Journal of Fungi

Article Aspergillus niger Decreases Bioavailability of Arsenic(V) via Biotransformation of Oxide into Biogenic Oxalate

Bence Farkas 1, Marek Kolenˇcík 2,3, Miroslav Hain 4, Edmund Dobroˇcka 5, Gabriela Kratošová 3, Marek Bujdoš 1, Huan Feng 6 , Yang Deng 6, Qian Yu 7, Ramakanth Illa 8, B. Ratna Sunil 9, Hyunjung Kim 10 , Peter Matúš 1 and Martin Urík 1,*

1 Institute of Laboratory Research on Geomaterials, Faculty of Natural Sciences, Comenius University in Bratislava, 84215 Bratislava, Slovakia; [email protected] (B.F.); [email protected] (M.B.); [email protected] (P.M.) 2 Department of Soil Science and Geology, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, 949 76 Nitra, Slovakia; [email protected] 3 Nanotechnology Centre, VŠB—Technical University of Ostrava, 70833 Ostrava, Czech Republic; [email protected] 4 Institute of Measurement Science, Slovak Academy of Sciences in Bratislava, 84104 Bratislava, Slovakia; [email protected] 5 Institute of Electrical Engineering, Slovak Academy of Sciences in Bratislava, 84104 Bratislava, Slovakia; [email protected] 6 Department of Earth and Environmental Studies, Montclair State University, Montclair, NJ 07043, USA; [email protected] (H.F.); [email protected] (Y.D.) 7 School of Ecology and Environmental Science, Yunnan University, Kunming 650091, China; [email protected] 8 Department of Chemistry, Rajiv Gandhi University of Knowledge Technologies, AP IIIT, Nuzvid 521202, India; [email protected] 9 Department of Mechanical Engineering, Bapatla Engineering College, Bapatla 522101, India; [email protected] 10 Department of Resources and Energy Engineering, Jeonbuk National University, 567, Baekje-daero, Deokjin-gu, Jeonju, Jeonbuk 54896, Korea; [email protected] * Correspondence: [email protected]; Tel.: +421-290-149-392

 Received: 19 October 2020; Accepted: 6 November 2020; Published: 9 November 2020 

Abstract: The aim of this work was to evaluate the transformation of (hausmannite) by microscopic filamentous fungus Aspergillus niger and the effects of the transformation on mobility and bioavailability of arsenic. Our results showed that the A. niger strain CBS 140837 greatly affected the stability of hausmannite and induced its transformation into biogenic crystals of manganese oxalates—falottaite and lindbergite. The transformation was enabled by fungal acidolysis of hausmannite and subsequent release of manganese ions into the culture medium. While almost 45% of manganese was bioextracted, the arsenic content in manganese precipitates increased throughout the 25-day static cultivation of fungus. This significantly decreased the bioavailability of arsenic for the fungus. These results highlight the unique A. niger strain’s ability to act as an active geochemical factor via its ability to acidify its environment and to induce formation of biogenic minerals. This affects not only the manganese speciation, but also bioaccumulation of potentially toxic metals and metalloids associated with manganese oxides, including arsenic.

Keywords: arsenic; filamentous fungi; bioextraction; bioaccumulation; manganese minerals

J. Fungi 2020, 6, 270; doi:10.3390/jof6040270 www.mdpi.com/journal/jof J. Fungi 2020, 6, 270 2 of 12

1. Introduction Microbially driven processes are essential for mobilization and availability of elements in soils and sediments [1]. Filamentous fungi naturally mediate the mobilization of metals and metalloids by affecting the stability of soil particles’ and mineral surfaces via extrusion of chelating, redox active, and acidic metabolites [2], while directly disturbing the mineral structure mechanically via turgor pressure [3]. Simultaneously, however, the pre-concentration of metabolites at the cell–mineral interfaces or near the hyphae can result in formation of new thermodynamically stable biogenic minerals [4,5]. Thus, the microbially induced transformation actively contributes to immobilization of metals and metalloids in the biogenic minerals’ structure, while effectively changing the sorptive properties of these newly formed phases [6]. These processes are especially significant for environmental scavengers of elements, such as manganese oxides [7]. Manganese oxides are considered as a significant component of the natural geochemical barrier due to their ability to efficiently adsorb and immobilize metals and metalloids [4,8–10]. The reactive property of natural manganese oxides, including (Mn2+Ca, Mg)Mn4+ O H O, cryptomelane K(Mn4+, 3 7· 2 Mn2+) O , hausmannite Mn2+Mn3+ O and Na Mn3+ Mn4+ O 9H O, affects geochemical 8 16 2 4 4 6 8 27· 2 destiny of various potentially toxic metals and metalloids in the environment [11]. Hausmannite is usually found in metamorphosed or hydrothermal manganese ores, and its mineral structure is occupied by both Mn2+ and Mn3+ cations [12]. The presence of both divalent and trivalent Mn cations renders hausmannite a unique model for studying the reactivity, stability and microbially induced transformation of manganese oxides. One of the naturally occurring hazardous elements, whose behavior is affected by redox and sorptive properties of manganese oxides [13], and which was classifies by World Health Organization (WHO) as one of the most significant environmental contaminant, is arsenic. Depending on its physical, chemical and biological factors, arsenic exists in different oxidation states 3, 0, +3, +5 [14–16], − which significantly affect arsenic geochemical distribution and abundance, biological availability and toxicity [11,15]. As indicated, the environmental migration of arsenic is directly influenced by manganic geochemical barriers [8,9]. However, the influence of microbial activity on arsenic immobilization from manganese oxides and hydroxides is not well studied or understood. Therefore, this work focuses on evaluation of biotransformation processes occurring during mutual interaction of heterotrophic common soil fungal strain Aspergillus niger, arsenic and its natural scavenger and one of the rock-forming manganese oxide minerals—hausmannite. Extent and significance of microbial activity on manganese and arsenic biogeochemistry is also discussed and supported by experimental results that confirm the ability of microorganisms to form new stable biogenic mineral phases and, thus, influence the mobility of arsenic in the environment.

