Fungal Bioweathering of Mimetite and a General GeomycologicalModel for Lead Apatite Mineral Biotransformations

Andrea Ceci,a,b Martin Kierans,c Stephen Hillier,d,e Anna Maria Persiani,b Geoffrey Michael Gadda,f

Geomicrobiology Group, College of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdoma; Laboratorio Biodiversità dei Funghi, Dipartimento diBiologia Ambientale, Sapienza Università di Roma, Rome, Italyb; Electron Microscopy, Central Imaging Facility, Centre for Advanced Scientific Technologies, College of LifeSciences, University of Dundee, Dundee, Scotland, United Kingdomc; The James Hutton Institute, Craigiebuckler, Aberdeen, Scotland, United Kingdomd; Department ofSoil and Environment, Swedish University of Agricultural Sciences, Uppsala, Swedene; Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute ofEcology and Geography, Chinese Academy of Sciences, Urumqi, People’s Republic of Chinaf

Fungi play important roles in biogeochemical processes such as organic matter decomposition, bioweathering of minerals androcks, and metal transformations and therefore influence elemental cycles for essential and potentially toxic elements,e.g., P, S, Pb, and As. Arsenic is a potentially toxic metalloid for most organisms and naturally occurs in trace quantities in soil,rocks, water, air, and living organisms. Among more than 300 arsenic minerals occurring in nature, mimetite [Pb5(AsO4)3Cl] isthe most stable lead arsenate and holds considerable promise in metal stabilization for in situ and ex situ sequestration andremediation through precipitation, as do other insoluble lead apatites, such as pyromorphite [Pb5(PO4)3Cl] and vanadinite[Pb5(VO4)3Cl]. Despite the insolubility of mimetite, the organic acid-producing soil fungus Aspergillus niger was able to solubi-lize mimetite with simultaneous precipitation of lead oxalate as a new mycogenic biomineral. Since fungal biotransformation ofboth pyromorphite and vanadinite has been previously documented, a new biogeochemical model for the biogenic transforma-tion of lead apatites (mimetite, pyromorphite, and vanadinite) by fungi is hypothesized in this study by application of geochemi-cal modeling together with experimental data. The models closely agreed with experimental data and provided accurate simula-tion of As and Pb complexation and biomineral formation dependent on, e.g., pH, cation-anion composition, and concentration.A general pattern for fungal biotransformation of lead apatite minerals is proposed, proving new understanding of ecologicalimplications of the biogeochemical cycling of component elements as well as industrial applications in metal stabilization, biore-mediation, and biorecovery.

Fungi actively contribute to many important geological pro-cesses (1–5). Biotransformations and the biogeochemical

cycling of elements, metal and mineral transformations, or-ganic matter decomposition, bioweathering, and soil and sed-iment formation are some of their most important geoactiveroles (1–6). Fungal involvement in biogeochemical cycling ofessential and potentially toxic elements (e.g., carbon, nitrogen,phosphorus, sulfur, metals, and metalloids) is clear and inter-linked with their abilities to adopt a variety of growth, meta-bolic, and morphological strategies (1–3, 6–12). Fungi are par-ticularly involved in metal biogeochemistry, with a variety ofprocesses determining mobility, and therefore bioavailability,and immobility, leading to formation of secondary mineralsand metal stabilization (1–7, 11–13).

Arsenic is an element belonging to group V-A and is a metal-loid possessing both metallic and nonmetallic properties. Arsenicis widely distributed in the Earth’s crust, with an average concen-tration of 2 to 5 mg kg�1, and is associated primarily with igneousand sedimentary rocks in the form of inorganic arsenic com-pounds (14–17). Arsenic therefore naturally occurs in trace quan-tities in the lithosphere, hydrosphere, and atmosphere and in allliving organisms, where it can be acutely toxic because of its sim-ilarity to inorganic phosphate and its affinity for protein thiols(14–16, 18, 19). Arsenate (AsO4

3�) is an analogue of essentialphosphate (PO4

3�) and can be taken up via phosphate transportsystems in most organisms, and it may replace phosphate in sev-eral energy transfer phosphorylation reactions (14–16). Arsenite(AsO3

3�) has a high affinity for protein thiol groups and cantherefore inactivate many enzymes (14–16). The spread and ubiq-

uity of As in the environment, its biological toxicity, and its en-hanced redistribution due to anthropogenic activities are causes ofmajor public concern (14–16, 18, 19). Anthropogenic arsenicsources include the use of agricultural pesticides, wood preserva-tives, and medicines, oil and coal burning for energy production,waste incineration and disposal, and industrial activities associ-ated with metal acquisition and processing from mineral ores(18, 19).

More than 300 arsenic minerals occur in nature, and of these,�60% occur as arsenates, 20% as sulfides and sulfosalts, 10% asoxides, and the rest as arsenites, arsenides, the native element, andmetal alloys (14, 16, 18–20). In primary arsenic-bearing minerals,arsenic is present as the anion arsenide (As3�) or diarsenide(As2

6�) or as sulfarsenide (AsS3�) in sulfidic ores in the form ofarsenides of Fe, Cu, Co, and Ni (15, 20). The most commonlyfound ores are arsenopyrite (FeAsS), enargite (Cu3AsS4), cobaltite

Received 4 March 2015 Accepted 11 May 2015

Accepted manuscript posted online 15 May 2015

Citation Ceci A, Kierans M, Hillier S, Persiani AM, Gadd GM. 2015. Fungalbioweathering of mimetite and a general geomycological model for lead apatitemineral biotransformations. Appl Environ Microbiol 81:4955–4964.doi:10.1128/AEM.00726-15.

Editor: D. Cullen

Address correspondence to Geoffrey Michael Gadd, g.m.gadd@dundee.ac.uk.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00726-15

August 2015 Volume 81 Number 15 aem.asm.org 4955Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

(CoAsS), niccolite (NiAs), orpiment (As2S3), and realgar (As4S4)(15, 16). The weathering of arsenic-containing rocks on exposureto the atmosphere and water results in conversion of the primaryminerals to secondary minerals, such as arsenic oxides, arsenite(AsO3

3�), and arsenate (AsO43�) minerals complexed with

mono-, di-, and trivalent cations (14, 19, 20). Secondary arseniteminerals are rare in natural environments, usually occurring asthe products of hydrothermal alterations under mildly reducingconditions (20). Conversely, secondary arsenate compoundscomprise a large class of minerals that have been found in manyoxidized environments, e.g., soils, mine tailings, and former in-dustrial sites, and among them, mimetite [Pb5(AsO4)3Cl] andschultenite (PbHAsO4) are probably the most common (20, 21).From the end of the 19th century, lead arsenates, e.g., schultenite,were employed for decades as insecticides in agriculture, increas-ing the concentration of arsenic in soils (20, 22, 23). Schultenite isa very rare mineral but is very stable in acidic solutions (20, 21, 24).At pH values above 5, mimetite is the most stable lead arsenatephase, and it is even formed in weakly saline solutions (20, 21,24, 25).

