining p ocesses (Ting e al., 2000; Jadha e al., 2013;
Xeno on os e al., 2015; Singh e al., 2018; Chakanka e al., 2019). Howe e , sca ce da a
ha e been epo ed abou he in luence o his pa ame e on bac e ial immobiliza ion
and on he ime needed by he ac i e ma e ial o Fe2+ bio-oxida ion in subsequen
s ages. In his s udy wo shaking speeds we e es ed a he op imum ope a ing
condi ions (NMV:ESA a io = 1:0.6, [Fe2+] = 9 g L-1): 130 and 170 pm. Highe shaking
speed alues we e no e alua ed since excessi ely igo ous shaking could damage he
in eg i y o he ma e ial and, he e o e, educe he li espan o A-BC.
In his s udy, A-BC p epa a ion ime signi ican ly dec eased om 63.7±2.3 o 44±0.1 h
when he shaking speed was inc eased om 130 o 170 pm (Table 2.5). This esul was
a ibu ed o a mo e e icien oxygena ion and con ac be ween he suppo ma e ial
and he cul u e medium. As a as biobased ma e ials a e conce ned, Gia eno e al.
(2008) ha e epo ed a Fe2+ bio-oxida ion ime o 60 h (g aphically in e ed) in he i s
coloniza ion s age o A. e ooxidans DSM11477 on c oss-linked chi osan beads a 180
pm (9 g Fe2+ L-1, 30 °C, pH = 1.8). When co on gauze was used, i ook 48 h o
success ully immobilize A. e ooxidans CCTCC M2013102 a 165 pm (9 g Fe2+ L-1, 30 °C,
pH = 2.0). Zhu e al. (2017) ha e concluded ha his suppo ma e ial needed a sho e
immobiliza ion ime han zeoli e and ac i a ed ca bon because he biocompa ibili y and
bioso p ion capaci y o cellulose a o ed he p ocess (Zhu e al., 2017). Thus, he lowe
oxida ion imes shown in Table 2.5 could be a ibu ed o he s uc u al and pa icula
p ope ies o BC in compa ison wi h plan cellulose (de Oli ei a e al., 2021). A e
bac e ial immobiliza ion, he Fe2+ bio-oxida ion ime dec eased compa ed o he A-BC

Chap e 2. Ope a ion wi h immobilized biomass
125
p epa a ion s ep, p obably due o he o ma ion o a dense bio ilm du ing he i s bio-
oxida ion s age (Table 2.5).
Table 2.5. In luence o shaking speed on bio-oxida ion ime du ing bo h A-BC
p epa a ion and A-BC use in wo consecu i e s ages.
Shaking speed ( pm)
Fe2+ bio-oxida ion ime (h)
p epa a ion
use
130
63.7±2.3
47.5±2.0
170
44.0±0.1
40.0±0.6
The ad an ageous esul s and he ac ha ma e ial in eg i y was no a ec ed du ing
he expe imen s a 170 pm suppo ed he selec ion o his shaking speed o u he
expe imen s.
2.3.3.3. In luence o dissol ed coppe concen a ion on bac e ial
immobiliza ion and subsequen i on bio-oxida ion s ages
Se e al s udies ha e epo ed ha he inhibi o y e ec o dissol ed coppe in he
medium a ies be ween unadap ed and adap ed bac e ia, as well as among A.
e ooxidans s ains (Liang e al., 2010; Ma inez-Bussenius e al., 2016; Liang e al.,
2018). In his s udy, he in luence o dissol ed coppe on A. e ooxidans ac i i y was
s udied du ing he A-BC p epa a ion and he subsequen bio-oxida ion s ages (S1-S4)
unde he p e iously selec ed ope a ing condi ions (1:0.6 NMV:ESA a io, 9 g Fe2+ L-1,
170 pm o bi al shaking).
The ac i e ma e ial was p epa ed in se e al bio eac o s in he p esence o di e en
coppe concen a ions anging om 5 o 20 g Cu2+ L-1. The absence o dissol ed coppe
was used as he con ol sample.