2. Materials and Methods

2.1. Chemicals and Reagents All reagents used in this study were of analytical grade (Na HAsO 7H O, MnSO 4H O, NaOH, 2 4· 2 4· 2 HCl and HNO3) and were obtained from Centralchem (Bratislava, Slovak Republic) or Sigma-Aldrich (Darmstadt, Germany). For inoculation, fungal growth and other cultivation related purposes, the Sabouraud Dextrose Broth and Sabouraud Dextrose Agar culture media (HiMedia, Mumbai, India) were used.

2.2. Preparation of Manganese Oxide

2+ 3+ Artificial manganese oxide hausmannite (Mn Mn 2O4) was synthesized by alkaline precipitation 1 of 1 L 0.5 mol L− MnSO4 using 40 g of NaOH. The mixture was then kept on a rotary shaker at 100 rpm (Unimax 2010, Heidolph, Schwabach, Germany) for 24 h at 25 ◦C in dark, and subsequently heated under reflux for 5 h. Synthesized precipitate was cooled, filtrated, washed with redistilled J. Fungi 2020, 6, 270 3 of 12

water, sterilized in a hot air oven at 80 ◦C for 24 h and kept in a sealed plastic bottle. It was further sterilized for 1 h in a hot air oven at 95 ◦C right before the experiment.

2.3. Fungal Strain The fungus Aspergillus niger strain CBS 140837 was obtained from the fungal collection of the Department of Mycology and Physiology at the Institute of Botany, Slovak Academy of Sciences. The fungal strain was maintained on the Sabouraud agar plates at 25 ◦C.

2.4. Fungal Cultivation in Presence of Manganese Oxides with Pre-Adsorbed Arsenic All cultivation experiments using the A. niger strain were performed in 100 mL sterile Erlenmeyer flasks with the mixture of 50 mL Sabouraud Dextrose Broth culture medium (HiMedia, Mumbai, India) 1 with the initial 9.1 mg L− As(V) concentration which was prepared from a stock solution of Na HAsO 7H O diluted in redistilled water. The 0.1 mL of spore suspensions, prepared by washing 2 4· 2 the surface of a 10-day old A. niger culture with sterile water and diluted to approximately 106 CFU 1 mL− , were transferred into growth media under aseptic conditions to inoculate the media with filamentous fungus. Prior to inoculation, the 0.25 g of synthetized manganese oxide in an 100 mL Erlenmeyer flask were sterilized by dry heating and, after sterilization, the culture medium with As(V) content was added to the sterile flask with the manganese oxide and stirred on a rotatory shaker for 24 h at 130 rpm (Unimax 2010, Heidolph, Schwabach, Germany). There were also manganese oxide-free treatment and arsenic-free treatments which were performed as control experiments. All experiments were carried out in triplicate under laboratory conditions during 25-day static cultivation that allowed fungus to reach the stationary growth phase. During the cultivation, the pH of culture medium, biomass weight, as well as dissolved, coprecipitated and bioaccumulated arsenic and manganese were determined on 3rd, 5th, 10th, 15th, 20th and 25th cultivation day. To evaluate arsenic and manganese distribution in cultivation system, the biomass grown on the surface of the culture medium was separated at the designated cultivation period, weighed and digested in the mixture of HCl and HNO3 for further analytical analysis. The spent medium was filtered, and the pH was recorded. The collected precipitates were also digested and along other components they were analyzed for total arsenic and manganese using flame atomic absorption spectrometry (F-AAS) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Furthermore, structural and morphological properties of mineral phases were characterized at the end of the cultivation.

2.5. Analytical Procedures For determination of arsenic and manganese in the liquid medium, digested biomass and precipitated phases, ICP-MS (iCap Q, Thermo Scientific, Waltham, MA, USA) and F-AAS (AAS Perkin Elmer Model 1100, Waltham, MA, USA) were used, respectively [17–20]. The ICP-MS was in KED (He) mode with 103Rh as an internal standard. Calibration standards were prepared from As standard stock 1 solution (1000 mg L− , CertiPur, Merck, Darmstadt, Germany). The deuterium background was used for correction on F-AAS for manganese determination. Calibration standards were prepared from 1 manganese standard stock solution (1000 mg L− , CertiPur, Merck, Darmstadt, Germany). For precise determination of crystal symmetry of manganese minerals, the X-ray diffractometer Bruker D8 DISCOVER equipped with an X-ray tube with a rotating Cu anode operating at 12 kW (40 kV/300 mA) was applied. All measurements were performed in parallel beam geometry with a parabolic Goebel mirror in the primary beam. The diffraction patterns in the angular range 20–80◦ of 2θ were recorded in a grazing incidence set-up with the angle of incidence α = 1.5◦. A parallel plate collimator with the angular acceptance at 0.35◦ was inserted in the diffracted beam. For morphology determination and particle size distribution, a Scanning Electron Microscope (SEM) QUANTA 450 FEG (FEI Company, Hillsboro, OR, USA) equipped with an Energy Dispersive Spectrometer (EDS) was used. Analysis was done at an accelerating voltage of 15 keV, and the samples were covered by a layer of carbon for better sample conduction. J. Fungi 2020, 6, x FOR PEER REVIEW 4 of 12

