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Bioleaching of Heavy Metals from Mine Tailings byAspergillus fumigatusBahi Jalili Seh-Bardan a , Radziah Othman a b , Samsuri Ab Wahid a , Aminudin Husin a &Fardin Sadegh-Zadeh aa Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia,Serdang, Selangor, Malaysiab Institute of Tropical Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

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Bioremediation Journal, 16(2):57–65, 2012Copyright ©c 2012 Taylor and Francis Group, LLCISSN: 1088-9868DOI: 10.1080/10889868.2012.665958

Bioleaching of Heavy Metals from MineTailings by Aspergillus fumigatus

Bahi Jalili Seh-Bardan,1

Radziah Othman,1,2

Samsuri Ab Wahid,1

Aminudin Husin,1 andFardin Sadegh-Zadeh1

1Department of LandManagement, Faculty ofAgriculture, Universiti PutraMalaysia, Serdang, Selangor,Malaysia2Institute of TropicalAgriculture, Universiti PutraMalaysia, Serdang, Selangor,Malaysia

ABSTRACT The bioleaching experiment was conducted for the removal ofheavy metals from mine tailings. A fungal strain was isolated from the goldmine tailings and it has been identified as Aspergillus fumigatus based on its 18SrDNA analysis. Bioleaching using A. fumigatus was carried out in bioleachingstep processes (one-step and two-step) at various tailings concentrations (1%,2%, 4%, and 8% [w/v]). In the one-step bioleaching process where fungi werecultivated in the presence of the tailings, concentration of oxalic acid was thehighest among the organic acids produced. On the other hand, in the two-step bioleaching process where the metabolic products of fungal growth, whichhave been separated from its biomass, were used, citric acid was dominant.In the one-step process, the highest As (62%), Fe (58%), Mn (100%), and Zn(54%) removals were observed at the lowest tailings concentration (1%). Theremoval of Pb at 1% tailings concentration in the one-step process was 56%,whereas 88% removal was achieved in the two-step process where citric acid wasdominant. In general, heavy metals removal efficiency decreased with increasedtailings of the concentration in both bioleaching processes. This study showsthe possibility of using A. fumigatus to bioleach hazardous heavy meals fromgold mine tailings.

KEYWORDS Aspergillus fumigatus, bioleaching, heavy metals, mine tailings, organicacids

INTRODUCTIONMine tailings are a major source of contamination by As and heavy metals

in soils and groundwater (Wang and Mulligan 2009). Decontamination of themine tailings is necessary to protect the environment. Conventional methodsto remediate heavy metal–contaminated sites are excavation and solidifica-tion/stabilization (Yanga et al. 2009). Although these techniques are suitable tocontrol the contamination, they do not permanently remove the heavy metalsbecause the metals will still remain in the tailings. Chemical extraction withstrong acids, chelators, or organic acids is considered as one of the most suit-able methods to remove heavy metals from contaminated sites (Peters 1999).Organic acids are more suitable to be used as metal extractants in the chemicalextraction method because they are less toxic, readily biodegradable, relatively

Address correspondence to RadziahOthman, Department of LandManagement, Faculty of Agriculture,Universiti Putra Malaysia, 43400,Serdang, Selangor, Malaysia. E-mail:radziah@agri.upm.edu.my

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inexpensive, form moderately stable metal complexes,and the extraction can be performed at mildly acidicconditions (pH 3–5) (Peters 1999). It is well known thatvarious organic acids such as citric, oxalic, and gluconicacids can be produced by fungi in the presence of heavymetals (Arwidsson et al. 2010). Evidence provided bycommercial processes to produce carboxylic acids fromsugar indicates that fungal species of genus Aspergillusand Penicillium sp. are the most efficient producersof these acids (Tzeferis 1994). Bioleaching with fungiis based on the acidolysis, complexolysis, redoxolysis,and bioaccumulation in the presence of organic carbonsource. Recent advances in the application of bioleach-ing of non-sulfidic ores and minerals for metal extrac-tion and detoxification of metal contamination havebeen reviewed by Jain and Sharma (2004). Several stud-ies have reported the ability of fungi to bioleach metalsfrom minerals (Anjum et al. 2010; Hosseini et al. 2007),ore (Mulligan, Kamali, and Gibbs 2004; Mulliganand Kamali 2003), and wastes (Amiri, Yaghmaei, andMousavi 2011; Dacera and Babel 2008; Wu and Ting2006; Wang et al. 2009; Aung and Ting 2005). Accord-ing to these studies, bio-produced organic acids suchas citric, oxalic, and gluconic acids, which have com-plexing capacities for heavy metals notably by actingas chelating or reducing agents, could leach the metalsduring microbial leaching. However, there is insuffi-cient information available regarding heavy metal re-moval from mine tailings by Aspergillus fumigatus. Thisstrain was chosen because it produces a high concen-tration of organic acids in the presence of mine tailingsbased on our preliminary study. This study was car-ried out to investigate the potential of A. fumigatus tobioleach As, Fe, Mn, Pb, and Zn using one-step andtwo-step bioleaching processes.