The ime equi ed o he comple e bio-oxida ion du ing he immobiliza ion pe iod
inc eased linea ly (R2 = 0.9839) as he dissol ed coppe concen a ion inc eased om 5
o 20 g Cu2+ L-1 (Figu e 2.18). Thus, he bio-oxida ion ime in he p esence o 20 g Cu2+
L-1 was 3.1- old longe han o he con ol sample du ing bac e ial immobiliza ion s age.
Chap e 2. Ope a ion wi h immobilized biomass
126
Figu e 2.18. Fe2+ bio-oxida ion ime du ing A-BC p epa a ion and subsequen bio-
oxida ion s ages a di e en Cu2+ concen a ions (a) and e olu ion o Fe2+ concen a ion
in he eac o wi h [Cu2+] = 10 g L-1 (b).
I is no ewo hy ha when he same A-BC pieces we e used o ou consecu i e bio-
oxida ion s ages (S1-S4) he a e age p ocess ime dec eased conside ably compa ed o
he A-BC p epa a ion ime (Figu e 2.18), which was a ibu ed o bac e ial adap a ion o
he me al. Sligh a ia ions we e eco de among he A-BC5, A-BC10 and A-BC15
samples, and he a e age alue (46.4±4.3 h) was only 14 % highe han ha eco ded in
he coppe - ee sample (con ol sample). Ano he salien esul ob ained in hese
samples is ha he bio-oxida ion ime o he ou cycles was e y simila in all cases, as
shown in uppe Figu e 2.18 o he concen a ion o 10 g Cu2+ L-1 as a ep esen a i e
example.
As a as he esul s o he A-BC sample wi h 20 g Cu2+ L-1 a e conce ned, he ime o
comple e Fe2+ bio-oxida ion g adually dec eased un il 64 % educ ion was eco ded in
s ages S2-S4, compa ed o he A-BC p epa a ion s ep (Figu e 2.18). This esponse was
a ibu ed o he ac i i y inhibi ion ha migh be due o he p esence o 20 g Cu2+ L-1.
Chap e 2. Ope a ion wi h immobilized biomass
127
Bac e ial inac i a ion was e en mo e se e e in he p esence o 30 and 40 g Cu2+ L-1, as
he amoun o oxidized Fe2+ du ing BAM30 and BAM40 p epa a ion was only 30 % a e
a p olonged pe iod o 140 h (when he expe imen was concluded).
Figu e 2.19 shows he aspec o he cul u e medium wi h A-BC be o e and a e Fe2+ bio-
oxida ion in he p esence o di e en concen a ions o dissol ed coppe (0-20 g Cu2+
L-1).
Figu e 2.19. Reac o s wi h A-BC be o e (a) and a e (b) Fe2+ bio-oxida ion in he
p esence o di e en concen a ions o dissol ed coppe (0-20 g Cu2+ L-1).
Based on he p omising esul s wi h he A-BC samples, he abili y o immobilized A.
e ooxidans o adap o he p esence o e en highe concen a ions o dissol ed coppe
was explo ed by exposing he sample A-BC15 o Cu2+ concen a ions up o 30 g Cu2+ L-1
in se e al consecu i e bio-oxida ion s eps.
Thus, bac e ial esis ance and adap a ion we e e i ied when he A-BC15 sample was
inally able o success ully oxidize all he Fe2+ in he p esence o 30 g Cu2+ L-1 in 89.0±1.0
h (Table 2.6). Al hough he bio-oxida ion ime inc eased wi h he dissol ed coppe
concen a ion ( ime = 7.5·[Cu2+] + 66.2, R2 = 0.9941, [Cu2+] ange = 20-30 g Cu2+ L-1), he
alues we e signi ican ly lowe han hose ob ained in he A-BC p epa a ion s ep using
unadap ed bac e ia in he p esence o 20 g Cu2+ L-1. These esul s a e consis en wi h
hose epo ed by Liang e al. (2018), who ha e concluded ha adap ed A. e ooxidans
has a ela i ely highe Fe2+ oxida ion a e du ing he p ocess han unadap ed bac e ia.