Mimetite is a very stable mineral at low dissolved Pb and Asconcentrations, and its stability field covers the pH range ofmost natural waters, even when they contain dissolved carbon-ate and sulfate (20, 24). Mimetite belongs to the apatite familyof minerals, which is described by the general formula{[A(1)4][A(2)6][(BO4)6X2]}, with complete or partial filling of theA site by a cationic species of Na, Mg, Sr, Ba, Pb, Ca, La, or Ce, theB site by an anionic species of V, P, As, S, Si, Ge, Cr, or B, andthe X site commonly by a halide such as Cl, F, I, Br, or the hydroxylion (26–28). Calcium apatite [Ca10(PO4)6(F,Cl,OH)2], pyromor-phite [Pb10(PO4)6Cl2], mimetite [Pb10(AsO4)6Cl2], and vana-dinite [Pb10(VO4)6Cl2] are the most common apatite prototypesin nature, being thermodynamically stable and relatively insolubleover a wide variety of environmental conditions (26). The apatitegroup of minerals hold considerable promise for metal stabiliza-tion, e.g., by P addition in soil to stabilize Pb by pyromorphiteformation, and also in the recycling of industrial and nuclearwaste and remedial treatments of lead-contaminated soil and wa-ter (28–37). Although mimetite has been shown to reduce theaqueous arsenic concentration (25, 38), it has been suggested thatlow-molecular-weight organic acids (LMWOA) in the rhizo-sphere can potentially liberate Pb and As(V) from mimetitethrough ligand-promoted organic complexation (21).

The aim of this research was to evaluate the potential of amodel fungus, Aspergillus niger, to transform natural samples ofmimetite in order to better understand the stability of lead apatitesin the environment and to shed further light on the relationshipsof fungal species to arsenic geomicrobiology and mineralogy.Moreover, a general model of biotransformation of lead apatiteminerals is hypothesized, supported by geochemical modeling(39–43), providing new understanding of ecological implicationsfor the biogeochemical cycling of component elements, as well asindustrial applications in metal stabilization, bioremediation, andbiorecovery.

MATERIALS AND METHODSOrganism, media, and growth conditions. The organism used for thebioleaching tests was Aspergillus niger van Tieghem (ATCC 201373), fromthe culture collection of the Geomicrobiology Group (University ofDundee). A. niger was chosen because it has been shown to have signifi-

cant geoactive properties in mineral bioweathering of the lead apatitespyromorphite and vanadinite (1, 2, 39, 40, 44–53). Moreover, this fungusis a common soil saprotroph and can produce substantial amounts oforganic acids, e.g., oxalic, citric, and gluconic acids (51, 54, 55). A. nigerwas routinely maintained on �25 cm3 malt extract agar (MEA) (Lab MLtd., Bury, Lancashire, United Kingdom) in 90-mm-diameter petri dishesand grown at 25°C in the dark. Tests were carried out at least in triplicateat 25°C in the dark using Czapek-Dox agar medium containing the fol-lowing (g liter�1 Milli-Q water): glucose, 30; NaNO3, 3; Na2HPO4, 1;MgSO4·7H2O, 0.5; KCl, 0.5; FeSO4·7H2O, 0.01, and agar no. 1 (LabM,Bury, United Kingdom), 15. Prior to autoclaving, the medium was ad-justed to pH 5.5 using concentrated HCl. A. niger was grown for 7 days onCzapek-Dox agar at 25°C in the dark prior to subculture for experiments.Inoculation of experimental media was achieved using 5-mm-diameterdisks of mycelium cut from actively growing margins of colonies using asterile cork borer. A. niger was grown on 25 cm3 of Czapek-Dox agaramended with the addition of mimetite to a 0.2% (wt/vol) final concen-tration in 90-mm-diameter petri dishes.

Preparation of natural mimetite-amended plates and inoculation. Apure sample of yellow natural mimetite [Pb5(AsO4)Cl] originated fromOjuela mine, Mapimi, Durango, Mexico. It was ground to a powder, usinga pestle and mortar (Milton Brook, Dorset, United Kingdom), which wassifted through two meshes (150- and 100-�m mesh). The portion withmesh size between 100 and 150 �m was used in all the experiments. Afterbeing oven dried at 105°C for 48 h, the powdered natural mimetite wasadded to autoclaved Czapek-Dox medium to a final concentration of0.2% (wt/vol) at a temperature of around 50°C prior to pouring. Prior toinoculation, 84-mm-diameter disks of sterile cellophane membranes (Fo-cus Packaging and Design Ltd., Louth, United Kingdom) were placedaseptically on the surface of the agar in each petri dish. The cellophanemembranes separate the fungal biomass species from the medium butallow the passage of nutrients or metabolites between the agar and thecolony, and they provide a convenient means of removing the mycelium(46). Growth of A. niger was evaluated by diametric extension of colonygrowth, as well as by biomass measurement (56). After 12 days growth,fungal biomass was removed from the cellophane membranes and ovendried at 105°C until maintaining a constant weight for at least 48 h. Resultswere expressed in terms of a tolerance index (TI) based on the dry weights(DW) of fungal biomass as follows: TIDW � (DW of treated myceli-um/DW of control mycelium) � 100 (57). After removal of both mem-brane and mycelium, the surface pH of the agar was measured at specificintervals across the diameter of the petri dish using an Orion 3 Star bench-top pH meter (Thermo Fisher Scientific Inc., Loughborough, UnitedKingdom) fitted with a flat-tip electrode (VWR International, Lutter-worth, England, United Kingdom). Statistical analysis was performed us-ing SigmaPlot 12 (Systat Software). One-way analysis of variance(ANOVA) tests on means were performed for data of dry weight anddiametric growth, and at least three replicate determinations were used inexperiments.

Purification of crystals produced by Aspergillus niger in the pres-ence of mimetite. Crystals were formed in the agar under fungal colonieswhen A. niger was grown on Czapek-Dox medium amended with naturalmimetite. Crystals were extracted from the medium by gently homoge-nizing the agar with Milli-Q water at 80°C in a crystallizing dish. Crystalswere dried at least overnight in a vacuum desiccator prior to examinationby scanning electron microscopy (SEM), energy dispersive X-ray analysis(EDXA), and X-ray diffraction (XRD).

XRF. The elemental composition of natural mimetite was determinedby X-ray fluorescence spectroscopy (XRF) using a Philips PW2424 se-quential spectrometer with an RhK� source, calibrated with certifiedstandard materials. The samples to be tested were placed in a 32-mm-diameter pellet mold and compacted under loads of 75 kN for 5 min and150 kN for a further 10 min prior to analysis. The specimen cups had a27-mm-diameter viewing aperture. The results are expressed as oxides.

Ceci et al.

4956 aem.asm.org August 2015 Volume 81 Number 15Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

SEM and EDXA. Samples were examined using light microscopy andscanning electron microscopy (SEM). They were mounted using carbonadhesive tape and allowed to dry in a desiccator at room temperature forat least 48 h. Energy-dispersive X-ray analysis (EDXA) was carried outbefore coating the samples for SEM in order to exclude the Au/Pd peak,which can overlap Pb/Cl peaks. A Phoenix EDXA analysis system embed-ded within a Philips XL30 ESEM, operating at an accelerating voltage of 20kV, was used to acquire elemental spectra. Specimens were coated with30-nm Au/Pd by using a sputter coater (Cressington 208HR; CressingtonScientific Instruments Ltd., Watford, United Kingdom) and then exam-ined using a Philips XL30 scanning electron microscope at an acceleratingvoltage of 15 kV.

XRD. X-ray diffraction (XRD) was used to identify the secondaryminerals and purity of the natural mimetite samples. Specimens werecompacted in the 15- by 20-mm window of a 2-mm-thick aluminum slide(or, in the case of small quantities, sprinkled onto a glass platelet posi-tioned in the window), and diffraction patterns were recorded from 3 to60° 2� using Ni-filtered Cu K-alpha radiation, counting for 3 s per 0.01°step, using an HB-62 diffractometer made by Hiltonbrooks Ltd. Mineralphases were identified by reference to patterns in the International Centrefor Diffraction Data (ICDD) Powder Diffraction File (PDF). The stan-dards used were mimetite (PDF-19-683) and lead oxalate (PDF-14-803).