MATERIALS AND METHODSCharacterization of Mine TailingsMine tailings used in this study were collected from a

gold mine located in the Peninsular Malaysia. The tail-ings samples were taken from the top 30 cm of the sur-face at 25 locations using an auger. All samples werestored at 4◦C and thoroughly mixed prior to use. Onethird of the sample was used for the microbial experi-ment, whereas the rest was air dried in the laboratory for3 days, sieved through 2 mm of mesh, homogenized,and stored in plastic bags prior to use. The electricalconductivity (EC) and pH of the tailings were mea-

sured in a sample with a double-distilled water ratio of1:2.5. The acid neutralization capacity (ANC) was de-termined according to Shu et al. (2001). The amountof CaCO3 in the tailings was determined by the acidneutralization method (Allison and Moodie 1965) andthe total content of heavy metals were determined bythe aqua regia method (HNO3:HCl = 1:3).

Sequential ExtractionsThe sequential extraction procedures were per-

formed according to Leleyter and Probst (1999). Detailsof the sequential extraction procedures are shown inTable 1. All suspensions were centrifuged at 3000 rpmfor 10 min and filtered through a 0.45-µm membranefilter prior to analysis.

Isolation and Identification ofIndigenous Fungi of Gold Mine

TailingsThe fungal strain used in this study was isolated from

the gold mine tailings and then cultivated on Petriplates having potato dextrose agar (PDA; 3.9% w/v)as a culture medium as described by Bousshard et al.(1996). The plates were incubated at 30◦C for 72 h.

The fungal isolate was identified through its genomicsequence by extracting the fungi by using the DNeasyPlant Mini Kit (69104; Qiagen, Inc., CA, USA). Next, aportion of the 18S–28S rDNA was amplified using thePCR (polymerase chain reaction) with a forward primer(5′-GGAAGTAAAAGTCGTAACAAGG-3′) and a re-verse primer (5′-TCCTCCGCTTATTGATATGC-3′).The PCR amplification was performed in a thermalcycler (Thermo PCYL220; Thermo Fisher Scientific,Waltham, MA, USA). The PCR amplification productswere purified using an EZ-10 Spin Column and a PCRPurification Kit (BS364; Bio Basic, Inc., CA, USA), andsubsequently, the sequences were compared with datasets from GenBank and deposited into the GenBankDNA sequence database (accession no. HQ248184).The results showed that the fungi isolate sequence wasa 100% match of the A. fumigatus BV strain.

Spores and Inoculums PreparationSpores from 1-week-old cultures were washed with

a sterile solution of 0.2% (w/v) Tween 80. The num-ber of spores was determined with the hemocytome-ter and a spore suspension with a concentration of

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TABLE 1 Procedure of the Sequential Extraction Technique

Fraction Procedure†

Dissolved with water 1 g tailings + 40 ml deionized water, shaking for 16 min at 25◦CExchangeable Residue + 40 ml NH4OAC (pH 4.5), shaking for 2 h at 25◦CBound to carbonates Residue + 40 ml 0.1 M acetic acid, shaking for 16 h at 25◦CBound to manganese oxide Residue + 40 ml 0.01 M hydroxylammonium chloride (pH 2), shaking for 30 min at 25◦CBound to amorphous iron oxide Residue + 40 ml 0.25 M NH2OH·HCl in 25% acetic acid, shaking for 16 h in the darkBound to crystalline iron Residue + 40 ml 0.2 M ammonium oxalate + 0.1 M ascorbic acid (pH 3.25), shaking for

30 min at 96◦C (water bath)Residual Residue + 10 ml 30% H2O2, shaking for 1 h at 25◦C+ 1 h at 85◦C for evaporation + 10

ml 30% H2O2 + 1 h 85◦C for evaporation + 50 ml 1 M CH3COONH4 (pH 2) + shakingfor 16 h at 25◦C

†Analysis were repeated 6 times.