Spectrometer (EDS) was used. Analysis was done at an accelerating voltage of 15 keV, and the J. Fungi 2020, 6, 270 4 of 12 samples were covered by a layer of carbon for better sample conduction. Spatial distribution of newly formed thermodynamically stable biominerals incorporated in biomassSpatial were distribution observed by of 3D newly X-ray formed microscopy thermodynamically and computed microtomography stable biominerals (microCT). incorporated For the in biomassanalysis, werethe microtomograph observed by 3D X-ray Nanotom microscopy 180 (GE and Phoenix, computed Wunstorf microtomography, Germany) (microCT). was used Forat the analysis,Institute of the Measurement microtomograph Science, Nanotom Slovak 180 Academy (GE Phoenix, of Sciences. Wunstorf, Nanotom Germany) 180 was is equipped used at the with Institute point ofsource Measurement of X-ray radiation Science, Slovakwith nano-focusation Academy of Sciences. (transmission Nanotom tungsten 180 is target equipped); maximum with point acceleration source of X-rayvoltage radiation is 180 kV with and nano-focusation energy 15 W. (transmission The applied tungstenscintillation target); type maximum detector (CsI) acceleration with a voltagematrix isphotodetector 180 kV and energyhas image 15 W. resolution The applied of 2300 scintillation × 2300 pi typexels, detector and the (CsI) size withof one a matrix pixel is photodetector 50 × 50 µm. hasMinimal image achieved resolution voxel of 2300 resolution2300 pixels, after and 3D the reconstruction size of one pixel is isdown 50 50to µ0.5m. Minimalµm. During achieved the × × voxelmeasurements, resolution an after acceleration 3D reconstruction voltage of is down150 kV to and 0.5 µam. current During of the90 mA measurements, were applied, an the acceleration detector voltageintegration of 150 time kV was and set a current to 500 ms. of 90 mA were applied, the detector integration time was set to 500 ms. The determined determined concentrations concentrations of of arsenic arsenic and and manganese manganese in biomass, in biomass, culture culture media media and non- and non-dissolveddissolved residue, residue, as well as well as biomass as biomass dry dry weight weight and and culture culture media media pH pH recorded recorded during the cultivation werewere compared compared among among the treatmentsthe treatments using using two-sample two-samplet-test assuming t-test assuming unequal variancesunequal invariances an extension in an extension Analysis ToolPak Analysis in ToolPak Microsoft in ExcelMicrosoft (Redmond, Excel (Redmond, WA, USA). WA, USA).

3. Results

3.1. Characterization of Synthesized Manganese Oxide Synthesized manganesemanganese oxide oxide formed formed fine and fine coarse and aggregates coarse withaggregates imperfect with pseudooctaedric imperfect single-grainpseudooctaedric morphology single-grain with morphology particle size with distribution particle size between distribution 1 µm andbetween 200 µ 1m. µm EDS and chemical 200 µm. analysisEDS chemical revealed analysis the dominant revealed contentsthe dominant of manganese contents and of manganese oxygen (the and carbon oxygen is attributed (the carbon to the is conductiveattributed to carbon the conductive layer) and carbon highlighted layer) theand chemical highlighted purity the of chemical crystals purity (Figure of1). crystals (Figure 1).

Figure 1. ScanningScanning electron electron micrograph of manganese ox oxideide before fungal treatment.treatment. The inset shows energy dispersive X-ray analysis (EDX)(EDX) of synthesizedsynthesized manganesemanganese oxide.oxide.

X-ray didiffractionffraction analysis verifiedverified tetragonal crystal symmetry with chemical formula of 2+ 3+ Mn2+MnMn3+2O2O4, 4the, the hausmannite. hausmannite. Individual Individual crys crystaltal spatial spatial parameters parameters such such as as aa, ,cc-axes-axes are are shown shown in Table1 1..

Table 1. Crystallographic parameters and symmetry of synthesized manganese oxide hausmannite Table 1. Crystallographic parameters and symmetry of synthesized manganese oxide hausmannite 2+ 3+ [Mn Mn 2O4] before biotransformation. [Mn2+Mn3+2O4] before biotransformation.

Manganese OxideManganese Oxide crystal symmetrycrystal symmetry tetragonal tetragonal a (Å) a (Å) 5.763 (2) 5.763 (2) c (Å) c (Å) 9.459 (4) 9.459 (4) α = β = γ α = β = γ 90◦ 90°

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3.2. Immobilization of Arsenic in the Manganese Mineral Phase(s)

J. FungiThe2020 process, 6, 270 of adsorption of dissolved arsenic(V) onto hausmannite was necessary to quantify5 of 12 its sorption properties. As depicted in Figure 2a, while the initial arsenic concentration in the hausmannite-free culture medium was 9.1 mg L−1, only 7.6 mg L−1 of dissolved arsenic was detected in3.2. the Immobilization culture medium of Arsenic after in 24 the h Manganese pre-adsorption Mineral of Phase(s) arsenic(V) onto the hausmannite. Thus, the manganeseThe process oxide of managed adsorption to ofadsorb dissolved as much arsenic(V) as 16.4% onto of hausmannite arsenic. This was represents necessary a to0.3 quantify mg g−1 sorptionits sorption capacity properties. of hausmannite As depicted for arsenic(V). in Figure 2a, while the initial arsenic concentration in the 1 1 hausmannite-freeThe amount cultureof arsenic medium immobilized was 9.1 mg in L −the, onlynon-dissolved 7.6 mg L− ofresidue dissolved of arsenicmanganese, was detected however, in increasedthe culture during medium cultivation after 24 h of pre-adsorption fungus. This ofis depicted arsenic(V) in onto Figure the 2b hausmannite. which shows Thus, increasing the manganese content 1 ofoxide total managed arsenic bound to adsorb to the as muchmanganese as 16.4% mineral of arsenic. phase( Thiss) from represents initially a 0.07 0.3 mg mg g to− finallysorption 0.32 capacity mg at theof hausmannite end of the fungal for arsenic(V). cultivation.

