F on ie s in Mic obiology | www. on ie sin.o g 1 Decembe 2021 | Volume 12 | A icle 802926
ORIGINAL RESEARCH
published: 22 Decembe 2021
doi: 10.3389/ micb.2021.802926
Edi ed by:
Raymond J. Tu ne ,
Uni e si y o Calga y, Canada
Re iewed by:
E ika Ko he,
F ied ich Schille Uni e si y Jena,
Ge many
E ol Duncan Cason,
Uni e si y o he F ee S a e,
Sou hA ica
*Co espondence:
E elyn K awczyk-Bä sch
e.k awczyk-bae sch@hzd .de
Sebas ian Schae e
s.schae e @unsw.edu.au
†P esen add ess:
Sebas ian Schae e
School o Chemical Enginee ing,
Uni e si y o New Sou h Wales,
Sydney, NSW, Aus alia
Special y sec ion:
This a icle was submi ed o
An imic obials, Resis ance and
Chemo he apy,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 27 Oc obe 2021
Accep ed: 22 No embe 2021
Published: 22 Decembe 2021
Ci a ion:
Schae e S, S eud ne R, Hübne R,
K awczyk-Bä sch E and
Me oun ML (2021) E ec o
Tempe a u e and Cell Viabili y on
U anium Biomine aliza ion by he
U anium Mine Isola e Penicillium
simplicissimum.
F on . Mic obiol. 12:802926.
doi: 10.3389/ micb.2021.802926
E ec o Tempe a u e and Cell
Viabili y on U anium
Biomine aliza ion by he U anium
Mine Isola e Penicillium
simplicissimum
Sebas ianSchae e
1*†, RobinS eud ne
1, RenéHübne
2, E elynK awczyk-Bä sch
1
* and
MohamedL.Me oun
3
1 Ins i u e o Resou ce Ecology, Helmhol z-Zen um D esden-Rossendo , D esden, Ge many, 2 Ins i u e o Ion Beam Physics
and Ma e ials Resea ch, Helmhol z-Zen um D esden-Rossendo , D esden, Ge many, 3 Depa men o Mic obiology,
Uni e si y o G anada, G anada, Spain
The emedia ion o hea y-me al-con amina ed si es ep esen s a se ious en i onmen al
p oblem wo ldwide. Cu en ly, cos - and ime-in ensi e chemical ea men s a e usually
pe o med. Bio emedia ion by hea y-me al- ole an mic oo ganisms is conside ed a mo e
eco- iendly and compa a i ely cheap al e na i e. The ungus Penicillium simplicissimum
KS1, isola ed om he looding wa e o a o me u anium (U) mine in Ge many, shows
p omising U bio emedia ion po en ial mainly h ough biomine aliza ion. The adap ion o
P. simplicissimum KS1 o hea y-me al-con amina ed si es is indica ed by an inc eased
U emo al capaci y o up o 550 mg U pe g d y biomass, compa ed o he non-hea y-
me al-exposed P. simplicissimum e e ence s ain DSM 62867 (200 mg U pe g d y
biomass). In addi ion, he e ec o empe a u e and cell iabili y o P. simplicissimum KS1
on U biomine aliza ion was in es iga ed. While iable cells a 30°C emo ed U mainly
ex acellula ly ia me abolism-dependen biomine aliza ion, a dec ease in empe a u e o
4°C o use o dead-au ocla ed cells a 30°C e ealed inc eased occu ence o passi e
bioso p ion and bioaccumula ion, as con i med by scanning ansmission elec on
mic oscopy. The p ecipi a ed U species we e assigned o u anyl phospha es wi h a
s uc u e simila o ha o au uni e, ia c yo- ime- esol ed lase luo escence spec oscopy.
The majo in ol emen o phospha es in U p ecipi a ion by P. simplicissimum KS1 was
addi ionally suppo ed by he obse a ion o inc eased phospha ase ac i i y o iable
cells a 30°C. Fu he mo e, iable cells ac i ely sec e ed small molecules, mos likely
phospho yla ed amino acids, which in e ac ed wi h U in he supe na an and we e no
de ec ed in expe imen s wi h dead-au ocla ed cells. Ou s udy p o ides new insigh s in o
he in luence o empe a u e and cell iabili y on U phospha e biomine aliza ion by ungi,
and u he mo e highligh he po en ial use o P. simplicissimum KS1 pa icula ly o U
bio emedia ion pu poses.
Keywo ds: biomine aliza ion, bio emedia ion, ungal biomass, u anium, was e wa e , Penicillium simplicissimum
Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 2 Decembe 2021 | Volume 12 | A icle 802926
INTRODUCTION
As a esul o o me u anium (U) mining and milling ac i i ies,
la ge amoun s o was ewa e con aining high concen a ions
o U and o he hea y me als ha e been gene a ed, wi h he
po en ial isk o con amina ing he su ounding en i onmen .
Once disposed in o he en i onmen , U could e en ually each
he op o he ood chain and be inges ed by humans, causing
heal h isks like se e e kidney and li e damage (Kei h e  al.,
2013). The e o e, i is necessa y no only o clean up con amina ed
si es, bu also o ea U-con amina ed was ewa e in o de o
p e en hea y me al elease o he en i onmen . The o me
U mine in Königs ein (Ge many) ep esen s such a con amina ed
si e. Be ween 1984 and 1990, he adionuclide U was ex ac ed
om he ock ma e ial, mainly composed o sands one, by
in-si u leaching – i.e., injec ion o sul u ic acid in o he
unde g ound ock. The esul ing U-bea ing, acidic liquid was
collec ed and u he p ocessed o inally eco e he hea y
me al (Zeißle e  al., 2006). Since he closu e o U mining
ac i i ies in Ge many, he sub-su ace o he mine has been
emedia ed by con olled looding o p e en he con amina ion
o aqui e s. A p esen , he looding wa e is s ill cha ac e ized
by ela i ely high concen a ions o U (~8–9 mg/L) and a low
pH o 2.9 owing o he acidic leaching p ocess (Kassahun
e  al., 2015). Fu he mo e, he concen a ion o hea y me als
like cadmium, nickel, and zinc a e ele a ed (Zi ns ein, 2015).