Chap e 2. Ope a ion wi h immobilized biomass
128
Table 2.6. Fe2+ bio-oxida ion ime o sample A-BC15 when exposed o highe dissol ed
coppe concen a ions.
S age
[Cu2+]
(g L-1)
Fe2+ bio-oxida ion ime
(h)
Immobiliza ion
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 inhibi o y coppe concen a ion achie ed in his s udy o A. e ooxidans DSM
14882 is highe o simila o hose epo ed in li e a u e o o he s ains. Fo example,
Myky czuk e al. (2011) ob ained ha he coppe ole ance o six A. e ooxidans s ains
cul i a ed in suspension anged om 0.3 o 9.5 g Cu2+ L-1. Simila ly, O ellana and Je ez
(2011) epo ed ha a 50 % inhibi ion o g ow h was egis e ed when unadap ed
ATCC23270 cells we e g own in 0.6 g Cu2+ L-1 and, con e sely, an equi alen pe cen age
o inhibi ion was obse ed o ATCC53993 a a concen a ion o 6.5 g Cu2+ L-1.
Addi ionally, Oe ike e al. (2018) epo ed ha s ain ATCC 53993 s ill ac i ely
exp essed he p o eins ela ed o he RND e lux sys ems a 12.7 g Cu2+ L-1. Highe
inhibi o y concen a ions we e achie ed by No o e al. (2000) who epo ed an oxygen
up ake inhibi ion in he 40-80 % ange o eigh A. e ooxidans s ains cul i a ed in he
p esence o 25.4 g Cu2+ L-1.
2.3.3.4. Ma e ial in eg i y
The main enance o he ma e ial´s physical in eg i y a e successi e bio-oxida ion
s ages is c ucial o ensu e p ocess e iciency and a oid issues in i s long- e m use.
Con en ionally, bio-based suppo ma e ials ha e a sho e li espan and de e io a e
mo e easily han ino ganic ones in biological sys ems (Leb e o e al., 2014).
Ne e heless, hey ha e he ad an age o being biodeg adable, which con ibu es o he
p ocess sus ainabili y and was e educ ion. In his s udy, he ma e ial main ained i s
physical in eg i y (co obo a ed by isual inspec ion) despi e being subjec ed o ex eme
acidic condi ions (pH 1.8) and igo ous shaking (170 pm) du ing se e al successi e
s ages.
Du ing he p ocess, a p ecipi a e appea ed on he su ace o he A-BC (Figu e 2.20a),
which was iden i ied as ja osi e by FTIR analysis acco ding o he mos dis inc i e bands
in he FTIR spec um (Figu e 2.20b) (Gia eno e al., 2008). Zhu e al. (2017) ha e
GENERAL CONCLUSIONS
AND FUTURE OVERLOOK

Gene al Conclusions and Fu u e O e look
233
GENERAL CONCLUSIONS
This hesis was ocused on imp o ing he p ocess o mic oo ganism-assis ed
mobiliza ion o me als o wo applica ions: he biomachining o coppe pieces o
eng a ing mic os uc u es and he bioleaching o coppe om p in ed ci cui boa ds.
Bo h applica ions ha e been s udied using he same bac e ium (A. e ooxidans). This
e sa ile, esis an and sa e mic oo ganism was able o success ully oxidize Fe2+,
ende ing a bio egene a ed oxidan o bo h applica ions.
The main gene al conclusions a e summa ized below.
• The 9K solu ion (con aining Fe2+ o Fe3+) was ound o be he mos sui able medium
o bo h A. e ooxidans g ow h and me al oxida ion. Coppe solubiliza ion was
no iceably inc eased by he p esence o mic oo ganisms in compa ison o he
abio ic sys em.
• An al e na e biop ocess wi h he egene a ion and coppe solubiliza ion s eps in
consecu i e s ages was p oposed. The SMRR peaked du ing he i s hou o
ea men and he amoun o coppe mobilized in i e consecu i e solubiliza ion
s eps (471.6 mg cm-1) was 52.4 % highe han he amoun ob ained in he
con inuous ope a ion o he same ea men ime (15 h) (224.6 mg cm-1). This
s a egy allowed he educ ion o bo h he ea men ime and he amoun o
deple ed solu ion, which enhances he sus ainabili y o he p ocess by p olonging
he solu ion li espan.