Geochemical modeling. In order to inform a geochemical model forbiotransformation of mimetite and the other lead apatites, including va-nadinite and pyromorphite, the computer programs for geochemicalmodeling PHREEQC, version 3 (58), and the Geochemist’s Workbench(GWB), version 10.0.3 (Rockware Inc., Golden, CO), were used, respec-tively, to study speciation and chemical equilibria of simulated solubiliza-tion reactions between lead apatites and oxalic acid and to calculate min-eral stability diagrams for lead apatites and Ca hydroxyapatite.Solubilization reactions by PHREEQC consisted of simulations in which 1mM mimetite/vanadinite/pyromorphite in pure water at 25°C was re-acted with additions of 50 mM oxalic acid in 25 steps. These simulationsprovided calculated values of concentrations for forming chemical speciesin solution and remaining mineral species at equilibrium. Stability dia-grams for lead and calcium apatites were calculated as a function of pHand log of oxalate activities. The available Minteq v4 database in PHREEQCwas integrated with critically selected stability constants of complexesbetween metals and organic acids (oxalic, citric, and gluconic acids) fromthe database of the National Institute of Standards and Technology(NIST), version 8 (59), and available thermodynamic data in the literature(60), while the available thermo_minteq database was used with GWB.With regard to differences in PHREEQC and GWB, stability diagramswere also generated by PHREEQC using the graphical supporting soft-ware PHREEPLOT (61). Thermodynamic data for mimetite (25), vana-dinite (62), and pyromorphite (63, 64) were used for the thermodynamiccalculations. Moreover, thebioleachingeffectofoxalicacidondifferentAsmin-erals/compounds {orpiment (As2S3), realgar (AsS), arsenolite (As4O6), claudetite(As4O6), As2O5, AsI3, AlAsO4·2H2O, Zn3(AsO4)2·2.5H2O, Cu3(AsO4)2·2H2O,Ag3AsO3, Ag3AsO4, Ni3(AsO4)2·8H2O, Co3(AsO4)2, FeAsO4·2H2O, Mn3

(AsO4)2·8H2O,Ca3(AsO4)2·4H2O,Ba3(AsO4)2, scorodite(FeAsO4·2H2O),sym-plesite [Fe3(AsO4)2·8H2O], bukovskýite [Fe2(AsO4)(SO4)(OH)·7H2O], haid-ingerite [Ca(AsO3OH)·H2O], pharmacolite [Ca(HAsO4)·2H2O], weilite (Ca-HAsO4), adamite [Zn2(AsO4)(OH)], annabergite [Ni3(AsO4)2·8H2O], austinite[CaZn(AsO4)(OH)], clinoclase [Cu3(AsO4)(OH)3], conichalcite [CaCu(AsO4)(OH)], cornubite [Cu5(AsO4)2(OH)4], erythrite [Co3(AsO4)2·8H2O],euchroite [Cu2(AsO4)(OH)·3H2O], köttigite [Zn3(AsO4)2·8H2O], legrandite[Zn2(AsO4)(OH)·H2O], mansfieldite (AlAsO4·2H2O), olivenite [Cu2(AsO4)(OH)], and sterlinghillite [Mn3(AsO4)2·4H2O]} was simulated by PHREEQCusing the available Minteq v4 database in PHREEQC and available thermody-namic data in the literature (20, 65). Among these, the natural lead arsenates{beudantite [PbFe3(AsO4)(SO4)(OH)6], bayldonite [PbCu3(AsO4)2

(OH)2], duftite [PbCu(AsO4)(OH)], fornacite [Pb2Cu(AsO4)(CrO4)(OH)], and schultenite [Pb(AsO3OH)]} and synthetic species[PbHAsO4·2H2O, Pb5(AsO4)3OH·H2O, Pb3(AsO4)2, and Pb8As2O13]

were of special relevance. As oxalic acid was considered to be the mainbioweathering agent of mimetite and lead apatites, its chemical reactivityon all relevant As minerals/compounds could be tested by PHREEQC. Inparticular, the stability of As minerals/compounds at 25°C (pure water,initial pH 5.5, 1 atm) was evaluated in simple reaction simulations of 1mM concentrations of each of them with 25 mM oxalic acid, which was avalue chosen taking account of total oxalic acid found to be excreted by A.niger after 12 days in preliminary tests on organic acid production (resultsnot shown).

RESULTSGrowth on and solubilization of natural mimetite by Aspergillusniger. The representative elemental composition of the naturalmimetite mineral used in this study was determined by X-ray flu-orescence (XRF). This confirmed the dominance of lead, arsenic,and chlorine in the samples and minor proportions of other met-als, e.g., Al, Mg, Ca, Rb, Zr, Ag, as well as P, probably related toother associated components and impurities (Table 1). X-ray dif-fraction (XRD) showed that mimetite was the main mineral com-ponent, while no other associated minerals were detected, pre-sumably because of detection limits (Fig. 1G). Under controlconditions, mimetite was stable in the agar medium, and scanningelectron microscopy (SEM) revealed a typical hexagonal crystalhabit with smooth and regular surfaces (Fig. 1A and B). Energy-dispersive X-ray microanalysis (EDXA) of the crystals showedlead, arsenic, and chlorine peaks (Fig. 1E). New crystal formationwas observed after approximately 3 to 5 days growth of A. niger at25°C. The new crystals were white, acicular, slender, and tapered,with needle- and platy-type habits (Fig. 1C and D). The purifiedcrystals showed an XRD pattern for pure lead oxalate (Fig. 1H).EDXA revealed an elemental spectrum completely different fromthat of control mimetite (Fig. 1E), one in which peaks of arsenicand chlorine completely disappeared and only those of lead, car-bon, and oxygen were present (Fig. 1F).

Growth and toxicity assessment of A. niger and surface pHanalysis. Incorporation of 0.2% (wt/vol) natural mimetite in Cza-pek-Dox medium had no visible effect on the growth of Aspergillusniger at 25°C in the dark. A. niger was able to grow in the presenceof the natural mimetite, and mycelium development and sporu-

TABLE 1 Mineral ore composition of mimetite determined using X-rayfluorescence

Component % by massa

PbO 67.22As2O3 17.87Al2O3 6.40Cl 2.14MgO 1.04CaO 0.49Rb2O 0.20ZrO2 0.19Ag2O 0.11P2O5 0.08CdO 0.07CuO 0.07ZnO 0.06MnO 0.05NiO 0.04SiO2 0.04Total 96.07a Data shown are average values from two determinations.

Fungal Biotransformation of Mimetite

August 2015 Volume 81 Number 15 aem.asm.org 4957Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

Ceci et al.

4958 aem.asm.org August 2015 Volume 81 Number 15Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

lation appeared normal, although a yellow pigmentation was ob-served underneath colonies during the experiments. Colony ex-pansion rates were measured for 12 days during fungal growth; thecontrol growth rate of A. niger was 9.84 0.75 mm day�1 over 7days incubation at 25°C (average of three replicates standarderror of the mean). There was almost no effect on colony exten-sion with and without mimetite (data not shown). Kruskal-Wallisone-way analysis of variance on ranks and all pairwise multiple-comparison procedures with Dunn’s method revealed no statisti-cally significant difference between fungal extension in the pres-ence of mimetite and the control. Tolerance indices (TI) were usedto compare the biomass yields of control and test conditions; thebiomass yield was not strongly reduced (TI � 97%) by the pres-ence of mimetite, and there was no statistically significant differ-ence between dry weight values from control and test conditions.Table 2 shows the pH values of Czapek-Dox medium, both priorto fungal growth (abiotic) and after growth of A. niger. The pH ofthe medium decreased after fungal growth, with medium valuesall being acidic (pH 3.5).