The wa e consequen ly has o be pumped o he su ace and
is cu en ly ea ed by a con en ional, chemical was ewa e
ea men plan .
Such chemical ea men s a e ime- and cos -in ensi e,
howe e (Azubuike e  al., 2016; Ve ma and Kuila, 2019).
Depending on he on-si e si ua ion, chemis y-based echniques
o en gene a e haza dous was e and become less e icien a
dec easing pollu an concen a ions (Azubuike e  al., 2016;
Ve ma and Kuila, 2019). Fo se e al yea s, science has been
conce ned wi h al e na i e bio emedia ion app oaches.
Bio emedia ion aims o use sui able mic oo ganisms o
p ospec i ely suppo o ou pe o m chemical ea men .
Mic oo ganisms used in bio emedia ion should ul ill se e al
c i e ia including: (i) high ole ance o hea y me als and
adionuclides; (ii) me abolic e sa ili y; and (iii) abili y o educe
solubili y and mobili y o he ino ganic con aminan s. Mic obial
in e ac ion mechanisms wi h hea y me als a e mainly clus e ed
in o passi e and ac i e p ocesses, based on hei dependence
on ac i e cell me abolism. In he passi e bioso p ion p ocess,
he ca ionic hea y me al, o example U(VI), binds o componen s
o he ungal cell wall, e.g., phospho yla ed polysaccha ides
and in acellula ly o nega i ely cha ged unc ional g oups like
phospha e o ca bona e g oups (Tsezos and Volesky, 1982;
González-Muñoz e  al., 1997; Lloyd and Macaskie, 2002;
Limcha oensuk e  al., 2015; Kulka ni e  al., 2016; Bano e  al.,
2018; Lopez-Fe nandez e al., 2018; Seg e in e al., 2018). Ac i e
mic obial in e ac ion mechanisms a e u he subdi ided in o
anae obic enzyma ic educ ion, biomine aliza ion, and
bioaccumula ion. Bioaccumula ion desc ibes he ac i e, con olled
up ake o hea y me als (e.g., ia side opho es) and hei
subsequen in acellula p ecipi a ion, which is s ill unde
in es iga ion (Limcha oensuk e  al., 2015; Ge be e  al., 2018;
Seg e in e al., 2018). Mic oo ganisms can also sec e e nega i ely
cha ged me aboli es, such as hyd ogen phospha es, hyd ogen
ca bona es, oxala es, o hyd oxides. This p ocess is called
biomine aliza ion and leads o ex acellula p ecipi a ion and
de oxi ica ion (Me oun e  al., 2011; Kaewdoung e  al., 2016;
Chandwadka e  al., 2018).
Besides bac e ia, a ious ungal species ha e been de ec ed
a u anium mining si es and a e known o hei ele a ed
hea y-me al adap ion and ole ance (De Silóniz e  al., 2002;
Anahid e  al., 2011; Zi ns ein e al., 2012; Ge be e  al., 2018;
GRAPHICAL ABSTRACT |
Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 3 Decembe 2021 | Volume 12 | A icle 802926
Glukho a e  al., 2018; S ępniewska e  al., 2020; Coelho e  al.,
2020a). The e o e, hey a e conside ed as pu a i e candida es
o bio emedia ion app oaches o emo e hea y me als om
con amina ed soil o was ewa e (Song e  al., 2019; Coelho
e  al., 2020b). In e ms o ungal biomine aliza ion and
bioso p ion o U, phospha es and ex acellula phospha ase
ac i i y ha e been epo ed o be he key playe s (Liu e  al.,
2010; Gün he e  al., 2014; Liang e  al., 2015, 2016; Vázquez-
Campos e  al., 2015; Zheng e  al., 2017; Wollenbe g e  al.,
2021). While di e en physico-chemical pa ame e s like pH
o backg ound medium composi ion ha e been assessed o
hei in luence on he biomine aliza ion o U, he impac o
me abolic ac i i y is no ye ully in es iga ed. In he p esen
s udy, he ungal s ain Penicillium simplicissimum, isola ed
om he looding wa e o he o me U mine in Königs ein
(Ge many), was in es iga ed owa d i s po en ial use o
bio emedia ion pu poses, pa icula ly o U-con amina ed si es.
We ocused on changes in he u anium bio emo al by
P. simplicissimum KS1 depending on empe a u e and cell
iabili y o un a el me abolic eliance. Addi ionally, he abili y
o P. simplicissimum KS1 o e ec i ely emo e U was compa ed
o he P. simplicissimum e e ence s ain DSM 62867 o in es iga e
an adap ion o U-con amina ed en i onmen s.
MATERIALS AND METHODS
Mic oo ganisms and Cul u e Condi ions
The ungus P. simplicissimum KS1 was isola ed om he looding
wa e o he o me U mine in Königs ein (Ge many) by
cul u e-dependen me hods using Sabou aud-Dex ose (SD,
bac o-pep one 5.0 g/L, casein pep one 5.0 g/L, glucose 40.0 g/L,
and Ca l Ro h) medium (Ge be e  al., 2015), adap ed om
Odds (1991). As a compa a i e ungal species, P. simplicissimum
DSM 62867 was pu chased om DSMZ (Leibniz Ins i u e
DSMZ-Ge man Collec ion o Mic oo ganisms and Cell Cul u es).
Bo h s ains we e g own in SD medium a 30°C and 130 pm
(The moshake EA2, C. Ge ha d ) o 72 h and s o ed a 4°C
on SD aga pla es a e g ow h a 30°C o 72 h.
