Effect of Temperature and Cell Viability on Uranium Biomineralization by the Uranium Mine Isolate Penicillium simplicissimum

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.