12 (a) 0.4 (b) ** ** 0.35 10 *** *** *** 0.3 8 *** *** ) 0.25 -1 6 0.2 (mg L medium (mg) 0.15

4 residue 0.1 2 0.05

As concentration in culture culture in As concentration 0 0

0102030 Mn nondissolved in As content 0102030 cultivation period cultivation period (day) (day)

Figure 2.2. ChangesChanges in in arsenic arsenic concentration concentration in hausmannite-freein hausmannite-free (open (open circles) circles) and hausmannite-treated and hausmannite- (blacktreated solid (black circles) solid culturecircles) mediaculture supplemented media supplemented with arsenic(V) with arsenic(V) (a) and its (a content) and its in content non-dissolved in non- dissolvedmanganese manganese residue (b) residue during a(b 25-day) during period a 25-day of A. period niger cultivation. of A. niger Resultscultivation. represent Results the represent mean values the meanof three values independent of three independent experiments experiments and error bars and show error the bars standard show the deviation. standard Asterisks deviation. indicate Asterisks the indicatesignificant the diff erencessignificant between differences the hausmannite-free between the and hausmannite-free hausmannite-treated and experiments hausmannite-treated (** p < 0.01, ***experimentsp < 0.001). (** p < 0.01, *** p < 0.001).

The amount of arsenic immobilized in the non-dissolved residue of manganese, however, increased 3.3. Manganese Bioextraction and Bioaccumulation by Fungus during cultivation of fungus. This is depicted in Figure2b which shows increasing content of total arsenicDuring bound the to fungal the manganese cultivation, mineral the pH phase(s) of culture from media initially changes 0.07 mg due to to finally the A. 0.32 niger mg strain’s at the endacidic of metabolitethe fungal cultivation.exudation (Figure 3). While arsenic(V) did not have any significant effect on pH development in comparison to hausmannite- and arsenic-free treatment, the presence of hausmannite and3.3. Manganesesubsequent Bioextraction release of andmanganese Bioaccumulation ions (Figure by Fungus 4a) resulted in statistically significantly less acidificationDuring theof the fungal culture cultivation, medium theby fungus. pH of culture Theref mediaore, the changes lowest pH due values to the A.detected niger strain’s were 3.5 acidic and 2.4metabolite for manganese-treated exudation (Figure and3). manganes While arsenic(V)e-free culture did not media, have any respectively. significant e ffect on pH development in comparisonAs indicated, to hausmannite- the acidification and arsenic-freeof hausmannite-treated treatment, the culture presence media of hausmannite triggered the and dissolution subsequent of −1 manganeserelease of manganese mineral. Within ions (Figure the 10th4a) resultedday of cultivation, in statistically up significantlyto 1250 mg L less of acidification manganese ofwas the detected culture inmedium the culture by fungus. medium Therefore, of each the treatment lowest pH (Figure values 4a). detected The presence were 3.5 andof arsenic 2.4 for manganese-treateddid not affect the dissolutionand manganese-free rate. However, culture the media, increase respectively. in pH (Figure 3) coincides well with the significant decrease in manganese dissolved in the medium. Most of it was most likely bioaccumulated by A. niger (Figure 4b) since the fungal uptake resulted in manganese mycelial concentrations up to 104 mg g−1. However, this value also included manganese that was immobilized in newly formed biogenic minerals closely associated with fungal biomass.

J. Fungi 2020, 6, x FOR PEER REVIEW 6 of 12 J. Fungi 2020, 6, 270 6 of 12 J. Fungi 2020, 6, x FOR PEER REVIEW 6 of 12 6.5 ** 6 ns 6.5 ns 5.5 ** 6 ns 5 ns 5.5 ** 4.5 *** 5 ns pH 4 *** ** 4.5 ** *** *** ns ns pH 3.5 *** 4 ns ** *** 3 * ns 3.5 ns ns 2.5 ns 3 * ns ns 2.52 2 0 102030 0cultivation 102030 period (day) cultivation period (day) Figure 3. Changes in culture media pH in hausmannite-free (open circles) and hausmannite-treated Figure 3. Changes in culture media pH in hausmannite-free (open circles) and hausmannite-treated (blackFigure solid 3. Changes circles) inculture culture media media supplemented pH in hausmannite with arsenic(V),-free (open and circles) hausmannite- and hausmannite-treated and arsenic-free (black solid circles) culture media supplemented with arsenic(V), and hausmannite- and arsenic-free controls(black solid (gray circles) triangles) culture during media 25-day supplemented static cultivation with arsenic(V), of an A. andniger hausmannite- strain. Results and represent arsenic-free the controls (gray triangles) during 25-day static cultivation of an A. niger strain. Results represent the mean meancontrols values (gray of triangles)three independent during 25-day experiments static cultivation and error bars of an show A. niger the standard strain. Results deviation. represent Asterisks the values of three independent experiments and error bars show the standard deviation. Asterisks indicate indicatemean values the ofsignificant three independent differences experiments between and the error hausmannite-free bars show the standard or hausmannite-treated deviation. Asterisks the significant differences between the hausmannite-free orns hausmannite-treated experiments and experimentsindicate the and significant controls (* differencesp < 0.05, ** p

) 1000

1200 )

-1 ns -1 800 ns 6080 **

) 1000 )