DNA Isola ion and Sange Sequencing o
he Fungal Isola e KS1
The DNA o KS1 was isola ed by ollowing he p o ocol o
alkaline DNA ex ac ion (Bi nboim and Doly, 1979). A
pu i ica ion and concen a ion s ep we e pe o med acco ding
o he ins uc ions o he DNA Clean & Concen a o ™-5 Ki
(Zymo Resea ch). A ungal-cha ac e is ic DNA agmen o
he in e nal ansc ibed space (ITS) egion o he 18S RNA
gene was ampli ied by PCR using he p ime s ITS5 and ITS4
(bo h The mo Fishe Scien i ic), acco ding o Ma in and
Rygiewicz (2005). The ob ained PCR p oduc s we e pu i ied
(DNA Clean & Concen a o ™-5 Ki ) and sequenced by Sange
sequencing pe o med by GATC Bio ech. The ob ained sequences
we e aligned and compa ed o hose in he nucleo ide-nucleo ide
Basic Local Alignmen Sea ch Tool (blas n) da abase o he
Na ional Cen e o Bio echnology In o ma ion (NCBI).1 The
Sange sequencing esul s a e a ailable on NCBI GenBank®
unde accession numbe SAMN22830865.
Fungal U Remo al Capaci y S udies
To in es iga e he emo al capaci y o U, he ungal cells we e
g own in SD medium o 72 h. A e wa ds he cells we e
sepa a ed om he medium and washed wice by s e ile il a ion
and esuspension in s e ile- il e ed ap wa e (pH = 5.0). Fi e
millili e s cul u e we e subsequen ly dilu ed in 45 ml s e ile-
il e ed ap wa e (pH = 5.0) o each a inal d y biomass (DBM)
o 0.10 ± 0.02 g/L. A u anyl s ock solu ion [UO2(NO3)2] was
added o a inal concen a ion o 0.1 mM. The samples we e
incuba ed o 52 h wi h agi a ion a 130 pm a 4 and 30°C,
using p e- empe ed chemicals. S e ile- il e ed samples, each
wi h a olume o 500 μl, we e egula ly aken. To each sample,
5 μl o concen a ed ni ic acid was added immedia ely. The
samples we e s o ed a 4°C and used o de e mina ion o he
U concen a ion by means o induc i ely coupled plasma mass
spec ome y (ICP-MS) using a NexION 350X (Pe kinElme ).
To de e mine he e ec o cell iabili y on U emo al, g own
ungal cells in SD medium we e au ocla ed o 30 min a 121°C.
The au ocla ed cell cul u e was cen i uged and washed wice
wi h s e ile- il e ed ap wa e (pH = 5.0), hen u he ea ed
as desc ibed abo e. The DBM was de e mined a e pe o ming
he espec i e expe imen . The eby, cells we e sepa a ed om
he medium by s e ile il a ion on a p e-d ied, weighed il e .
The biomass on he il e was subsequen ly d ied o e nigh
a 80°C be o e inal weighing.
De e mina ion o O hophospha e
Concen a ion and Acid-Phospha ase
Ac i i y
In o de o de e mine he o hophospha e concen a ion and
he acid-phospha ase ac i i y in ol ed in ungal U emo al,
washed ungal cells (DBM 0.10 ± 0.02 g/L) we e ei he suspended
in 100 ml SD medium o in 100 ml s e ile- il e ed ap wa e
(pH = 5.0). The cells in SD medium we e incuba ed o 52 h
a 30°C and 130 pm. The cells in s e ile- il e ed ap wa e
(pH = 5.0) we e u he p epa ed and incuba ed as desc ibed
in sec ion “Fungal U Remo al Capaci y S udies.” Samples o
each expe imen we e s e ile- il e ed a e 52 h, and 1 ml o
each sample was analyzed o i s o hophospha e concen a ion.
To his end, an ion ch oma og aph sys em Dionex™ In eg ion™
HPIC™ (The mo Fishe Scien i ic) was u ilized wi h he ollowing
equipmen : analy ical column (Dionex IonPac, AS23 – 4 μm,
RFIC, 2x 250mm), gua d column (Dionex IonPac, AG23 – 4 μm,
RFIC, 2x 50 mm), and eluen 4.5 mM Na2CO3/0.8 mM NaHCO3.
Addi ionally, 1 ml o he samples was analyzed o i s acid-
phospha ase ac i i y ollowing he ins uc ions o he Acid
Phospha ase Ac i i y Fluo ome ic Assay Ki (Sigma-Ald ich):
200 μl o each sample and con ol solu ion we e pipe ed in
a 96-well pla e and analyzed o luo escence using he mic opla e
luminescence eade Mi h as 2 (Be hold Technologies), equipped
1
h ps://blas .ncbi.nlm.nih.go /Blas .cgi (Accessed May 18, 2017).
Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 4 Decembe 2021 | Volume 12 | A icle 802926
wi h 355 × 40 exci a ion and 460 × 25 emission il e , o 30 s
wi h a coun ing ime o 0.1 s and a lamp ene gy o 40%. All
expe imen s we e pe o med in iplica e.
Scanning Elec on Mic oscopy
Fo SEM measu emen s, ungal cells o he wo P. simplicissimum
s ains KS1 and DSM 62867 we e ea ed wi h U o 52 h, as
desc ibed in sec ion “Fungal U Remo al Capaci y S udies.” The
cells we e eco e ed by cen i uga ion (10 min, 13,793 g, 4°C).
The supe na an was emo ed, and he pelle was u he p ocessed
o SEM a he Cen o de Ins umen ación Cien í ica (Uni e si y
o G anada, Spain), acco ding o Ande son (1951). The specimens
we e imaged using a S-4800 mic oscope (Hi achi) ope a ed a
an accele a ing ol age o 10 kV. Fo quali a i e chemical analysis,
ene gy-dispe si e X- ay spec oscopy (EDXS) was ca ied ou a
30 keV using a con en ional Si(Li) de ec o wi h a S-UTW window.