• The s a egy o immobilizing he biomass on a suppo ma e ial was sea ched by
selec ing biocellulose (BC), poly inyl alcohol (PVA), cellulose iace a e (CTA), and
chi osan (CH) o be es ed o ha pu pose. Only BC and PVA hyd ogels ul illed he
sui abili y p o ocol, and, inally he BC was selec ed o u he expe imen s based
on i s no el y and i s ou s anding p ope ies such as i s biodeg adabili y, highly
po ous ne wo k s uc u e and chemical s abili y.
• The bes ope a ing condi ions o biomass immobiliza ion we e es ablished o he
BC ma e ial. Unde hese condi ions (1:0.6 o he NMV:ESA a io, 170 pm o he
o bi al shaking, and 9 g Fe2+ L-1), he ime equi ed o he oxidan egene a ion was
educed by 30 % when compa ed wi h he cell suspension. The immobiliza ion ime
was a ec ed by he p esence o inc easing concen a ions o dissol ed coppe .
Ne e heless, when he ac i e ma e ial was consecu i ely exposed o highe
concen a ions o he me al, i was inally able o success ully oxidize all he Fe2+ in
he p esen o up o 30 g Cu2+ L-1.
• The biomachining o coppe pieces o eng a ing s uc u es equi es he e icien
p o ec ion he me al su ace no o be exposed o he oxidan solu ion. The
combina ion o one common lacque ( ed ligh bulb lacque ) and a PSA adhesi e
Gene al Conclusions and Fu u e O e look
234
was ound o be a easible and e icien al e na i e o he selec i e p o ec ion o
su aces wi hou impai ing bac e ial ac i i y.
• The a e age speci ic me al emo al a e (SMRRa ) in he biomachining assays was
maximum in he 0-1 h ange (20-24 mg h-1 cm-2) and, a e wa ds, i dec eased in a
loga i hmic end. The equa ions o p edic ing he ea men ime equi ed o he
selec ed heigh o be machined we e p oposed.
• Al hough simila SMRRa and heigh alues we e achie ed when suspended o
immobilized biomass we e used in he biomachining expe imen s, sho e
egene a ion imes we e needed in he p esence o he ac i e biocellulose. In
addi ion, o he bene i o using immobilized mic oo ganisms would lie in he easily
handling ( eeding and eplacing) o he biomass in la ge-scale ope a ion.
• The epea abili y o he p ocess when eusing he biomachining solu ion is
pa icula ly ema kable o his applica ion, as ex ends i s li espan and inc eases he
sus ainabili y o he p ocess.
• As a as mic oo ganism-assis ed solubiliza ion o me als om PCBs is conce ned,
he en i e pieces wi hou he epoxy co e we e selec ed o he expe imen s,
because g inding p e- ea men en ailed an impo an cos and gene a ed
pa icula e ma e in he en i onmen . In addi ion, he use o he en i e PCBs
acili a ed he managemen and sepa a ion o he pieces om he leacha e, as well
as p o iding a “cleane ” medium o mic obial ac i i y.
• The al e na e ( wo s ep) bioleaching expe imen wi h he en i e PCBs ende ed a
o al amoun o 82 % o coppe dissol ed by he end o he assay (300 hou s), which
was a ibu ed o he ele an con ibu ion o he biomass in egene a ing he
oxidan .
• The bioleaching and biomachining p ocesses gene a ed liquid esidues wi h high
me al concen a ions. Two al e na i es we e s udied o eco e ing coppe om a
syn he ic solu ion: p ecipi a ion and elec odeposi ion. The expe imen al esul s
e ealed ha bo h p ocedu es ende ed high me al eco e y (98 %) a a easonable
cos and equi ed he p elimina y oxida ion and p ecipi a ion o i on, which
ob iously implied he addi ional consump ion o eagen s and longe ope a ion
ime.