-1 ns -1 (mg (mg L

medium 600 ns 800 (mg g 4060 400 ns (mg (mg L

medium 600 ns (mg g 40 ns 20 ns 200 ns 400 ns

Mn concentration in culture culture in concentration Mn 0 200 ns 200 Mn concentration inbiomass ns 0 102030 0ns 102030 Mn concentration in culture culture in concentration Mn 0 0 Mn concentration inbiomass 0cultivation 102030 period 0cultivation 102030 period cultivation(day) period cultivation(day) period Figure 4. Changes in manganese(day) concentration in arsenic-free (gray solid circles)(day) and arsenic-treated Figure(black 4. solid Changes circles) in culturemanganese media concentration (a) and its in content arsenic in-free the (gray fungal solid mycelium circles) and (including arsenic-treated mineral (blackmanganeseFigure solid 4. Changes phasescircles) in associatedculture manganese media with concentration ( thea) and biomass) its contentin (arsenicb) during in-free the a (grayfungal 25-day solid mycelium period circles) of A.(includingand niger arsenic-treatedcultivation. mineral manganeseResults(black solid represent phases circles) the associat culture mean valuesed media with of the( threea) andbiomass) independent its content (b) during experimentsin the a 25-dayfungal and periodmycelium error of bars A. (includingshow niger thecultivation. standardmineral Resultsdeviation.manganese represent Asterisks phases the associat mean indicate valuesed the with significantof threethe biomass) independ differences (bent) during experiments between a 25-day the and arsenic-free period error barsof A. and show niger arsenic-treated the cultivation. standard ns deviation.experimentsResults represent Asterisks (** p the< 0.01,indicate mean valuesnot the significant). significant of three independ differencesent experimentsbetween the andarsenic-free error bars and show arsenic-treated the standard experiments (** p < 0.01, ns not significant). Asdeviation. indicated, Asterisks the acidification indicate the significant of hausmannite-treated differences between culture the mediaarsenic-free triggered and arsenic-treated the dissolution of experiments (** p < 0.01, ns not significant). 1 manganeseDuring mineral. the cultivation Within the experi 10th dayment, of cultivation,the filamentous up to 1250 fungus mg L −A.of niger manganese was able was detectedto extract in the culture medium of each treatment (Figure4a). The presence of arsenic did not a ffect the dissolution approximatelyDuring the 45% cultivation of manganese experi fromment, its originalthe filamentous content in fungushausmannite A. niger and wasredistribute able to itextract in the rate. However, the increase in pH (Figure3) coincides well with the significant decrease in manganese cultureapproximately medium 45% (8%) of manganeseand mycelium from (37%). its original Arseni contentc did innot hausmannite have any significant and redistribute effect iton in thethe dissolved in the medium. Most of it was most likely bioaccumulated by A. niger (Figure4b) since the manganeseculture medium redistribution (8%) and in myceliumthe cultivation (37%). system Arseni, andc did the notfungus have did any not significant manage to effect increase on thethe fungal uptake resulted in manganese mycelial concentrations up to 104 mg g 1. However, this value mobilitymanganese of arsenic redistribution (Figure in2b). the cultivation system, and the fungus did not −manage to increase the also included manganese that was immobilized in newly formed biogenic minerals closely associated mobility of arsenic (Figure 2b). 3.4.with Effects fungal of biomass.Manganese Mineral Phase(s) on Arsenic Bioaccumulation During the cultivation experiment, the filamentous fungus A. niger was able to extract 3.4. Effects of Manganese Mineral Phase(s) on Arsenic Bioaccumulation approximatelyThe fungal 45%growth of manganese was not affected from itsby originalthe arsenic content presence; in hausmannite however, manganese and redistribute obviously it in inhibitedthe cultureThe initialfungal medium growth growth (8%) phase was and notof mycelium A. affected niger (Figure (37%).by the 5a). Arsenicarsenic In the presence; did latter not ca have se,however, the any apparent significant manganese fungal eff ectobviously biomass on the weightmanganeseinhibited increased initial redistribution growth during phase the in late the of growth cultivationA. niger phase (Figure system, in 5a).comparison andIn the the latter fungus to hausmannite-free case, did the not apparent manage media; fungal to increase however, biomass the this could be attributed to additional weight of manganese minerals intimately associated with fungal mobilityweight increased of arsenic during (Figure the2b). late growth phase in comparison to hausmannite-free media; however, biomass.this could be attributed to additional weight of manganese minerals intimately associated with fungal biomass.

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3.4. Effects of Manganese Mineral Phase(s) on Arsenic Bioaccumulation The fungal growth was not affected by the arsenic presence; however, manganese obviously inhibited initial growth phase of A. niger (Figure5a). In the latter case, the apparent fungal biomass weight increased during the late growth phase in comparison to hausmannite-free media; however, this could be attributed to additional weight of manganese minerals intimately associated withJ. Fungi fungal 2020, 6, biomass. x FOR PEER REVIEW 7 of 12

0.7 0.04 (a) *** (b) 0.6 0.035 *** *** ** 0.03 0.5 ns ns 0.025 *** 0.4 ns ns 0.02 *** (g) ** 0.3 ns (mg) 0.015 ** 0.2 ns *** biomass weight biomass ns 0.01

0.1 * biomass in As content 0.005 ns 0 0 0 102030 0102030 cultivation period cultivation period (day) (day)