Addi ional s udies we e pe o med using a GEMINI FESEM
mic oscope (Ca l Zeiss) ope a ed a an accele a ing ol age o 20 kV.
High-Angle Annula Da k-Field Scanning
T ansmission Elec on Mic oscopy
(HAADF-STEM)
Fo HAADF-STEM measu emen s, U in e ac ion expe imen s
wi h P. simplicissimum KS1 o DSM 62867 we e pe o med a
30°C and, in he case o P. simplicissimum KS1, addi ionally
a 4 and 30°C wi h au ocla ed, non- iable cells (as desc ibed
in sec ion “Fungal U Remo al Capaci y S udies”). A e 52 h,
he cells we e immedia ely cen i uged (10 min, 13,793 g, 4°C).
The supe na an was emo ed, he pelle was washed h ee
imes wi h s e ile- il e ed ap wa e (pH = 5.0) and subsequen ly
ixed wi h glu a dialdehyde a 1% ( / ) om 50% s ock solu ion
( / ) and s o ed a 4°C. The P. simplicissimum DSM 62867
sample was u he p ocessed o STEM analysis a he Cen o
de Ins umen ación Cien í ica (Uni e si y o G anada, Spain),
acco ding o Renau Pique as and Megias Megias (1998).
Penicillium simplicissimum KS1 samples we e u he p epa ed
a he Ad anced Imaging/Elec on Mic oscopy acili y o he
Cen e o Molecula and Cellula Bioenginee ing (Technische
Uni e si ä D esden, Ge many). HAADF-STEM imaging and
spec um imaging analysis based on EDXS we e pe o med a
200 kV wi h a Talos F200X mic oscope equipped wi h an X-FEG
elec on sou ce and a Supe -X EDX de ec o sys em (FEI).
P io o STEM analysis, he specimen – moun ed on a high-
isibili y low-backg ound holde – was placed o 2 s inside
Model 1,020 Plasma Cleane (E. A. Fischione Ins umen s Inc.).
C yo-TRLFS Measu emen s
Fo he de e mina ion o po en ial U(VI) species o med by
he P. simplicissimum s ain KS1, ime- esol ed lase -induced
luo escence spec oscopy (TRLFS) was used. The de ec ion
limi o aqueous U is cu en ly 0.2 μg/L (Be nha d and Geipel,
2007). The c yo-TRLFS samples we e p epa ed as desc ibed
in sec ion “Fungal U Remo al Capaci y S udies” using a ungal
DBM o a ound 0.25 g/L. The eby, P. simplicissimum KS1 was
s udied in he p esence o 0.1 mM U(VI) a 4 and 30°C.
Addi ionally, au ocla ed cells we e in es iga ed a 30°C a an
ini ial U(VI) concen a ion o 0.1 mM. As con ol samples, P.
simplicissimum KS1 was p epa ed wi hou U(VI), and
0.1 mM U(VI) solu ions wi hou ungal biomass we e measu ed
a e an incuba ion a 30°C. All samples we e incuba ed o
48 h. A e he in e ac ion expe imen s, he cell pelle s we e
sepa a ed om he supe na an by cen i uga ion a 5445 g
o 20 min. The pelle s we e washed wice wi h s e ilized ap
wa e (pH 5.0), and bo h supe na an and ungal biomass
we e sepa a ely shock- ozen in plas ic cu e es by liquid
ni ogen and s o ed a −80°C. The U(VI) luminescence a
153 K was measu ed a e exci a ion wi h lase pulses a 266 nm
(Minili e high-ene gy solid-s a e lase ; Con inuum) and an
a e age pulse ene gy o 300 mJ. The emission o he samples
was eco ded using an iHR550 spec og aph (HORIBA Jobin
Y on) and an ICCD came a (HORIBA Jobin Y on) in he
425.0–625.0 nm wa eleng h ange by a e aging 100 lase pulses
and using a ga e ime o 10 ms. TRLFS spec a we e analyzed
and decon olu ed by means o pa allel ac o analysis
(PARAFAC) using he N-way oolbox wi h Ma lab R2015a
(Ande sson and B o, 2000). PARAFAC is known o be a
aluable ool o luminescence da a decon olu ion, since
PARAFAC da a p ocessing deli e s in o ma ion abou specia ion,
indi idual emission spec a, and luminescence decays in bo h
chemical and biological sys ems (Bade e  al., 2016, 2019;
D obo e  al., 2016).
RESULTS AND DISCUSSION
Isola ion and Physiological
Cha ac e iza ion o he Fungal Isola e
Penicillium simplicissimum KS1
Using cul u e-dependen me hods, P. simplicissimum KS1 was
p e iously isola ed on SD medium om he looding wa e
o he o me U mine in Königs ein (Ge many; Ge be e  al.,
2015). Compa ed o he o he isola ed euka yo ic and p oka yo ic
s ains, P. simplicissimum KS1 displayed a high U emo al
capaci y (Ge be e  al., 2015) and was he e o e chosen o
u he s udies. By sequencing he ITS 18S RNA gene and
compa ison wi h blas n (NCBI), P. simplicissimum KS1 (accession:
SAMN22830865) displayed a maximum phylogene ic iden i y
wi h P. simplicissimum (accession: MH856014.1; 100% que y
co e ; 98.49% iden i y) and he axonomical synonymous
Penicillium pul ilo um (accession: KF624805.1; 100% que y
co e ; 98.35% iden i y). The mic obial di e si y in he looding
wa e is known o be domina ed by i on- and sul u -oxidizing
bac e ia, as well as i on- educing bac e ia (Zi ns ein, 2015;
Ge be , 2019). Howe e , a chaea and euka yo es, including
ungal species, we e de ec ed as well (Zi ns ein e  al., 2012;
Zi ns ein, 2015; Ge be , 2019). P e iously, ou g oup isola ed
ano he hea y me al- ole an ungal species om he looding
wa e o Königs ein ha belongs o he di ision o Basidiomyco a
(Ge be e  al., 2018) – in con as o P. simplicissimum KS1,
which is an ascomyce ous ungus.