• Al hough he p ecipi a ion me hod was mo e a o dable (al hough ime-consuming)
han he elec o eco e y, he inal p oduc ob ained (CuO) is less a ac i e han Cu0
o he s ock ma ke . Bea ing in mind ha he wo ld demand o coppe (and he
LME o icial p ices) has been on he ise du ing he las h ee mon hs and is
expec ed o go u he up, he me al eco e y om hese deple ed solu ions can be
an en ep eneu ial oppo uni y in eg a ed in he ci cula economy.
Gene al Conclusions and Fu u e O e look
235
FUTURE OVERLOOK
The esul s o his hesis sugges ed ha u he esea ch on se e al addi ional aspec s
would bene i he inal p oduc i e implemen a ion o bo h applica ions. Thus, he u u e
o e look could include:
• To sea ch o a conso ium o sa e mic oo ganisms (ins ead o one single bac e ium
ype) ha could ace and mi iga e he medium inc easing oxici y caused by coppe
solubiliza ion h oughou he p ocess.
• To assess he economical and en i onmen al easibili y o he biomachining in
de ail, in o de o dissemina e he sui abili y o ha biop ocess o manu ac u ing
molds o o he addi ional p oduc i e applica ions.
• To in es iga e how o apply he p oposed al e na e bioleaching p ocess o o he
elec onic was es o me al eco e y. In addi ion, he sus ainable and a o dable
bioex ac ion o o he p ecious me als (Ag, Au, P …) could be an a ac i e
oppo uni y o en ep eneu s.
• To explo e he au oma iza ion o he manda o y oxida ion o i on p io o he
coppe elec odeposi ion in was e solu ions o ope a ion cos educ ion.
• To design and ins all a pilo -scale plan o he bioleaching o en i e PCBs in o de
o ob ain echnical in o ma ion and make a decision abou he implemen a ion a
highe scale.
Gene al Conclusions and Fu u e O e look
236

APPENDIX 1
239
LIST OF PUBLISHED ARTICLES
San aolalla, A., Gu ie ez, J., Gallas egui, G., Ba ona, A., Rojo, N., 2021. Immobiliza ion o
Acidi hiobacillus e ooxidans in bac e ial cellulose o a mo e sus ainable bioleaching
p ocess. Jou nal o En i onmen al Chemical Enginee ing. 9 (4), 105283.
San aolalla, A., Ga cía J., Rojo, N., Ba ona, A., Gallas egui, G., 2020. Viabili y o wo
al e na i es o ea ing was e solu ions om he biomachining p ocess. Jou nal o
Cleane P oduc ion. 270, 122549.
San aolalla, A., Rojo, N., Gu ie ez, J., Ba ona, A., 2020. Immobiliza ion o
Acidi hiobacillus Fe ooxidans on Two Hyd ogels. Chemical enginee ing ansac ions. 79,
7-12.
San aolalla, A., Al a ez-B aña, Y., Beni o-Lopez, F., Basabe-Desmon s, L., Ba ona, A.,
Gallas egui, G., Rojo, N. Biomachining: an en i onmen ally iendly echnique o he
ab ica ion o PDMS mic o luidic de ices. Sen o Lab on a Chip.
LIST OF CONFERENCES
Rojo, N., Gallas egui, G., Díaz-Tena, E., San aolalla, A., Elías, A., Ba ona., A., 2017. Join
assessmen o biomachining and e-was e bio eco e y. 10 h Wo ld Cong ess o Chemical
Enginee ing. 1-5 Oc obe ; Ba celona, Spain. Type o p esen a ion: pos e .
San aolalla, A., Rojo, N., C espo, A., Díaz-Tena, E., Gallas egui, G., Ba ona, A., 2018.
T ea ing was e p in ed ci cui boa ds om mobile phones: coppe leaching in abio ic and
bio ic media. 23 d In e na ional Cong ess o Chemical and P ocess Enginee ing. 25-29
Augus ; P ague, Czech Republic. Pos e .
San aolalla, A., Rojo, N., Gallas egui, G., Ba ona, A., 2018. A sequen ial p ocess o
biomachining coppe pieces including oxidan egene a ion. 2nd In e na ional
Con e ence on Bio esou ce Technology o Bioene gy, Biop oduc s and En i onmen al
Sus ainabili y. 16-19 Sep embe ; Si ges, Spain. Type o p esen a ion: pos e .