Geochemical model. PHREEQC is software used to simulatemultiple geochemical equilibria in solution (complexes, minerals,solid solutions, gases, ion exchange, and surface complexes) (58,66). PHREEQC was used in this work to estimate the bioleachingeffect of oxalic acid on mimetite, other lead apatites (vanadiniteand mimetite), and other As minerals/compounds. The simula-tions of the chemical dissolution of mimetite and the other leadapatites by gradual oxalic acid addition showed increasing solubi-lization of each lead apatite and competition between severalchemical equilibria for the simultaneous formation of differentcomplexes or the precipitation of lead oxalate or other minerals.Moreover, the predominance of some chemical equilibria was ob-served. For instance, by calculating chemical reactions betweenmimetite and increasing oxalic acid additions to the system, it wasseen that the concentration of some oxalato complexes, e.g.,[Pb(C2O4)] and [Pb(HC2O4)]�, increased progressively at the ex-pense of the lead oxalate concentration, and this could dependmainly on the ligands present, ion activities, equilibrium con-stants, temperature, pH, and Eh. Furthermore, from the simula-tions we can see that the stabilities of the lead apatites to oxalic acidare different. Pyromorphite was the most stable of all the leadapatites in the presence of oxalic acid leaching, while vanadinite

was the most sensitive. Despite these differences, the stability dia-grams for lead apatites generated by the Geochemist’s Workbench(GWB) showed similar predominance ranges of pH and oxalicacid activity (Fig. 2A to C). The geochemical conditions underwhich lead oxalate are stable is at a minimum oxalate activity of�10�4.15 over pH values of 4.5 with respect to other lead min-erals (Fig. 2A to C). In contrast, for hydroxyapatite, the formationof whewellite was observed over wider geochemical conditions(minimum oxalate activity of �10�5.75 over pH values of �7.5)(Fig. 2D). It is worth mentioning that despite different thermody-namic databases being used in PHREEQC and GWB and theirdifferent simulation approaches, no topologically significant dif-ferences were observed in the stability diagrams that were gener-ated by PHREEQC and GWB. From the experimental results andsimulations, a general pattern of bioleaching of lead apatites isshown in Fig. 3. From the simulations on Pb mineral/compoundstability in reaction with 25 mM oxalic acid, oxalic acid was able todissolve, in different ways, all the tested As compounds, and thepH dropped to �1.8. It is worth mentioning that the majority ofthe As minerals/compounds were totally dissolved, with the ex-ceptions of orpiment, realgar, Ba3(AsO4)2, mimetite, schultenite,symplesite, fornacite, köttigite, Pb5(AsO4)3OH·H2O, andPb8As2O13. For each of these, the difference between the initial 1mM concentration and the final simulated concentration was cal-culated. The differences, in mM, were 3.9 � 10�9, 2.2 � 10�7, 7.8 �10�3, 2.7 � 10�4, 8.6 � 10�4, 3.3 � 10�8, 4.3 � 10�8, 2.6 � 10�8,5.7 � 10�4, and 2.3 � 10�19, respectively. Moreover, from thebioleaching simulations it was also possible to observe the simul-taneous formation of metal complexes and metal oxalates accord-ing to the specific composition of the As mineral/compounds. Forinstance, for all Pb arsenates, the formation of lead oxalate com-plexes and lead oxalate (PbC2O4) was observed, as well as forma-tion of whewellite (CaC2O4·H2O), weddellite (CaC2O4·2H2O),caoxite (CaC2O4·3H2O), and the calcium oxalate complex[CaC2O4] when Ca occurred in the As mineral/compounds, suchas haidingerite [Ca(AsO3OH)·H2O], pharmacolite [Ca(HAsO4)·2H2O], and weilite (CaHAsO4).

DISCUSSION

This work examined the stability of natural mimetite[Pb5(AsO4)3Cl] in the presence of the ubiquitous soil fungusAspergillus niger, which possesses well-known geoactive properties(1, 2, 39, 40, 44–53), including citric, gluconic, and oxalic acidproduction (51, 54, 55). Experiments clearly revealed the com-plete bioweathering of a pure natural mimetite by the fungus andthe simultaneous bioprecipitation of pure lead oxalate[Pb(C2O4)] as a new mycogenic biomineral (Fig. 1; Table 1). Thebioleaching of other lead apatites, such as pyromorphite and va-nadinite, by A. niger and other organic acid-producing fungi withthe simultaneous bioprecipitation of lead oxalate has been de-scribed in the literature (39, 44, 45). This suggests the existence ofa common pattern in fungal bioleaching of the main lead apatites

FIG 1 Scanning electron microscopy (SEM), energy-dispersive X-ray microanalysis (EDXA), and X-ray diffraction (XRD) patterns of mimetite and lead oxalate.(A and B) Control crystals of mimetite. Scale bars, 20 �m. (C and D) Lead oxalate crystals formed by Aspergillus niger in medium amended with 0.2% (wt/vol)natural mimetite after growth for 12 days at 25°C. Scale bars, 20 �m. (E) Typical EDXA spectrum of natural mimetite. (F) Typical EDXA spectrum of lead oxalate.(G) XRD pattern of mimetite. (H) XRD pattern of lead oxalate. Typical images/spectra/patterns are shown from one of several determinations, all of which gavesimilar results.

TABLE 2 Surface pH values of uninoculated agar and agar underneathfungal colonies growing on Czapek-Dox medium

Medium

Surface pH valuea of:

Uninoculated agar Agar with A. niger

Czapek-Dox 5.64 0.03 2.47 0.25Czapek-Dox � 0.2% (wt/vol)

mimetite6.27 0.02 3.49 0.24

a The surface pH values shown are averages from more than six measurements acrossthe agar plates, with relative standard deviations, after growth for 12 days at 25°C in thedark.

Fungal Biotransformation of Mimetite

August 2015 Volume 81 Number 15 aem.asm.org 4959Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

(Fig. 3), which has been supported here with the geochemicalsimulations.

Fungi, as well as other microbes (e.g., bacteria, algae, and pro-tists), can mediate chemical weathering of rocks and mineralsthrough several mechanisms, including acidolysis, complexolysis,redoxolysis, and metal accumulation in the biomass (1, 2, 39, 45,50, 56, 67–70). The excretion of organic acids (e.g., oxalic, citric,gluconic, and lactic acids) can be strongly influenced by growthconditions, such as the presence of toxic metal minerals, pH, buff-ering capacity, nutrient availability, and the C, P, S, and N sources(1, 2, 39, 45, 51, 52, 71–74). Organic acids can provide a source ofprotons and contribute to acidification, which can also result fromthe proton-translocating plasma membrane ATPase, nutrient up-take in exchange for H�, and respiration if carbonic acid is formed(39, 67). More importantly, organic acids provide a source of li-gands which can form complexes with metals and enhance theefficiency and rate of mineral dissolution (39, 44, 45). Further-more, organic acids can be a source of electrons which can reducemetals to lower oxidation states. New chemical equilibria and newcomplexes can subsequently be formed, which can considerably

increase such chemoorganotrophic bioleaching (1, 2, 39, 45). Allthese processes, along with metal accumulation in the fungal bio-mass, play important roles in the dissolution of lead apatites andthe formation of new biominerals. Mobilization and immobiliza-tion are the most important outcomes resulting from these inter-actions. From PHREEQC simulations of lead apatite bioleachingit is possible to identify the different groups of ionic species whichcan form in solution: (i) free ions, which arise mainly from apatitebioleaching, such as metal cations (e.g., Pb2�, VO2

�, and VO2�),anions (e.g., PO4

3�, HPO42�, H2PO4

�, VO43�, HVO4

2�,H2VO4

�, AsO43�, HAsO4

2�, H2AsO4�, HC2O4

�, and C2O42�),

halogens (e.g., Cl�, Br�, and I�), and (OH�); (ii) complexes be-tween anionic species of LMWOA and metal cations from the Asite of apatite {e.g., Pb(C2O4), [Pb(HC2O4)]�, [VO2(C2O4)]�,[VO2(C2O4)2]3�, VO(C2O4), and [VO(C2O4)2]2�}; (iii) com-plexes between halogen anions and OH�, originating from the Xsite of apatites and metal cations {e.g., (PbCl)�, PbCl, (PbCl2)�,and [Pb(OH)]�); and (iv) complexes between inorganic anions ofP, As, V, originating mainly from the B site of apatite or anions in