To u he cha ac e ize he ungal isola e, sui able ca bon
sou ces o he en ichmen o he ungus we e in es iga ed
Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 5 Decembe 2021 | Volume 12 | A icle 802926
(Supplemen a y Table1). Penicillium simplicissimum KS1 showed
good g ow h in he p esence o glucose and uc ose, and
medium g ow h wi h galac ose, mannose, saccha ose, and xylose,
whe eas no g ow h was obse ed in e hanol, lac a e, oxalic
acid, and sodium ace a e. The o al o ganic con en o he
looding wa e a he mining si e in Königs ein is below 1 mg/L
(Zi ns ein, 2015). I would he e o e ha e o be en iched wi h
ca bon sou ces and he biomass i sel o in-si u bio emedia ion.
Al e na i ely, he p esence and g ow h o mic oo ganisms in
he looding wa e may beob ained h ough he biodeg ada ion
o unde g ound wood cons uc ions, which leads o a
decomposi ion in o mono- and polysaccha ides (e.g., a abinose,
glucose, xylose, and galac ose; Ba aniak e  al., 2002).
In addi ion, he ole ance o P. simplicissimum KS1 owa d
selec ed hea y me als in solu ion was s udied o e alua e i s
sui abili y o bio emedia ion applica ions (Supplemen a y
Table 2). The highes ole ance was obse ed owa d ch omium
(>22 mM) and zinc (>15 mM), whe eas nickel and U inhibi ed
he g ow h o P. simplicissimum KS1 a concen a ions o 0.2
and 0.7 mM, espec i ely. The highes oxici y o nickel bu
lowe o zinc is in good ag eemen wi h he obse a ions o
Anahid e  al. (2011). The epo ed dis inc ly highe ole ance
concen a ions by Anahid e  al. (2011) migh be caused by
he u iliza ion o a P. simplicissimum s ain ha could bemo e
ole an o hea y me als due o (i) a po en ially a i icially
inc eased hea y-me al adap ion o he ungal s ain ia
sub-cul u ing p io o he me al ole ance es o (ii) a na u ally
s onge adap ion o hea y me al – e.g., due o a hea y-me al-
exposed place o o igin. Also, he hea y-me al ole ance was
in es iga ed on solid media in con as o liquid media which
was used in he p esen wo k. The ungal yeas Rhodospo idium
o uloides was also isola ed om he looding wa e o he
o me U mine in Königs ein and likewise showed ele a ed
hea y-me al ole ance (Ge be e  al., 2018). While his ungal
s ain was mo e ole an owa d U (up o 6 mM), i s ole ance
owa d ch omium, coppe , cadmium, and zinc is low compa ed
o P. simplicissimum KS1 (Ge be e  al., 2018). This inding
highligh s he sui abili y o P. simplicissimum KS1 o
bio emedia ion pu poses in ol ing a ious hea y me als.
U Bio-Associa ion S udies: E ec o
Tempe a u e and Cell Viabili y
The in luence o empe a u e and cell iabili y on he U emo al
capaci y o P. simplicissimum KS1 and he P. simplicissimum
e e ence s ain DSM 62867 was in es iga ed (Figu e 1).
Penicillium simplicissimum DSM 62867 was selec ed as a mos
likely e e ence s ain ha is no hea y-me al-adap ed; since
con a y o P. simplicissimum KS1, he s ain was isola ed om
p is ine soil samples in Ge many. Kine ic U emo al s udies
a di e en empe a u es (4 and 30°C) and cell iabili y o e
52 h sugges ed a h ee-phase U emo al o bo h ungal s ains
(Figu e1). The U concen a ion was se o 0.1 mM, ep esen ing
he U concen a ion ha could eme ge in he mining si e
esul ing om a p ospec i ely en isaged ise o looding le els.
Cu en ly, he U concen a ion in Königs ein (Ge many) anges
be ween ~0.03 and 0.04 mM. Two empe a u es we e chosen:
he op imal g ow h empe a u e o ungal species (30°C), plus
a lowe empe a u e (4°C), so as o s udy a possible me abolic
in luence on U in e ac ion.
Fi s , U may ha e been emo ed passi ely by bioso p ion
o P. simplicissimum as indica ed by a linea inc ease in U
emo al du ing he i s 5 h a 30°C (Figu e 1, s aigh ed
line). This linea inc ease was ollowed by a less-s eep inc ease
in U emo al a 30°C up o 24–30 h o incuba ion. This second
phase was signi ican ly educed wi h a empe a u e dec ease
o 4°C (Figu e 1, blue line) demons a ing ac i e me abolic
p ocesses addi ionally in ol ed in passi e bioso p ion du ing
he U emo al a 30°C ( iable P. simplicissimum KS1 30°C:
107 mg U/g DBM, 4°C: 27 mg U/g DBM; iable P. simplicissimum
AB
FIGURE1 | U emo al capaci y o Penicillium simplicissimum KS1 (A) and DSM 62867 (B) a ~0.1 g DBM/L o e 52 h a 4°C (blue) and 30°C ( ed) and 0.1 mM
ini ial U concen a ion. A 30°C, iable (s aigh line) and dead-au ocla ed cells (do ed line) we e s udied. SDs a e depic ed as e o ba s.

Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 6 Decembe 2021 | Volume 12 | A icle 802926
DSM 62867 30°C: 72 mg U/g DBM, 4°C: 20 mg U/g DBM).