Figure 5. Changes in biomass dry weight (a) and arsenic content in biomass of fungus A. niger (b) Figure 5. Changes in biomass dry weight (a) and arsenic content in biomass of fungus A. niger (b) cultivated in hausmannite-free (open circles) and hausmannite-treated (black solid circles) culture cultivated in hausmannite-free (open circles) and hausmannite-treated (black solid circles) culture media supplemented with arsenic(V) over 25 days. Gray triangles represent hausmannite- and arsenic-freemedia supplemented treatment. with The dataarsenic(V) of A. nigerover’s 25 biomass days. areGray fitted triangles using modifiedrepresent Gompertz’shausmannite- growth and equationarsenic-free [21 ].treatment. Results represent The data the of meanA. niger values’s biomass of three are independent fitted using experiments modified Gompertz’s and error bars growth show theequation standard [21]. deviation. Results represent Asterisks the indicate mean thevalues significant of three di independentfferences between experiments the hausmannite-free and error bars andshow/or the hausmannite-treated standard deviation. experimentsAsterisks indicate and controlsthe significant (* p < differences0.05, ** p < between0.01, *** thep < hausmannite-0.001, ns not ns significant).free and/or hausmannite-treated experiments and controls (* p < 0.05, ** p < 0.01, *** p < 0.001, not significant). Still, the bioavailability of arsenic in hausmannite-treated media was significantly reduced (FigureStill,5b). the While bioavailability the maximum of arsenic value ofin accumulated hausmannite-treated arsenic was media 0.035 was mg significantly in hausmannite-free reduced media,(Figure the5b). content While ofthe arsenic maximum in the valueA. niger of accumustrain’slated biomass arsenic did notwas change 0.035 mg significantly in hausmannite-free throughout themedia, cultivation the content period, of arsenic and it didin the not A. exceed niger strain’s a value biomass of 0.011 mg.did not This change was the significantly consequence throughout of arsenic absorptionthe cultivation in the period, newly and formed it did mineral not exceed phases, a valu ore due of 0.011 to its mg. adsorption This was onto the surfaces consequence of hausmannite of arsenic orabsorption biogenic in manganese the newly mineralsformed mineral (Figure phases,2b). or due to its adsorption onto surfaces of hausmannite or biogenic manganese minerals (Figure 2b). 3.5. Formation of Biogenic Manganese Oxalate 3.5. Formation of Biogenic Manganese Oxalate During the 25 days of A. niger cultivation in media supplemented with hausmannite, new biogenicDuring precipitates the 25 days were of formed. A. niger The cultivation content of in this media newphase supplem wasented after 20with days hausmannite, in quantities new that allowedbiogenic usprecipitates to identify were the manganese formed. The mineral content falottaite of this new [Mn(C phaseO )was3H afterO]. Later 20 days on, in by quantities the end of that the 2 4 · 2 cultivationallowed us experiment,to identify the it was manganese dehydrated mineral and partially falottaite transformed [Mn(C2O4)·3H into2O]. lindbergite Later on, [Mn(C by theO end) 2H of theO]. 2 4 · 2 Thecultivation microbially experiment, induced transformationit was dehydrated of hausmannite and waspartially confirmed transformed by X-ray diintoffraction lindbergite analysis in[Mn(C biomass2O4)·2H as well2O]. The as in microbially the residual induced solid phase transformation in the culture of mediumhausmannite (Figure was6). confirmed by X-ray diffraction analysis in biomass as well as in the residual solid phase in the culture medium (Figure 6).

before cultivation (a) 25 day (b) 20. day after cultivation 15. day falottaite lindbergite 10. day 10000 hausmannite 5. day logIntensity

1000 10 30 50 70 10 30 50 2Theta 2Theta

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0.7 0.04 (a) *** (b) 0.6 0.035 *** *** ** 0.03 0.5 ns ns 0.025 *** 0.4 ns ns 0.02 *** (g) ** 0.3 ns (mg) 0.015 ** 0.2 ns *** biomass weight biomass ns 0.01

0.1 * biomass in As content 0.005 ns 0 0 0 102030 0102030 cultivation period cultivation period (day) (day)

Figure 5. Changes in biomass dry weight (a) and arsenic content in biomass of fungus A. niger (b) cultivated in hausmannite-free (open circles) and hausmannite-treated (black solid circles) culture media supplemented with arsenic(V) over 25 days. Gray triangles represent hausmannite- and arsenic-free treatment. The data of A. niger’s biomass are fitted using modified Gompertz’s growth equation [21]. Results represent the mean values of three independent experiments and error bars show the standard deviation. Asterisks indicate the significant differences between the hausmannite- free and/or hausmannite-treated experiments and controls (* p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant).

Still, the bioavailability of arsenic in hausmannite-treated media was significantly reduced (Figure 5b). While the maximum value of accumulated arsenic was 0.035 mg in hausmannite-free media, the content of arsenic in the A. niger strain’s biomass did not change significantly throughout the cultivation period, and it did not exceed a value of 0.011 mg. This was the consequence of arsenic absorption in the newly formed mineral phases, or due to its adsorption onto surfaces of hausmannite or biogenic manganese minerals (Figure 2b).