FIG 2 Mineral stability diagrams of pH versus log of oxalate activity for lead apatites and calcium hydroxyapatite calculated using Geochemist’s Workbench(GWB). Activities for Pb2�, Ca2�, SO4

2�, CO32�, and Cl� were assumed to be 10�3 M, that for PO4

3� was assumed to be 10�9 M, and those for AsO43� and

HVO42� were assumed to be 10�7 M. (A) Stability field of mimetite in the system Pb, As, S, Cl, C, H, and O. (B) Stability field of vanadinite in the system Pb, V,

S, Cl, C, H, and O. (C) Stability field of pyromorphite in the system Pb, P, S, Cl, C, H, and O. (D) Stability field of lead oxalate in the system Ca, P, S, Cl, C, H,and O.

Ceci et al.

4960 aem.asm.org August 2015 Volume 81 Number 15Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

FIG 3 Fungal bioleaching model for lead apatite minerals. Fungi can mediate bioweathering of lead apatites through the excretion of organic acids (e.g., oxalic,citric, gluconic, and lactic acids), which provide a source of protons (acidolysis), ligands (complexolysis), and electrons (redoxolysis). Ligands, protons, andmetals are available from the environmental pool which can originate from microbial communities in the soil rhizosphere and abiotic geochemical processes.Mobilization results mainly from the formation of several chemical complexes and free ions, and immobilization results from (i) metal biosorption to structuralcomponents with nucleation and crystal growth of new minerals, (ii) metal bioaccumulation inside mycelia by transport, intracellular deposition, and seques-tration, and (iii) metabolite release by precipitation of, e.g., oxalates, carbonates, and phosphates. Secondary reprecipitation of lead minerals is possible ifLMWOA concentrations decrease or the ionic concentrations of inorganic anions and metals increase.

Fungal Biotransformation of Mimetite

August 2015 Volume 81 Number 15 aem.asm.org 4961Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

the environmental pool, such as hydroxide, sulfate, carbonate,and metal cations {e.g., [Pb(CO3)2]2�, and [Pb(H2PO4)]�}.

Complexation is therefore important, and ligand-promoteddissolution appears to be fundamental to the bioleaching of leadapatites. All fungal LMWOA may contribute to this process, al-though oxalic acid appears to be highly important because of itsstrong chelating activity as well as its relatively high first acid dis-sociation constant (pKa1 � �1.25 [59]) despite being a carboxylicacid. In fact, the medium pH noticeably decreases after inocula-tion, and all pH values underneath fungal colonies were acidic(Table 2). In this study, the PHREEQC simulations of chemicaldissolution of mimetite and the other lead apatites by oxalic acidshowed competition between several chemical equilibria for theformation of the different complexes or the precipitation of leadoxalate or other biominerals. Moreover, the predominance ofsome chemical equilibria was observed, and this could dependmainly on the ligands present, ion activities, equilibrium con-stants, temperature, pH, and Eh. Furthermore, from the simula-tions we could observe that the stabilities of the lead apatitesagainst oxalic acid attack were different. Pyromorphite was themost stable of all the lead apatites to oxalic acid leaching, whilevanadinite was the most sensitive, even though the vanadinitesolubility product (log Ksp � �86.1, S � 7.69 � 10�11 mol liter�1)is higher than that of pyromorphite (log Ksp � �84.4, S � 1.19 �10�10 mol liter�1) (39). Although mimetite (log Ksp � �76.35,S � 9.31 � 10�10 mol liter�1) (25) is more soluble than vana-dinite, it is more stable than vanadinite to oxalic acid attack be-cause arsenic is less sensitive to complexolysis, redoxolysis, andacidolysis than vanadium.

Metal immobilization is another important property of fungi,and this can occur through (i) biosorption to structural compo-nents of cell walls with accompanying nucleation and crystalgrowth of new minerals, (ii) metal bioaccumulation by transport,intracellular deposition, and sequestration, and (iii) metaboliterelease with precipitation of, e.g., oxalates, carbonates, phos-phates, and other biominerals (1, 2, 4, 5, 12, 40, 49–51, 63, 75–78)(Fig. 3). Insoluble lead oxalate was the predominant biomineralproduct found here after bioleaching of lead apatites in anhydrousor hydrated forms (39, 44, 45). Lead oxalate is highly insoluble(log Ksp � �8.02) (59), but it is unlikely that lead oxalate would bethermodynamically stable in many soils. According to the stabilitydiagrams generated by GWB, the geochemical conditions underwhich lead oxalate is stable are at a minimum oxalate activity of�10�4.15 over pH values of 4.5 with respect to other lead min-erals. Oxalate is common in many soils, and concentrations mayrange from 25 to 1,000 �M (44, 50). However, average oxalic acidconcentrations in soils are generally 1 mM, e.g., 10�3 mM inforest soil, but high concentrations may occur in local microenvi-ronments (50). This environmental range for oxalate activity istoo low for lead oxalate to be stable. Lead oxalate is most likely tobe stable in acidic environments with low sulfur activities thatsuppress the formation of anglesite, in the proximity of fungalhyphae, or in the rhizosphere, where oxalate concentrations maybe high (1, 2, 44, 50). It is important to note that the conversion oflead oxalate back to lead apatites (pyromorphite, mimetite, andchervetite/vanadinite) is possible over a wide pH range, encom-passing most of the normal pH range for soils even at low concen-trations of phosphorus (10�9 mM), arsenic (10�7 mM), and va-nadium (10�7 mM). The reverse conversion to pyromorphite canbe achieved in the presence of dilute solutions of phosphate and

chloride (44). This may occur for mimetite in the presence ofdilute solutions of As and Cl and for vanadinite with alkaline Vand Cl solutions. In the latter case, the formation of Pb2V2O7 orchervetite is also possible. After bioleaching of lead apatites andthe migration of lead complexes in soils, secondary reprecipita-tion of lead minerals (including lead apatites) in soil may be pos-sible, especially if LMWOA concentrations decrease and those ofother anions, such as phosphates, carbonates, and sulfates, in-crease (Fig. 3). It is worth mentioning that in the presence ofarsenic compounds, several fungal species can mediate the pro-duction of volatile methylated arsenic compounds (2, 79).

The transformation of lead apatites by fungi may have impor-tant consequences. As mentioned above, fungi can play importantroles in the biogeochemical cycling of metals through immobili-zation and/or mobilization. The solubilization of apatites by fungiis of importance when considering the application of this mineralgroup for the stabilization of potentially toxic metals and radio-nuclides, e.g., Pb and U. Furthermore, this pattern of fungal bi-oleaching of lead apatites can be extended to other metal apatites,such as calcium apatite [Ca5(PO4)3(OH,F,Cl)]. In this case, theformation of monohydrated (whewellite) and dihydrated (wedd-ellite) calcium oxalate can be mediated by many different fungalspecies (50, 80–83). This transformation is also confirmed by oursimulations with GWB, even if the whewellite seems to be morestable than lead oxalate, with an oxalate activity of �10�5.75 overpH values of �7 (Fig. 2D).