Fu he mo e, a decline in appa en cell iabili y a e 24 h a
30°C was obse ed o P. simplicissimum KS1 (Supplemen a y
Figu e 1), which o e laps wi h he dec ease in slope o U
emo al capaci y, as well as a pla eau a e a ound 30 h. This
sugges s a hi d p ocess en ailing passi e U bioso p ion o
dead ungal biomass (Pang e  al., 2011). Simila U emo al
p ocesses we e epo ed o o he U- ole an ungal species,
d i en by ac i e bioaccumula ion and passi e bioso p ion (Ge be
e  al., 2018; Wollenbe g e  al., 2021).
To suppo ou hypo hesis ha ac i e me abolic p ocesses a e
in ol ed in U emo al by P. simplicissimum KS1 and DSM 62867,
he U emo al capaci y o dead-au ocla ed ungal biomass a
30°C was also s udied (Figu e 1, do ed ed lines). Thus, iable
cells e ealed highe U accumula ion alues compa ed o hose
o dead-au ocla ed cells a e 2 days (P. simplicissimum KS1
iable: 107 mg U/g DBM, dead: 34 mg U/g DBM;
P. simplicissimum DSM 62867 iable: 72 mg U/g DBM, dead:
24 mg U/g DBM). U emo al by dead cells is d i en by immedia e
passi e bioso p ion, as epo ed o a ious ungal species (Pang
e  al., 2011; Ge be e  al., 2018; Wollenbe g e  al., 2021); and
i can e en su pass he U emo al o iable cells ia so p ion
o eleased o exposed compounds upon cell dea h, like
lipopolysaccha ides o phospha es, as seen o Coniochae a odinicola
(Vázquez-Campos e  al., 2015). Ou obse a ion implies a mo e
p ominen in ol emen o ac i e me abolic p ocesses in he U
emo al o P. simplicissimum KS1 – o ins ance biomine aliza ion
and in acellula accumula ion – han passi e bioso p ion. Fo
bo h s ains, dead-au ocla ed ungal biomass emo ed mo e U
om he solu ion when compa ed o iable cells a 4°C, which
may be explained by addi ional a ailable binding si es, bo h
in a- and ex acellula ly, due o damaged cell walls.
Mo eo e , P. simplicissimum KS1 emo ed mo e U om
he solu ion han P. simplicissimum DSM 62867 unde simila
en i onmen al condi ions (Figu e1) and P. simplicissimum KS1
showed a slowe esponse o he U s ess a 4°C. Bo h hese
obse a ions suppo an adap ion o he ungal isola e o hea y-
me al s ess, as compa ed o he e e ence s ain, which was
no exposed o hea y me als be o e isola ion.
Rema kably, P. simplicissimum KS1 was able o emo e up
o 80% o he ini ially in oduced U om solu ion a e 48 h,
depending on he ungal biomass concen a ion
(Supplemen a y Figu e2). Wi h an inc ease in ungal biomass
om 0.05 o 0.58 g/L, an exponen ial dec ease in U emo al
capaci y (no malized by he ac ual biomass) was obse ed
o bo h P. simplicissimum KS1 and DSM 62867. O e all, P.
simplicissimum DSM 62867 emo ed less U han P.
simplicissimum KS1, especially o a biomass o a ound 0.1 g/L
and lowe , as can be seen in Figu e 1 o a ixed DBM
a ound 0.1 g/L. The maximum U emo al capaci y o P.
simplicissimum KS1 o ~550 mg U/g DBM ou pe o med no
only he e e ence s ain P. simplicissimum DSM 62867
(~200 mg U/g DBM), bu also o he ungal species including
Saccha omyces ce e isiae, Rhizopus sp., and R. o uloides
(Supplemen a y Table3), which again p o es i s g ea po en ial
o bio emedia ion pu poses. Howe e , a di ec compa ison
o hose alues is di icul ; he expe imen al condi ions a y
be ween di e en s udies and, especially, he physicochemical
condi ions o he espec i e expe imen al se up (pH,
empe a u e, o biomass concen a ion) a e known o
emendously a ec he U emo al capaci y o mic oo ganisms
(Bus a d e al., 1997; Ge be e al., 2018; Zheng e al., 2018).
Fo his eason, only he maximum U emo al capaci ies,
obse ed espec i ely, a e compa ed in Supplemen a y Table3.
HAADF-STEM Cha ac e iza ion o U
Biomine aliza ion by P. simplicissimum
KS1 Cells
HAADF-STEM imaging combined wi h EDXS-based elemen
dis ibu ion analysis was pe o med o in es iga e he e ec
o empe a u e and cell iabili y on he cellula localiza ion
o U complexes and he unde lying in e ac ion mechanisms
o U wi h he ungus P. simplicissimum KS1 (Figu e 2).
Me abolically ac i e ungal cells we e incuba ed wi h 0.1 mM U
o 48 h a 4 and 30°C, in addi ion o dead-au ocla ed cells,
which we e only incuba ed a 30°C o he same ime and a
he same U concen a ion.
Spec um imaging analysis o he samples showed signi ican
di e ences in he amoun o accumula ed U and i s cellula
localiza ion. Fo me abolically ac i e and iable cells a 30°C
(Figu e 2, op ow), la ge ex acellula U p ecipi a ions we e
de ec ed, in addi ion o low in acellula amoun s o U
accumula ions. The ex acellula U p ecipi a ions showed an
amo phous na u e, and hus di e ed s uc u ally om he
needle-like objec s obse ed in acellula ly. Addi ional SEM
s udies combined wi h EDXS analysis (Supplemen a y
Figu es 3, 4) con i med ha he emo ed U is localized
ex acellula ly by P. simplicissimum KS1 (and DSM 62867).
The s uc u e o he U accumula ions e oked a biomine aliza ion-
media ed p ecipi a ion (Liang e  al., 2015).