3.5. Formation of Biogenic Manganese Oxalate During the 25 days of A. niger cultivation in media supplemented with hausmannite, new biogenic precipitates were formed. The content of this new phase was after 20 days in quantities that allowed us to identify the manganese mineral falottaite [Mn(C2O4)·3H2O]. Later on, by the end of the cultivation experiment, it was dehydrated and partially transformed into lindbergite [Mn(C2O4)·2H2O]. The microbially induced transformation of hausmannite was confirmed by X-ray

J.diffraction Fungi 2020, 6analysis, 270 in biomass as well as in the residual solid phase in the culture medium (Figure8 of 12 6).

before cultivation (a) 25 day (b) 20. day after cultivation 15. day falottaite lindbergite 10. day 10000 hausmannite 5. day logIntensity

1000 10 30 50 70 10 30 50 2Theta 2Theta Figure 6. X-ray diffraction spectra of the residual solid manganese phases analyzed in pre-defined intervals (a). The XRD patterns indicate a transformation of the initial hausmannite into biogenic falottaite [Mn(C2O4) 3H2O] and lindbergite [Mn(C2O4) 2H2O] (b). · · The observed mineralogical transformation explained dynamics of manganese concentration in culture media (Figure4a). After 10 days of cultivation, the extruded oxalate chelated dissolved manganese cations and ultimately formed biogenic manganese oxalates. The formed biogenic mineral phase lindbergite showed orthorhombic crystal symmetry with individual crystallographic parameters suchJ. Fungi as 2020 the, 6a, ,x b,FOR c-axes PEER shownREVIEW in Table2. It forms prismatic needles with long-column morphology8 of 12 (Figure7a) and imperfect surfaces (Figure7b). Its size distribution ranged from 1 µm to 1 cm. Figure 6. X-ray diffraction spectra of the residual solid manganese phases analyzed in pre-defined Table2.intervalsCrystallographic (a). The XRD patterns parameters indicate and symmetry a transformation of biogenic oflindbergite the initial [Mn(ChausmanniteO ) 2H intoO] identified biogenic 2 4 · 2 falottaite [Mn(C2O4)·3H2O] and lindbergite [Mn(C2+ 32+O4)·2H2O] (b). after biotransformation of hausmannite [Mn Mn 2O4] by an A. niger strain.

The observed mineralogical transformation explainedManganese dynamics Oxalate Hydrate of manganese concentration in culture media (Figure 4a). Aftercrystal 10 days symmetry of cultivation, the extruded orthorhombic oxalate chelated dissolved manganese cations and ultimately formed biogenica (Å) manganese oxalates.10.524 The (2) formed biogenic mineral phase lindbergite showed orthorhombic bcrystal(Å) symmetry with individual6.614 (2) crystallographic parameters such as the a, b, c-axes shown in Table 2. It formsc (Å) prismatic needles with9.769 long-column (3) morphology (Figure 7a) and α = β = γ 90 imperfect surfaces (Figure 7b). Its size distribution ranged from 1 ◦µm to 1 cm.

(a) (b)

Figure 7.7. ScanningScanning electron electron microgra micrographph indicating indicating the the typical typical morphology of lindbergitelindbergite 2+ 3+ [Mn(C2OO4)·2H) 2H2O]O] (a ()a )that that resulted resulted from from fungal fungal biot biotransformationransformation of hausmannite of hausmannite [Mn [Mn2+Mn3+Mn2O4]. BothO ]. 2 4 · 2 2 4 Bothminerals minerals can be can identified be identified in the in SEM the SEMimage image (b), where (b), where the needle-like the needle-like lindbergite lindbergite is associated is associated with withsmall small grains grains of hausmannite. of hausmannite.

Table 2. Crystallographic parameters and symmetry of biogenic lindbergite [Mn(C2O4)·2H2O]

identified after biotransformation of hausmannite [Mn2+Mn3+2O4] by an A. niger strain.

Manganese Oxalate Hydrate crystal symmetry orthorhombic a (Å) 10.524 (2) b (Å) 6.614 (2) c (Å) 9.769 (3) α = β = γ 90°

Formation of oxalate as a restricting mechanism for manganese availability is highlighted by the presence of biogenic manganese phases directly in fungal biomass. Lindbergite 3D visualization highlighted its spatial distribution, and surface morphology (Figure 8). It was identified in the biomass surfaces, as well as internally anchored to the biomass. While the most of the lindbergite crystals encapsulated in the biomass form smaller sized crystals with characteristic spherical and prismatic morphology, crystals situated on the biomass surfaces form larger sized crystals with needle-like morphology (Figure 8).

J. Fungi 2020, 6, 270 9 of 12

Formation of oxalate as a restricting mechanism for manganese availability is highlighted by the presence of biogenic manganese phases directly in fungal biomass. Lindbergite 3D visualization highlighted its spatial distribution, and surface morphology (Figure8). It was identified in the biomass surfaces, as well as internally anchored to the biomass. While the most of the lindbergite crystals encapsulated in the biomass form smaller sized crystals with characteristic spherical and prismatic morphology, crystals situated on the biomass surfaces form larger sized crystals with needle-like J.morphology Fungi 2020, 6, x (Figure FOR PEER8). REVIEW 9 of 12

(a) (b)

(c) (d)