1 × 107 spores/ml was prepared. One milliliter of thespore suspension was inoculated on 100 ml of sucrosemedium, consisted of (g/L): sucrose (100), NaNO3

(1.5), KH2PO4 (0.5), MgSO4·7H2O (0.025), KCl(0.025), and yeast extract (1.6) (Bosshard, Bachofen, andBrandl 1996).

Bioleaching ExperimentsAll samples were incubated on a shaker at 150 rpm at

30◦C. Sterilization of the samples and media was doneby autoclaving at 121◦C for 15 min. All experimentswere performed in triplicates. Two different techniquesof bioleaching were carried out, a one-step and a two-step process. In the one-step process, the fungi werecultivated in the presence of the tailings, whereas inthe two-step process, the bioleaching was carried outusing the metabolic products of fungal growth that wereseparated from their biomass.

The one-step bioleaching process was conductedin sterilized 250-ml Erlenmeyer flasks containing a1-ml spore suspension inoculated with 100 ml sucrosemedium. Sterilized tailings samples were added to theflask at 1%, 2%, 4%, or 8% (w/v). The two-step pro-cess was also carried out in a 250-ml Erlenmeyer flaskfilled with a sterilized 100-ml sucrose medium inocu-lated with 1 ml of fungal spore suspension. However,the tailings samples were not added at the beginningof the incubation. After 15 days of incubation, the cul-ture was centrifuged and filtered through a 0.22-µmmembrane filter and the filtrate was mixed with dif-ferent amounts of sterilized tailings (1%, 2%, 4%, and8% [w/v]). The control treatment was prepared using afresh sucrose medium without fungi at 1%, 2%, 4%, and8% (w/v). Two milliliters of solution were taken fromeach flask at regular intervals for pH measurement and

the volume in each flask was topped up to 100 ml withthe sterile sucrose medium. When there was no changein the percentage of metal ions removed, the flaskswere centrifuged and the supernatant was then filteredthrough a 0.45-µm membrane filter and analyzed formetals and organic acids.

Chemical Leaching ExperimentThe chemical leaching was carried out with oxalic,

citric, and gluconic acids and a mixture of these acidsat concentrations equal to the acid concentrations pro-duced in the one-step bioleaching process at 1% tailingsconcentration. Reagent-grade citric and oxalic acids(Sigma-Aldrich, St. Louis, MO, USA) were used in thisexperiment. One gram of the tailings was mixed with100 ml of organic acid in a 250-ml Erlenmeyer flask.The solution was shaken for 48 h at 30◦C and 150 rpmin a shaker. The samples were then filtered through the0.45-µm membrane filters and the heavy metal concen-trations in the filtrates were determined.

Analytical Methods to DetermineConcentrations of Heavy Metals and

Organic AcidsThe concentrations of heavy metals were analyzed

using a graphite furnace atomic absorption spectropho-tometer (GFAAS, Model Z5000, Perkin-Elmer, Ger-many) and an atomic absorption spectrophotometer(AAS, Model 5100 PC, Perkin-Elmer, Germany). Theorganic acids (analyzed as citrate, oxalate, gluconate)were determined using a high-performance liquid chro-matography (HPLC) system (HP 1100 series) equippedwith the ultraviolet (UV) detectoron Aminex HPX-87H, 300 mm × 7.8 mm (Bio-Rad) column with

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microguard cation H (Bio-Rad) pre-column attached,5 mM H2SO4 as the mobile phase, and a flow rate of0.5 ml/min at ambient temperature, and the detectionwas made at 210 nm. The retention times were 9.5, 6.15,and 12.3 min for citric acid, oxalic acid, and gluconicacid, respectively.

Bioleaching YieldThe percentage of bioleaching yield, i.e. metal re-

moval was calculated according to the following equa-tion:

% Removal

= metal concentration in leachate × volume of leachatetotal metal content in tailings × tailings concentration

× 100

Statistical AnalysesAll experimental data were subjected to the analysis

of variance (ANOVA) procedure and the differencesbetween means were determined using Tukey’s test at a5% confidence level. The statistical analyses were per-formed with the Statistix statistical software package(Analytical Software 2007).