Wi h a dec ease in empe a u e o 4°C (Figu e 2, cen e
ow), P. simplicissimum KS1 appea ed o accumula e U a
he cell su ace and in acellula ly. La ge ex acellula
accumula ions, as de ec ed a 30°C, we e no obse ed a
4°C. These esul s indica e ha he la ge ex acellula U
accumula ions we e d i en by a me abolically ac i e p ocess,
i.e., biomine aliza ion. Biomine aliza ion elies on he ac i i y
o enzymes, such as phospha ases, o deg ade o ganic
phospha es, gi ing ise o he gene a ion o o hophospha e.
Biomine aliza ion is he e o e ba ely obse able a lowe
empe a u es and me abolically inac i e cells (Liang e  al.,
2015). Due o cell dea h a e 24 h a 30°C and pu a i e
damage o he ungal cell wall, U pe haps en e ed he cells;
his would ha e been ollowed by passi e bioso p ion by
nega i ely cha ged unc ional g oups and migh ha e been
bound o he elease o cellula compounds, plus cell wall,
and memb ane agmen s (Vázquez-Campos e  al., 2015).
Inac i a ion o P. simplicissimum KS1 by au ocla a ion, wi h
incuba ion a 30°C (Figu e2, bo om ow), led o U p ecipi a ions
ha we e mainly isible in acellula ly, along wi h mino U
bioso p ion a he cell su ace. Conside ing he con ol samples
o un ea ed iable and un ea ed dead-au ocla ed cells
(Supplemen a y Figu e 5), he dead-au ocla ed cells showed
Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 7 Decembe 2021 | Volume 12 | A icle 802926
pa ial de achmen s o he cell wall, possibly o e ing addi ional
binding si es o U and acili a ing he in lux o he hea y
me al and subsequen passi e in acellula U bioso p ion. The
use o dead ungal biomass o bio emedia ion o U-con amina ed
was ewa e is an al e na i e app oach (Vázquez-Campos e  al.,
2015; Coelho e al., 2020b). Unde he expe imen al condi ions
chosen by Vázquez-Campos e al. (2015), dead ungal biomass
emo ed mo e U compa ed o he iable cells. Ye , o he ungal
species – P. simplicissimum KS1 (Figu e1) in he p esen wo k,
o R. o uloides (Ge be e al., 2018) – emo ed ele a ed amoun s
o U by iable cells. Hence, he ungal isola e P. simplicissimum
KS1 could ep esen a sou ce o bo h bio emedia ion app oaches
o emo e U om was ewa e – exploi ing iable o dead cells.
EDXS-based elemen mapping in Figu e 2 e ealed U
associa ion wi h phospho us, which indica es biomine aliza ion
and bioso p ion o U phospha es ex a- and in acellula ly.
The con ibu ion o phospho us in U biop ecipi a ion has been
p e iously epo ed (Liu e  al., 2010; Gün he e  al., 2014;
Liang e  al., 2015, 2016; Vázquez-Campos e  al., 2015; Zheng
e  al., 2017; Wollenbe g e  al., 2021). Howe e , phospho us
was no solely de ec ed supe imposed upon he U signal
(Supplemen a y Figu e 6). O he elemen s, such as ni ogen,
could beexplained by pu a i e bioso p ion o U by biopolyme s
( o example, chi in, cellulose and i s de i a i es) a e damaging
he ungal cell wall and passi e bioso p ion o amino
unc ionali ies (Galun e  al., 1984; Zhao e  al., 2016).
Fo compa ison, P. simplicissimum DSM 62867 and i s
in e ac ion wi h U a 30°C we e s udied by means o HAADF-
STEM and SEM as well (Supplemen a y Figu es4, 7). Simila
o P. simplicissimum KS1, SEM e ealed ex acellula U
biomine aliza ion, al hough spec um imaging analysis displayed
some mino di e ences. Mos no ably, he amoun o in acellula
U accumula ion inc eased signi ican ly. The di e ences be ween
P. simplicissimum KS1 and DSM 62867 indica ed an adap a ion
o P. simplicissimum KS1 o U. U anium is no as p ominen ly
p esen in acellula ly in he ungal isola e, which may ha e
adap ed i s me abolic esponse o hea y me al en i onmen al
s ess, as obse ed p e iously in inc eased hea y me al esis ance
and U emo al om solu ion.
De e mina ion o Ex acellula
O hophospha e and Phospha ase Ac i i y o
P. simplicissimum KS1in he P esence o U
Based on he mic oscopic da a, phospha es appea o bec ucially
ele an o he U emo al ia ac i e biomine aliza ion by
P. simplicissimum KS1. Since his migh ha e been media ed
by phospha ase ac i i y, he quan i ica ion o o hophospha e
concen a ion and phospha ase ac i i y o P. simplicissimum
KS1 and DSM 62867 we e s udied in s e ile- il e ed ap wa e
wi h an ini ial U concen a ion o 0.1 mM and wi hou U in
SD medium (Figu e 3).
O e all, P. simplicissimum KS1 showed a subs an ially highe
phospha ase ac i i y and ex acellula o hophospha e
concen a ion han P. simplicissimum DSM 62867, independen
o he s udied media. This pi o al obse a ion is in good
ag eemen wi h he HAADF-STEM esul s (Figu e 2;
Supplemen a y Figu es 6, 7). The e, P. simplicissimum KS1
FIGURE2 | HAADF-STEM mic og aphs o iable P. simplicissimum KS1 a 30 and 4°C ( op and cen e ows) and dead-au ocla ed cells a 30°C (bo om ow)
oge he wi h EDXS-based elemen dis ibu ions o u anium (magen a) and phospho us (g een). The ungal isola e was incuba ed in 0.1 mM U (backg ound
elec oly e: s e ile- il e ed ap wa e pH 5.0) o 48 h. The scale ba s indica e 1 μm.
Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 8 Decembe 2021 | Volume 12 | A icle 802926
demons a ed a highe ex acellula amoun o phospho us,
o e lapping wi h he U signal. Fu he mo e, he phospha ase
ac i i y dec eased wi h a dec easing amoun o nu ien s (i.e.,
in p esence o ap wa e ), dec easing empe a u e, and dead-
au ocla ed cells. As shown be o e, such esul s poin o he
majo ole o ac i e me abolic p ocesses o U emo al.
In addi ion, a empe a u e decline o samples wi h dead
cells led o a dec easing amoun o ex acellula phospha e.
These esul s a e in line wi h hose ob ained by HAADF-
STEM (Figu e 2), whe e iable P. simplicissimum KS1 cells
a 30°C showed p ominen biomine aliza ion, p obably d i en
by phospha ases, dec easing wi h empe a u e declining o
4°C, and no de ec ed wi h dead-au ocla ed cells. The
impo ance o phospha es and phospha ases in he emo al
o U by bac e ia (Beazley e al., 2007; Kulka ni e al., 2016)
and he con ibu ion o phospha e anspo e genes in U
ole ance o S. ce e isiae (Sakamo o e  al., 2012) ha e been
desc ibed in he li e a u e and come o suppo hei obse ed
in ol emen in U p ecipi a ion by P. simplicissimum KS1
and DSM 62867.
Iden i ica ion o Bio-Associa ed and
Ex acellula U Species: C yo-TRLFS
S udies
C yo-TRLFS was used o in es iga e he e ec o empe a u e
and cell iabili y on he luminescence p ope ies (i.e., emission
bands) o he U species associa ed wi h o p oduced by he
cells o he ungal isola e P. simplicissimum KS1. To his end,
he supe na an and ungal biomass we e measu ed sepa a ely
a e he incuba ion wi h 0.1 mM U(VI). Toge he wi h he
kine ic, mic oscopic, and spec opho ome ic expe imen s, he
ob ained da a would help o iden i y p ocesses by which he
ungal isola e in e ac s wi h he adionuclide.
PARAFAC s udies based on c yo-TRLFS spec a o he
U- ea ed ungal biomass (0.1 mM U o 48 h and a ungal DBM
o ~0.25 g/L) showed wo dominan U(VI) species
(Figu es 4A,B). A a ying empe a u e (4 and 30°C) and
depending on ungal cell iabili y ( iable o dead-au ocla ed),
he wo species we e de ec ed in di e en p opo ions
(Figu e 4B). The i s species (U species 1, g een) domina ed
all h ee samples and was cha ac e ized by h ee main emission
bands a 497.1, 519.0, and 540.2 nm, as shown in he luminescence
spec um (Figu e 4A). Wi h 92% (~64 mg U/g DBM) a 30°C
and iable cells, his U species was p opo ionally and
quan i a i ely mo e p ominen as compa ed o iable cells a
4°C (60%, ~14 mg U/g DBM) and dead-au ocla ed cells a 30°C
(87%, ~11 mg U/g DBM). Gi en his obse a ion and he lowe
ine s uc u e when compa ed o he second species,
p opo ionally less-p esen (U species 2, black), i was assumed
ha he domina ing species ep esen ed a bio-associa ed o ganic
U phospha e species ha was p oduced ac i ely and passi ely
by he ungal cells. The second species, ha ing a g ea e ine
s uc u e and shi ed emission bands (505.4, 527.6, and 551.6),
AB
CD
FIGURE3 | Phospha ase ac i i y (A,B) and ex acellula phospha e (C,D) de e mina ion o P. simplicissimum KS1 and DSM 62867 a e 48 h incuba ion wi hou U
in SD medium o ap wa e (A,C) and wi h 0.1 mM U in ap wa e (B,D).
Schae e e al. U anium Biomine aliza ion by Penicillium simplicissimum
F on ie s in Mic obiology | www. on ie sin.o g 9 Decembe 2021 | Volume 12 | A icle 802926
was assumed o co espond o a mo e homogenous, ino ganic
U(VI) phospha e species (Wang e  al., 2008), which could
be p oduced ac i ely by ungal phospha ase ac i i y.
In addi ion o he wo bio-associa ed U(VI) species
de ec ed, wo u he species (named U species 3 and 4)
we e calcula ed ia PARAFAC in he esul ing supe na an
a e he incuba ion o P. simplicissimum KS1in 0.1 mM U(VI;
Figu es4C,D). U species 3 (Figu e4C, blue) showed emission
bands a 481.4, 503.9, and 526.4 nm; U species 4 (Figu e4C,
o ange) a 491.6, 513.4, and 537.2 nm. Bo h species we e
mo e homogenous han he bio-associa ed species 1. As o
he bio-associa ed species, U species 3 and 4 we e de ec ed
in di e en p opo ions depending on cell iabili y and
empe a u e (Figu e 4D). U species 3 was dominan in he
supe na an o dead-au ocla ed cells (100%) and iable cells
a 30°C (87%), bu a 4°C, U species 4 (64%) su passed
species 3 (36%). U species 4 was only p esen o 10% in
iable cells a 30°C and no de ec able in he supe na an
o dead-au ocla ed cells a 30°C. Thus, U species 4 was
mos likely only sec e ed ac i ely by iable cells, displaying
inc eased p opo ions a low empe a u e (4°C). Rema kably,
u anyl ni a e, he ini ially added u anium species, was no
obse ed. This indica es ha all he u anium in solu ion
in e ac ed wi h biological ma e .
Iden i ica ion o he exac u anyl species based on he
emission bands o e e ence compounds is di icul due o
a ying expe imen al condi ions and hus di e en complexing
agen s and esul ing spec al shi s. All ou species can
AB
CD
FIGURE4 | Decon olu ed luminescence spec a (A,C) and species dis ibu ion (B,D) based on he PARAFAC analyses o P. simplicissimum KS1 biomass ( op)
and supe na an (bo om) a e 48 h incuba ion wi h 0.1 mM U(VI) a pH 5.0.