Figure 8. The 3D microscopy imaging ofof lindbergitelindbergite [Mn(C[Mn(C2O4)·2H) 2H2O]O] crystals crystals (indicated (indicated by by blue 2 4 · 2 color)color) thatthat are are intimately intimately associated associated with with fungal fung biomass.al biomass. The general The general view of biomassview of withbiomass lindbergite with lindbergite(scale bar = (scale2 mm) bar (a =); 2 micrograph mm) (a); micrograph of lindbergite of lindbergite biogenic crystalbiogenic (scale crystal bar (scale= 2 mm bar at= 2 x-axis) mm at ( bx-); axis)lindbergite (b); lindbergite crystals associatedcrystals associated with the with mycelium the my visualizedcelium visualized by 3D imagingby 3D imaging (scale bar(scale= 2bar cm = at2 cmx-axis) at x-axis) (c), and (c), cross and sectioncross section of biomass of biomass with visualized with visualized lindbergite lindbergite (scale bar(scale= 1bar mm) = 1 (mm)d). (d). 4. Discussion 4. Discussion The fungus Aspergillus niger has been shown to be capable of producing various chelating The fungus Aspergillus niger has been shown to be capable of producing various chelating metabolites, including oxalate, citrate and various siderophores [22,23]. This enables soil minerals’ metabolites, including oxalate, citrate and various siderophores [22,23]. This enables soil minerals’ dissolution and biologically accelerated deterioration of solid surfaces in the environment [24]. dissolution and biologically accelerated deterioration of solid surfaces in the environment [24]. Besides organic chelates, soil fungi acidify their natural habitat, which also effectively enhances the Besides organic chelates, soil fungi acidify their natural habitat, which also effectively enhances the natural weathering processes [25]. Fungal acidolysis and complexolysis of solid substrates were also natural weathering processes [25]. Fungal acidolysis and complexolysis of solid substrates were also successfully utilized in some biohydrometallurgical methods [26,27]. These unique abilities of fungal successfully utilized in some biohydrometallurgical methods [26,27]. These unique abilities of fungal consortia have manifested in our experiments in the potency of the fungal A. niger strain to decrease the consortia have manifested in our experiments in the potency of the fungal A. niger strain to decrease pH of culture media to values as low as 2.4 (Figure2b). However, natural soil components mitigate this the pH of culture media to values as low as 2.4 (Figure 2b). However, natural soil components effect, since we have observed that the presence of hausmannite in the culture medium alleviated the mitigate this effect, since we have observed that the presence of hausmannite in the culture medium production of acidic metabolites by the fungus. Still, the acidification was strong enough to disintegrate alleviated the production of acidic metabolites by the fungus. Still, the acidification was strong the of hausmannite and release almost 45% of manganese ions into the medium enough to disintegrate the crystal structure of hausmannite and release almost 45% of manganese (Figure4). ions into the medium (Figure 4). The proton- and ligand-mediated dissolution of manganese by filamentous fungi is well established The proton- and ligand-mediated dissolution of manganese by filamentous fungi is well process [28]. The excretion of oxalate has been found to be a key factor affecting the mobility of established process [28]. The excretion of oxalate has been found to be a key factor affecting the various potentially toxic metals in the environment via formation of mycogenic minerals [29,30]. In our mobility of various potentially toxic metals in the environment via formation of mycogenic minerals experiments, the extrusion of oxalate by the fungus A. niger and bioextraction of manganese ions from [29,30]. In our experiments, the extrusion of oxalate by the fungus A. niger and bioextraction of manganese ions from hausmannite’s crystal structure has led to a precipitation and crystallization of two stable manganese oxalate minerals—falottaite and lindbergite. This process was extremely time- demanding, since the falottaite was first detected by XRD only after 15 days of cultivation (Figure 5a). Nevertheless, we suggest that both oxalates coexist, and their distribution ratio is most likely linked to changes of the culture medium pH in the late fungal growth phase. As a significant amount of manganese oxalates were associated with the biomass (Figure 7), we hypothesize that the fragments of the fungal cell wall, or the hyphae themselves could serve as the nucleation sites via formation of saturated microdomains in the cell wall [31,32].

J. Fungi 2020, 6, 270 10 of 12 hausmannite’s crystal structure has led to a precipitation and crystallization of two stable manganese oxalate minerals—falottaite and lindbergite. This process was extremely time-demanding, since the falottaite was first detected by XRD only after 15 days of cultivation (Figure5a). Nevertheless, we suggest that both oxalates coexist, and their distribution ratio is most likely linked to changes of the culture medium pH in the late fungal growth phase. As a significant amount of manganese oxalates were associated with the biomass (Figure7), we hypothesize that the fragments of the fungal cell wall, or the hyphae themselves could serve as the nucleation sites via formation of saturated microdomains in the cell wall [31,32]. The outcome of hausmannite biodeterioration and transformation into mycogenic minerals have some implications not only in biogeochemistry of inorganic nutrients, but also potentially toxic metals and metalloids [6]. While we expected the decrease in arsenic content in solid manganese phases due to extensive leaching of manganese in the culture medium (Figure4), surprisingly, biosynthesis of mycogenic mineral phases increased affinity of arsenic towards the solid surfaces and the arsenic immobilization efficiency increased during fungal cultivation (Figure2b). It is very likely that the freshly precipitated biogenic manganese phases showed stronger affinity to adsorb and immobilize dissolved arsenic. To conclude our experiment, we suggest that our results highlighted the significant role of microscopic filamentous fungus A. niger as a geoactive factor in manganese transformation and arsenic mobility. Using laboratory-based research in a model designated cultivation system, we successfully simulated the natural process of fungal interactions with a hausmannite substrate in the presence of arsenic(V). Through metabolic activity, the fungus was capable of changing the conditions in the system to an extent that resulted in manganese oxide dissolution. This led to precipitation of manganese mycogenic minerals, and subsequently affected the amount of immobilized arsenic due to changes in sorptive interactions between arsenic and the surfaces of manganese phases.

Author Contributions: Conceptualization, M.K. and M.U.; methodology, M.U. and H.K.; software, M.K.; formal analysis, E.D., M.B. and G.K.; investigation, B.F. and M.H.; resources, Y.D. and P.M.; writing—original draft preparation, M.K. and B.F.; writing—review and editing, Q.Y., M.U., H.F. and R.I.; visualization, B.R.S.; supervision, M.U. and P.M.; funding acquisition, M.U. and G.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Scientific Grant Agency of the Ministry of Education, Science, Research and Sports of the Slovak Republic and the Slovak Academy of Sciences VEGA under contracts Nos. 1/0164/17, and 1/0146/18, and Cultural and Educational Agency of the Ministry of Education, Science and Sport of the Slovak Republic KEGA under contract No. 013SPU-4/2019. Conflicts of Interest: The authors declare no conflict of interest.

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