RESULTS AND DISCUSSIONPhysicochemical Characteristics of

the Mine TailingsSelected physicochemical properties of mine tailings

are as shown in Table 2. The pH was slightly alkaline(7.8), which is due to the addition of lime during wastewater treatment and this treated water is pumped back

TABLE 2 Some Properties and Concentration of Heavy Metalsof Mine Tailings

Parameter Values

pH 7.8 ± 0.1†

EC (dS m−1) 1.43 ± 0.22CaCO3 (mg kg−1) 58000 ± 3500ANC (kg H2SO4/t) 59 ± 6.4Total As (mg kg−1) 2030 ± 75Total Fe (mg kg−1) 10300 ± 18Total Mn (mg kg−1) 672.43 ± 14Total Zn (mg kg−1) 134.83 ± 16Total Pb (mg kg−1) 90.06 ± 8.3

†Mean of three replicates ± SD.

FIGURE 1 The distribution of As, Fe, Mn, Zn, and Pb in thesequentially extracted mine tailings.

into the tailing area. The ANC value of the tailingswas 59 kg H2SO4/t, which indicate the alkaline natureof the tailings. The results show a high concentrationof As (2030 mg kg−1), Fe (10300 mg kg−1), Mn (672mg kg−1), Pb (90 mg kg−1), and Zn (134 mg kg−1).The total content provided a poor indication aboutthe mobility or extractability of the metals. Figure 1shows the percentage of As, Fe, Mn, Pb, and Zn ineach fraction extracted from the mine tailings by theseven-step extraction scheme. The highest percentageof As was in the Fe-Mn–associated fraction (46%), fol-lowed by residual fraction (32%), exchangeable (13%),bound to carbonate (6%), and water soluble (3%). TheFe-Mn–associated fraction constitutes 58% of Fe, fol-lowed by residual (23%), exchangeable (10%), bound tocarbonate (9%), and water soluble (2%). Manganese wasfound mostly in the exchangeable form (67%), followedby bound to carbonate (18%), bound to Fe-Mn fraction(12%), residual (2.7%), and water soluble (0.3%). Theexchangeable fraction was the most dominant for Pb(63%), followed by bound to Fe-Mn oxide (27%), water-soluble form (5%), residual fraction (3%), and bound tocarbonate (2%). The residual fraction constitutes 77%for Zn, 11% is bound to Fe-Mn oxides, 10% is in ex-changeable form, and the remaining 2% is bound tocarbonate and in water-soluble form.

Bioleaching ExperimentsOrganic Acid Production

Acidolysis is the most important mechanism in thebioleaching process (Bosshard, Bachofen, and Brandl

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TABLE 3 Organic Acid Production in the One-Step and Two-Step Bioleaching Processes

Experiment Tailings (%) Oxalic acid (mM) Gluconic acid (mM) Citric acid (mM)

One-step bioleaching 1 216 ± 10.6† 21.1 ± 2.60 8.5 ± 0.82 138 ± 12.15 6.8 ± 1.62 3.3 ± 0.74 103.4 ± 5.4 0.82 ± 0.37 ND8 59.7 ± 7.4 0.06 ± 0.02 ND

Two-step bioleaching — 14.0 ± 2.3 ND 107 ± 8.7

Note. ND = not detected.†Mean of three replicates ± SD.

1996), therefore, it is necessary to determine the typeand concentration of organic acids produced in the bi-oleaching process. Table 3 shows the concentrations oforganic acids produced in the one-step and two-stepbioleaching processes. The results show that the con-centrations of organic acids produced were dependenton the amount of tailings added as well as the bioleach-ing type.

In the one-step bioleaching process, the highest con-centration of oxalic, gluconic, and citric acids were pro-duced at 1% tailings concentration. The presence oftailings enhanced the production of oxalic acid by thefungi in the one-step process. The high buffering ca-pacity of the tailings (Table 2) could be a reason for theoxalic acid excretion. Strasser, Burgstaller, and Schin-ner (1994) has reported that oxalic acid production byA. niger increased considerably in the medium with ahigher buffering capacity. The presence of tailings inthe one-step process also induced gluconic acid pro-duction. A similar trend was also reported by otherresearchers (Strasser, Burgstaller, and Schinner 1994;Burgstaller and Schinner 1993). The concentration ofcitric acid decreased as the tailings concentration in-creased. This could be due to the presence of tail-ings increased Mn concentration (as well as possiblyother elements) and the pH of the culture medium thathave been reported to reduce citric acid concentration(Podgorski and Lesniak 2000).