San aolalla, A., Rojo, N., Elías, A., Gallas egui, G., Beni o-Lopez, F., Basabe-Desmon s, L.,
Ba ona, A., 2019. Assessmen o he biomachining ime o he sus ainable eng a ing o
mic os uc u es on me al wo kpieces. 4 h G een and Sus ainable Chemis y Con e ence.
5-8 May; D esde, Ge many. Type o p esen a ion: pos e .
San aolalla, A., Rojo, N., Gu ie ez, J., Gallas egui, G., Ba ona, A., 2019. No el biosuppo
ma e ial o A. e ooxidans immobiliza ion. 5 h Eu opean Cong ess o Applied
Bio echnology (ECCE12-ECAB5). 15-19 Sep embe ; Flo ence, I aly. Type o p esen a ion:
o al communica ion.
San aolalla, A., Al a ez-B aña, Y., Gallas egui, G., Basabe-Desmon s, L., Rojo, N., Beni o-
Lopez, F., 2019. PDMS mic o luidic de ices ab ica ion by a cyclic biomachining p ocess.
240
The 23 d In e na ional Con e ence on Minia u ized Sys ems o Chemis y and Li e
Sciences (μTAS 2019). 27-31 Oc obe ; Basel, Swi ze land. Type o p esen a ion: pos e .
Gallas egui, G., San aolalla, A., Rojo, N., U bina, L., Ga cía, J., Ba ona, A.,2019. Op ions
o Reco e ing Coppe om Biomachining Was e Solu ions. 3 d In e na ional Cong ess
o Chemical Enginee ing (ANQUE - ICCE -CIBIQ). 19-21 June; San ande , Spain. Type o
p esen a ion: pos e .
San aolalla, A., Rojo, N., Gu ie ez, J., U eña, I., Gu ie ez, I., Ba ona, A., 2019. An Insigh
in o No el Ma e ials o A. e ooxidans Immobiliza ion. 3 d In e na ional Cong ess o
Chemical Enginee ing (ANQUE - ICCE -CIBIQ). 19-21 June; San ande , Spain. Type o
p esen a ion: pos e .
247
LIST OF TABLES
Table 1. Some chemoli ho ophic mic oo ganisms used in mic oo ganism-assis ed me al
mobiliza ion p ocesses epo ed in li e a u e............................................................................. 15
Table 2. Some he e o ophic mic oo ganisms epo ed in bibliog aphy o mic oo ganism-
assis ed me al mobiliza ion p ocesses. ....................................................................................... 17
Table 3. Some o he mic oo ganism conso iums employed in mic oo ganism-assis ed me al
mobiliza ion p ocesses. ............................................................................................................... 19
Table 4. Me al solubiliza ion e iciencies (%) epo ed in li e a u e employing A. e ooxidans
bac e ia. ...................................................................................................................................... 21
Table 5. Di e en suppo ma e ials epo ed in bibliog aphy o A. e ooxidans
immobiliza ion............................................................................................................................. 29
Table 6. Ad an ages and d awbacks o mic oo ganism-assis ed me al mobiliza ion p ocess
compa ed o o he me al ex ac ion p ocesses. ......................................................................... 34
Table 7. SMRR (mg h-1 cm-2) alues epo ed in li e a u e o di e en s ains o he A.
e ooxidans bac e ium. .............................................................................................................. 37
Table 8. Economical and en i onmen al assessmen compa ison o hyd ome allu gy and
biohyd ome allu gy me hods o PCB ea men (Sodha e al., 2020). ...................................... 42
Table 9. Pulp densi ies (g L-1) and pa icle sizes (µm) epo ed in li e a u e. ............................. 44
Table 10. Composi ion o cul u e medium acco ding o inal i on concen a ion. .................... 68
Table 11. Reagen s used in he colo ime ic me hod o e ous and o al i on de e mina ion.
..................................................................................................................................................... 71
Table 1.1. Expe imen al condi ions o bac e ial g ow h, me al mobiliza ion and abio ic assays.