The PHREEQC simulations also shed light on the possible sta-bility of the other As minerals/compounds in response to the bi-oleaching effect of oxalic acid. The majority of the tested sub-strates were completely dissolved by addition of 25 mM oxalicacid, and the formation of different metal complexes and oxalateminerals was observed, depending on the chemical compositionof the specific As mineral/compound examined. Even if these sim-ulations have simple assumptions without the interaction of otherLMWOA and are based only on equilibrium thermodynamicswithout considering the possible kinetics involved, it is possiblethat similar bioleaching patterns observed in the simulationscould also occur in the environment and under experimental con-ditions. In all the simulations, the same mechanisms of mobiliza-tion and immobilization were obvious, including the acidolysis,complexolysis, and redoxolysis phenomena described previouslyfor mimetite and lead apatites.

In conclusion, this study on the fungal bioleaching of mimetitehighlights the potential significance of fungi in arsenic geochem-istry and mineralogy. Lead apatite bioleaching by fungi can have aprofound ecological importance for arsenic cycling in ecosystemsand also poses some questions about the bioremedial feasibility ofusing the formation of lead apatites in metal stabilization. It alsocould inform alternative approaches for metal biorecovery fromabandoned mines, mineral ores, and mineral wastes. Fungi couldbe used as efficient biominers, tolerating high concentrations ofpotentially toxic elements.

ACKNOWLEDGMENTS

We gratefully acknowledge Laszlo Csetenyi (Concrete Technology Group,Department of Civil Engineering, University of Dundee, United King-dom) for assistance with XRD and Antonio Gianfagna (Dipartimento diScienze della Terra, Sapienza Università di Roma, Italy) for support in themineralogy of lead apatites. We also thank David Parkhurst (USGS, Den-ver, CO, USA) and David Kinniburgh (BGS, Wallingford, United King-

Ceci et al.

4962 aem.asm.org August 2015 Volume 81 Number 15Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

dom) for support in the thermodynamic calculations and graphical dis-play using PHREEQC and PHREEPLOT, respectively.

G. M. Gadd gratefully acknowledges an award under the 1000 TalentsPlan with the Xinjiang Institute of Ecology and Geography, Chinese Acad-emy of Sciences, Urumqi, China.

REFERENCES1. Gadd GM. 2007. Geomycology: biogeochemical transformations of

rocks, minerals, metals and radionuclides by fungi, bioweathering andbioremediation. Mycol Res 111:3– 49. http://dx.doi.org/10.1016/j.mycres.2006.12.001.

2. Gadd GM. 2010. Metals, minerals and microbes: geomicrobiology andbioremediation. Microbiology 156:609 – 643. http://dx.doi.org/10.1099/mic.0.037143-0.

3. Gadd GM. 2011. Geomycology, p 416 – 432. In Reitner J, Thiel V (ed),Encyclopedia of geobiology. Springer, Heidelberg, Germany.

4. Burford EP, Fomina M, Gadd GM. 2003. Fungal involvement in bio-weathering and biotransformation of rocks and minerals. Mineral Mag67:1127–1155. http://dx.doi.org/10.1180/0026461036760154.

5. Burford EP, Kierans M, Gadd GM. 2003. Geomycology: fungi in mineralsubstrata. Mycologist 17:98 –107. http://dx.doi.org/10.1017/S0269915X03003112.

6. Finlay R, Wallander H, Smits M, Holmstrom S, van Hees P, Lian B,Rosling A. 2009. The role of fungi in biogenic weathering in boreal forestsoils. Geomycology 23:101–106.

7. Gadd GM. 2004. Microbial influence on metal mobility and applicationfor bioremediation. Geoderma 122:109 –119. http://dx.doi.org/10.1016/j.geoderma.2004.01.002.

8. Rosling A, Lindahl BD, Taylor AFS, Finlay RD. 2004. Mycelial growthand substrate acidification of ectomycorrhizal fungi in response to differ-ent minerals. FEMS Microbiol Ecol 47:31–37. http://dx.doi.org/10.1016/S0168-6496(03)00222-8.

9. Rosling A, Roose T, Herrmann AM, Davidson FA, Finlay RD, GaddGM. 2009. Approaches to modelling mineral weathering by fungi. FungalBiol Rev 23:138 –144. http://dx.doi.org/10.1016/j.fbr.2009.09.003.

10. Adeyemi A, Gadd G. 2005. Fungal degradation of calcium-, lead- andsilicon-bearing minerals. Biometals 18:269 –281. http://dx.doi.org/10.1007/s10534-005-1539-2.

11. Fomina M, Burford EP, Hillier S, Kierans M, Gadd GM. 2010. Rock-building fungi. Geomicrobiol J 27:624 – 629. http://dx.doi.org/10.1080/01490451003702974.

12. Gadd GM, Raven JA. 2010. Geomicrobiology of eukaryotic microorgan-isms. Geomicrobiol J 27:491–519. http://dx.doi.org/10.1080/01490451003703006.

13. Gadd GM, Rhee YJ, Stephenson K, Wei Z. 2012. Geomycology: metals,actinides and biominerals. Environ Microbiol Rep 4:270 –296. http://dx.doi.org/10.1111/j.1758-2229.2011.00283.x.

14. Dhuldhaj U, Yadav I, Singh S, Sharma N. 2013. Microbial interactionsin the arsenic cycle: adoptive strategies and applications in environmentalmanagement. Rev Environ Contam Toxicol 224:1–38. http://dx.doi.org/10.1007/978-1-4614-5882-1_1.

15. Tamaki S, Frankenberger WT Jr. 1992. Environmental biochemistry ofarsenic. Rev Environ Contam Toxicol 124:79 –110.

16. Akter K, Owens G, Davey D, Naidu R. 2005. Arsenic speciation andtoxicity in biological systems. Rev Environ Contam Toxicol 184:97–149.

17. Mazziotti-Tagliani S, Angelone M, Armiento G, Pacifico R, CremisiniC, Gianfagna A. 2012. Arsenic and fluorine in the Etnean volcanics fromBiancavilla, Sicily, Italy: environmental implications. Environ Earth Sci66:561–572. http://dx.doi.org/10.1007/s12665-011-1265-8.

18. Moreno-Jiménez E, Esteban E, Peñalosa J. 2012. The fate of arsenic insoil-plant systems. Rev Environ Contam Toxicol 215:1–37. http://dx.doi.org/10.1007/978-1-4614-1463-6_1.

19. Garelick H, Jones H, Dybowska A, Valsami-Jones E. 2008. Arsenicpollution sources. Rev Environ Contam Toxicol 197:17– 60.

20. Drahota P, Filippi M. 2009. Secondary arsenic minerals in the environ-ment: a review. Environ Int 35:1243–1255. http://dx.doi.org/10.1016/j.envint.2009.07.004.

21. Bajda T. 2011. Dissolution of mimetite Pb5(AsO4)3Cl in low-molecular-weight organic acids and EDTA. Chemosphere 83:1493–1501. http://dx.doi.org/10.1016/j.chemosphere.2011.01.056.

22. Cancès B, Juillot F, Morin G, Laperche V, Alvarez L, Proux O, Haze-mann J-L, Brown GE, Calas G. 2005. XAS evidence of As(V) association

with iron oxyhydroxides in a contaminated soil at a former arsenical pes-ticide processing plant. Environ Sci Technol 39:9398 –9405. http://dx.doi.org/10.1021/es050920n.

23. Arai Y, Lanzirotti A, Sutton SR, Newville M, Dyer J, Sparks DL. 2006.Spatial and temporal variability of arsenic solid-state speciation in histor-ically lead arsenate contaminated soils. Environ Sci Technol 40:673– 679.http://dx.doi.org/10.1021/es051266e.

24. Magalhães MCF, Silva MCM. 2003. Stability of lead(II) arsenates. MonatshChem 134:735–743. http://dx.doi.org/10.1007/s00706-002-0581-9.

25. Bajda T. 2010. Solubility of mimetite Pb5(AsO4)3Cl at 5–55°C. EnvironChem 7:268 –278.

26. Gatta GD, Lee Y, Kao C-C. 2009. Elastic behavior of vanadinite,Pb10(VO4)6Cl2, a microporous non-zeolitic mineral. Phys Chem Miner36:311–317. http://dx.doi.org/10.1007/s00269-008-0279-6.

27. Dong ZL, White TJ, Sun K, Wang LM, Ewing RC. 2005. Electronirradiation induced transformation of (Pb5Ca5)(VO4)6F2 apatite toCaVO3 perovskite. J Am Ceram Soc 88:184 –190.

28. Dong Z, White TJ. 2004. Calcium-lead fluoro-vanadinite apatites. I.Disequilibrium structures. Acta Crystallogr B 60:138 –145. http://dx.doi.org/10.1107/S0108768104001831.

29. Ruby MV, Davis A, Nicholson A. 1994. In situ formation of lead phos-phates in soils as a method to immobilize lead. Environ Sci Technol 28:646 – 654. http://dx.doi.org/10.1021/es00053a018.

30. Ioannidis TA, Zouboulis AI. 2003. Detoxification of a highly toxic lead-loaded industrial solid waste by stabilization using apatites. J Hazard Ma-ter 97:173–191. http://dx.doi.org/10.1016/S0304-3894(02)00258-3.

31. Oelkers EH, Montel J-M. 2008. Phosphates and nuclear waste storage.Elements 4:113–116. http://dx.doi.org/10.2113/GSELEMENTS.4.2.113.

32. Manning DAC. 2008. Phosphate minerals, environmental pollution andsustainable agriculture. Elements 4:105–108. http://dx.doi.org/10.2113/GSELEMENTS.4.2.105.

33. Jeanjean J, Vincent U, Fedoroff M. 1994. Structural modification ofcalcium hydroxyapatite induced by sorption of cadmium ions. J SolidState Chem 108:68 –72. http://dx.doi.org/10.1006/jssc.1994.1010.

34. Cotter-Howells J. 1996. Lead phosphate formation in soils. Environ Pol-lut 93:9 –16. http://dx.doi.org/10.1016/0269-7491(96)00020-6.

35. Cotter-Howells J, Caporn S. 1996. Remediation of contaminated land byformation of heavy metal phosphates. Appl Geochem 11:335–342. http://dx.doi.org/10.1016/0883-2927(95)00042-9.

36. Oelkers EH, Valsami-Jones E. 2008. Phosphate mineral reactivity andglobal sustainability. Elements 4:83– 87. http://dx.doi.org/10.2113/GSELEMENTS.4.2.83.

37. Parsons SA, Smith JA. 2008. Phosphorus removal and recovery frommunicipal wastewaters. Elements 4:109 –112. http://dx.doi.org/10.2113/GSELEMENTS.4.2.109.

38. Twidwell LG, Plessas KO, Comba PG, Dahnke DR. 1994. Removal ofarsenic from wastewaters and stabilization of arsenic bearing waste solids:summary of experimental studies. J Hazard Mater 36:69 – 80. http://dx.doi.org/10.1016/0304-3894(93)E0054-6.

39. Ceci A, Rhee YJ, Kierans M, Hillier S, Pendlowski H, Gray N, PersianiAM, Gadd GM. Transformation of vanadinite [Pb5(VO4)3Cl] by fungi.Environ Microbiol, in press. http://dx.doi.org/10.1111/1462-2920.12612.

40. Liang X, Hillier S, Pendlowski H, Gray N, Ceci A, Gadd GM. Uraniumphosphate biomineralization by fungi. Environ Microbiol, in press. http://dx.doi.org/10.1111/1462-2920.12771.

41. Párraga J, Rivadeneyra MA, Martín-García JM, Delgado R, Delgado G.2004. Precipitation of carbonates by bacteria from a saline soil, in naturaland artificial soil extracts. Geomicrobiol J 21:55– 66. http://dx.doi.org/10.1080/01490450490253464.

42. Delgado G, Delgado R, Párraga J, Rivadeneyra MA, Aranda V. 2008.Precipitation of carbonates and phosphates by bacteria in extract so-lutions from a semi-arid saline soil. Influence of Ca2� and Mg2� con-centrations and Mg2�/Ca2� molar ratio in biomineralization. Geomi-crobiol J 25:1–13.

43. Delgado G, Párraga J, Martín-García JM, de las Angustias RivadeneyraM, Sánchez-Marañón M, Delgado R. 2012. Carbonate and phosphateprecipitation by saline soil bacteria in a monitored culture medium. Geo-microbiol J 30:199 –208.

44. Sayer JA, Cotter-Howells JD, Watson C, Hillier S, Gadd GM. 1999. Leadmineral transformation by fungi. Curr Biol 9:691– 694. http://dx.doi.org/10.1016/S0960-9822(99)80309-1.

45. Fomina M, Alexander I, Hillier S, Gadd G. 2004. Zinc phosphate and

Fungal Biotransformation of Mimetite

August 2015 Volume 81 Number 15 aem.asm.org 4963Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from

pyromorphite solubilization by soil plant-symbiotic fungi. GeomicrobiolJ 21:351–366. http://dx.doi.org/10.1080/01490450490462066.

46. Sayer JA, Gadd GM. 1997. Solubilization and transformation of insolubleinorganic metal compounds to insoluble metal oxalates by Aspergillus ni-ger. Mycol Res 101:653– 661. http://dx.doi.org/10.1017/S0953756296003140.

47. Magyarosy A, Laidlaw R, Kilaas R, Echer C, Clark D, Keasling J. 2002.Nickel accumulation and nickel oxalate precipitation by Aspergillus niger.Appl Microbiol Biotechnol 59:382–388. http://dx.doi.org/10.1007/s00253-002-1020-x.

48. Wei Z, Liang X, Pendlowski H, Hillier S, Suntornvongsagul K, Siha-nonth P, Gadd GM. 2013. Fungal biotransformation of zinc silicate andsulfide mineral ores. Environ Microbiol 15:2173–2186. http://dx.doi.org/10.1111/1462-2920.12089.

49. Wei Z, Hillier S, Gadd GM. 2012. Biotransformation of manganeseoxides by fungi: solubilization and production of manganese oxalatebiominerals. Environ Microbiol 14:1744 –1753. http://dx.doi.org/10.1111/j.1462-2920.2012.02776.x.

50. Gadd GM, Bahri-Esfahani J, Li Q, Rhee YJ, Wei Z, Fomina M, Liang X.2014. Oxalate production by fungi: significance in geomycology, biodete-rioration and bioremediation. Fungal Biol Rev 28:36 –55. http://dx.doi.org/10.1016/j.fbr.2014.05.001.

51. Gadd GM. 1999. Fungal production of citric and oxalic acid: importancein metal speciation, physiology and biogeochemical processes. Adv Mi-crob Physiol 41:47–92.

52. Sayer JA, Gadd GM. 2001. Binding of cobalt and zinc by organic acids andculture filtrates of Aspergillus niger grown in the absence or presence ofinsoluble cobalt or zinc phosphate. Mycol Res 105:1261–1267. http://dx.doi.org/10.1016/S0953-7562(08)61998-X.

53. Gharieb MM, Sayer JA, Gadd GM. 1998. Solubilization of natural gyp-sum (CaSO4.2H2O) and the formation of calcium oxalate by Aspergillusniger and Serpula himantioides. Mycol Res 102:825– 830. http://dx.doi.org/10.1017/S0953756297005510.

54. Magnuson J, Lasure L. 2004. Organic acid production by filamentousfungi, p 307–340. In Tkacz J, Lange L (ed), Advances in fungal biotech-nology for industry, agriculture, and medicine. Springer, New York,NY, USA.