..................................................................................................................................................... 81
Table 1.2. To al coppe amoun emo ed in he bio ic (BM1) and abio ic (SN1) expe imen s
a e he h ee-hou imme sion o a coppe piece. .................................................................... 87
Table 1.3. Maximum SMRR alues ob ained in he li e a u e and in his s udy when using
di e en ini ial concen a ions o Fe3+ a T= 30-31 °C). .............................................................. 89
Table 2.1. Expe imen al condi ions o he i on bio-oxida ion expe imen s using he A-BC
pieces a 31 °C and pH 1.8. ........................................................................................................ 110
Table 2.2. Coppe concen a ion in he medium du ing A-BC15 p epa a ion and subsequen
bio-oxida ion s ages wi h he sample A-BC15. ......................................................................... 111
Table 2.3. Selec ion o sui able ma e ials o A. e ooxidans immobiliza ion. ........................ 114
Table 2.4. Time equi ed o he bio-oxida ion o he Fe2+ du ing he immobiliza ion o A.
e ooxidans on di e en suppo ma e ials (pH = 1.6-2.0 and T = 30-31 °C). ......................... 120
Table 2.5. In luence o shaking speed on bio-oxida ion ime du ing bo h A-BC p epa a ion and
A-BC use in wo consecu i e s ages. ......................................................................................... 125
Table 2.6. Fe2+ bio-oxida ion ime o sample A-BC15 when exposed o highe dissol ed coppe
concen a ions. ......................................................................................................................... 128
Table 2.7. Op imum ope a ing condi ions o A-BC p epa a ion and use. ............................... 129
Table 2.8. Fe2+ oxida ion ime equi ed by A-BC a e 15 days o s o age (a e age o wo
successi e bio-oxida ion cycles). ............................................................................................... 130
Table 3. 1. Imme sion imes in he mold-e ching expe imen s. ............................................... 149
Table 3. 2. Expe imen al condi ions in me al emo al expe imen s when using A-BC and cell
suspension (con ol). ................................................................................................................. 151

248
Table 3. 3. SMRR (mg h-1 cm-2), s uc u e´s heigh (H , μm), and emo ed coppe (mCu, mg)
a ia ion acco ding o he wo ea men ime in e als (h) o he ci cula geome y. ......... 157
Table 3. 4. Heigh and dep hs alues (µm) epo ed in bibliog aphy by o he au ho s. .......... 160
Table 3. 5. Linea equa ions ha co ela e mold´s heigh (µm) and emo ed coppe mass
(mCu, g) wi h he numbe o mold-e ching s age (N). .............................................................. 164
Table 3. 6. Rela ionship be ween he egene a ion ime ( egen, h) and he concen a ion o
dissol ed coppe (Cu, g L-1). ...................................................................................................... 166
Table 3. 7. Linea equa ions ha co ela e he heigh o he s uc u e (H , μm) and he amoun
o coppe emo ed (mCu, g) wi h he numbe o mold-e ching s ages (N). ............................ 169
Table 4. 1. Me al concen a ion (mg g-1 PCB) o mobile’s PCBs epo ed in li e a u e and
ma ke p ice acco ding o he London Me al Exchange (LME, € kg-1). ..................................... 188
Table 4. 2. A e age me al con en in PCBs in his s udy (mg me al g-1 PCB). ........................... 189
Table 4. 3. Resul s o he EDXRF analysis o di e en a eas o he PCB powde ...................... 189
Table 4. 4. Resul s o he EDXRF analysis o di e en a eas o he en i e PCB. ....................... 190
Table 4. 5. Me al emo al alues ob ained o Cu, Zn, Ni, and Pb. .......................................... 192
Table 4. 6. Leaching e iciencies o se e al me als epo ed in he li e a u e when ea ing PCB
pieces. ....................................................................................................................................... 200
Table 5. 1. Pa ame e s measu ed in he SLR0, SLR1 and SLR2 samples. .................................... 222
Table 5. 2. Cos analysis o coppe elec odeposi ion a 10 V and pH 4.0. ............................. 226