55. Plassard C, Fransson P. 2009. Regulation of low-molecular weight or-ganic acid production in fungi. Fungal Biol Rev 23:30 –39. http://dx.doi.org/10.1016/j.fbr.2009.08.002.

56. Vodnik D, Byrne AR, Gogala N. 1998. The uptake and transport of leadin some ectomycorrhizal fungi in culture. Mycol Res 102:953–958. http://dx.doi.org/10.1017/S0953756297005959.

57. Sayer JA, Raggett SL, Gadd GM. 1995. Solubilization of insoluble metalcompounds by soil fungi: development of a screening method for solubi-lizing ability and metal tolerance. Mycol Res 99:987–993. http://dx.doi.org/10.1016/S0953-7562(09)80762-4.

58. Parkhust DL, Appelo CAJ. 2013. Description of input and examples forPHREEQC version 3—a computer program for speciation, batch-reaction, one-dimensional transport, and inverse geochemical calcula-tions. U.S. Geological Survey techniques and methods, book 6. U.S. Geo-logical Survey, Denver, CO, USA. http://pubs.usgs.gov/tm/06/a43/.

59. Smith RM, Martell AE, Motekaitis RJ. 2004. Critically selected stabilityconstants of metal complexes. NIST standard reference database 46, ver-sion 8.0. NIST, Gaithersburg, MD, USA.

60. Clever HL, Derrick ME, Johnson SA. 1992. The solubility of somesparingly soluble salts of zinc and cadmium in water and in aqueous elec-trolyte solutions. J Phys Chem Ref Data 21:941–1004. http://dx.doi.org/10.1063/1.555909.

61. Kinniburgh D, Cooper D. 2010. PhreePlot: creating graphical outputwith PHREEQC. http://www.phreeplot.org.

62. Gerke TL, Scheckel KG, Schock MR. 2009. Identification and distribu-tion of vanadinite (Pb5(V5�O4)3Cl) in lead pipe corrosion by-products.Environ Sci Technol 43:4412– 4418. http://dx.doi.org/10.1021/es900501t.

63. Rhee YJ, Hillier S, Pendlowski H, Gadd GM. 2014. Pyromorphiteformation in a fungal biofilm community growing on lead metal. EnvironMicrobiol 16:1441–1451. http://dx.doi.org/10.1111/1462-2920.12416.

64. Debela F, Arocena JM, Thring RW, Whitcombe T. 2010. Organicacid-induced release of lead from pyromorphite and its relevance to rec-lamation of Pb-contaminated soils. Chemosphere 80:450 – 456. http://dx.doi.org/10.1016/j.chemosphere.2010.04.025.

65. Liu H-L, Zhu Y-N, Yu H-X. 2009. Solubility and stability of lead arsenatesat 25°C. J Environ Sci Health 44:1465–1475. http://dx.doi.org/10.1080/10934520903217856.

66. Charlton SR, Parkhurst DL. 2011. Modules based on the geochemicalmodel PHREEQC for use in scripting and programming languages. Com-put Geosci 37:1653–1663. http://dx.doi.org/10.1016/j.cageo.2011.02.005.

67. Burgstaller W, Schinner F. 1993. Leaching of metals with fungi. J Bio-technol 27:91–116. http://dx.doi.org/10.1016/0168-1656(93)90101-R.

68. Fomina M, Hillier S, Charnock JM, Melville K, Alexander IJ, Gadd GM.2005. Role of oxalic acid overexcretion in transformations of toxic metalminerals by Beauveria caledonica. Appl Environ Microbiol 71:371–381.http://dx.doi.org/10.1128/AEM.71.1.371-381.2005.

69. Fomina MA, Alexander IJ, Colpaert JV, Gadd GM. 2005. Solubilizationof toxic metal minerals and metal tolerance of mycorrhizal fungi. Soil BiolBiochem 37:851– 866. http://dx.doi.org/10.1016/j.soilbio.2004.10.013.

70. Gadd GM. 2009. Biosorption: critical review of scientific rationale, envi-ronmental importance and significance for pollution treatment. J ChemTechnol Biotechnol 84:13–28. http://dx.doi.org/10.1002/jctb.1999.

71. Fomina M, Charnock JM, Hillier S, Alvarez R, Gadd GM. 2007. Fungaltransformations of uranium oxides. Environ Microbiol 9:1696 –1710.http://dx.doi.org/10.1111/j.1462-2920.2007.01288.x.

72. Fomina M, Charnock JM, Hillier S, Alvarez R, Livens F, Gadd GM.2008. Role of fungi in the biogeochemical fate of depleted uranium. CurrBiol 18:R375–R377. http://dx.doi.org/10.1016/j.cub.2008.03.011.

73. Gadd GM, Fomina M. 2011. Uranium and fungi. Geomicrobiol J 28:471–482. http://dx.doi.org/10.1080/01490451.2010.508019.

74. Sullivan TS, Gottel NR, Basta N, Jardine PM, Schadt CW. 2012. Firingrange soils yield a diverse array of fungal isolates capable of organic acidproduction and Pb mineral solubilization. Appl Environ Microbiol 78:6078 – 6086. http://dx.doi.org/10.1128/AEM.01091-12.

75. Gadd GM. 2001. Metal transformations, p 359 –382. In Gadd GM (ed),Fungi in bioremediation. Cambridge University Press, Cambridge,United Kingdom.

76. Rhee YJ, Hillier S, Gadd GM. 2012. Lead transformation to pyromor-phite by fungi. Curr Biol 22:237–241. http://dx.doi.org/10.1016/j.cub.2011.12.017.

77. Rhee YJ, Hillier S, Pendlowski H, Gadd GM. 2014. Fungal transforma-tion of metallic lead to pyromorphite in liquid medium. Chemosphere113:17–21. http://dx.doi.org/10.1016/j.chemosphere.2014.03.085.

78. Li Q, Csetenyi L, Gadd GM. 2014. Biomineralization of metal carbonatesby Neurospora crassa. Environ Sci Technol 48:14409 –14416. http://dx.doi.org/10.1021/es5042546.

79. Bentley R, Chasteen TG. 2002. Microbial methylation of metalloids:arsenic, antimony, and bismuth. Microbiol Mol Biol Rev 66:250 –271.http://dx.doi.org/10.1128/MMBR.66.2.250-271.2002.

80. Pinzari F, Tate J, Bicchieri M, Rhee YJ, Gadd GM. 2013. Biodegradationof ivory (natural apatite): possible involvement of fungal activity in bio-deterioration of the Lewis Chessmen. Environ Microbiol 15:1050 –1062.http://dx.doi.org/10.1111/1462-2920.12027.

81. Burford EP, Hillier S, Gadd GM. 2006. Biomineralization of fungalhyphae with calcite (CaCO3) and calcium oxalate mono- and dihydrate incarboniferous limestone microcosms. Geomicrobiol J 23:599 – 611. http://dx.doi.org/10.1080/01490450600964375.

82. Guggiari M, Bloque R, Aragno M, Verrecchia E, Job D, Junier P. 2011.Experimental calcium-oxalate crystal production and dissolution by se-lected wood-rot fungi. Int Biodeterior Biodegrad 65:803– 809. http://dx.doi.org/10.1016/j.ibiod.2011.02.012.

83. Pinzari F, Zotti M, De Mico A, Calvini P. 2010. Biodegradation ofinorganic components in paper documents: formation of calcium oxalatecrystals as a consequence of Aspergillus terreus Thom growth. Int BiodetBiodegrad 64:499 –505. http://dx.doi.org/10.1016/j.ibiod.2010.06.001.

Ceci et al.

4964 aem.asm.org August 2015 Volume 81 Number 15Applied and Environmental Microbiology

on August 18, 2018 by guest

http://aem.asm

.org/D

ownloaded from