In the two-step bioleaching process, A. fumigatus se-creted 107 and 14 mM of citric acid and oxalic acid,respectively, but no gluconic acid was produced. Thisfinding is in agreement with those of Burgstaller andSchinner (1993) and Bosshard, Bachofen, and Brandl(1996), which showed that low pH favors the produc-tion of citric acid, but not gluconic acid and oxalicacid.

Generally, the types and the concentrations of or-ganic acids excreted by fungi are influenced mainly by

the pH, the buffering capacity of the medium, carbonsource, and the presence or absence of certain heavymetals and trace elements (Burgstaller and Schinner1993). For example, in the presence of a spent refineryprocessing catalyst, A. niger produced a higher concen-tration of oxalic acid than gluconic and citric acids dueto the presence of heavy metals such as Ni and Mo(Santhiya and Ting 2005).

Effects of Tailings Concentration on pH ofthe Culture Medium

The change in the medium culture pH at differentconcentrations of tailings over time for two of the bi-oleaching processes is shown in Figure 2a and b. Ingeneral, pH changes were less drastic with increasingtailings concentration for both bioleaching processes.The pH of medium containing tailings was higher thanin the system without tailings. In the one-step bioleach-ing, the initial pHs were 7.45, 7.66, 7.88, and 8.16 formedium containing 1%, 2%, 4%, and 8% tailings, re-spectively, but dropped to 3.77 for media containing1% tailings, 4.24 for 2%, 4.82 for 4%, and 5.96 for8% tailings after 33 d of incubation (Figure 2a). ThepH of the culture medium in the two-step bioleach-ing process also increased with the increase in tailingsconcentration (Figure 2b). After tailings were added tothe flasks, the pH of the culture medium immediatelyincreased from 2.75 to 4.53, 5.35, 6.88, and 7.24 formedium containing 1%, 2%, 4%, and 8% tailings, re-spectively. However, after 3 d of incubation, the pHsof the medium dropped to 4.2, 4.95, 5.68, and 6.32 formedium containing 1%, 2%, 4%, and 8% tailings, re-spectively. No further changes in the pH were observeduntil the end of incubation time for all treatments. Theresults showed that in the two-step bioleaching process,pH change was not as drastic as what was observed inthe one-step bioleaching processes.

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FIGURE 2 Variations of pH during the bioleaching at varioustailings concentrations in (a) one-step bioleaching, and (b) two-step bioleaching.

Fungi are generally able to acidify the culturemedium in the presence of organic compounds by ex-cretion of protons, absorption of nutrients in exchangefor protons, excretion of organic acids, and carbondioxide formation (Burgstaller and Schinner 1993). Thelower pH values in the one-step compared to the two-step bioleaching process can be explained by the higherconcentration of organic acids produced in the one-steprather than the two-step bioleaching process (Table 3).The higher concentration of the tailings in the cul-ture medium, the higher buffering capacity, thereforechange was less drastic in the medium with a higherconcentration of tailings.

Heavy Metals Removal in BioleachingProcesses

The removal percentage of As, Fe, Mn, Pb, and Znby the one-step and two-step bioleaching processesand the control experiment at various tailings con-centration are shown in Table 4. In general, removalpercentage of heavy metals decreased with increasingtailings concentration. Both bioleaching processes wereable to increase removal of heavy metals compared tothe controls. The removal percentage of heavy metalswas negligible in the control treatments at all tailingsconcentration. Table 5 shows the comparison of heavymetals removal using the one-step bioleaching processat 1% tailings concentration between the present studyand the reported values from the literature. The val-ues obtained from the present study are comparable orconsiderably greater than the values reported in otherstudies.

The study showed that, in the one-step bioleachingprocess, oxalic acid was the main leaching agent pro-duced by fungi in the range of 216–260 mM at varioustailings concentration. Oxalic acid is a strong chelatingand reducing agent that can form inner-sphere ring-typesurface complexes with metals (Rodenas et al. 1997). Aresulting shift of electron density toward the centralmetal ion when a complex is formed weakens the linkbetween a metal and the solid lattice, thus promotingdetachment of the metal into a solution (Lin and Ben-jamin 1992). Oxalic acid is one of the strongest acidsamong the organic acids, therefore it donates protonseasily. Protonation is extremely important in the dis-solution process, as it reconstitutes the original surfacestructure so that dissolution can continue (Elliott andShastri 1999). Oxalic acid is extremely effective in re-leasing metals bound to Fe and Mn oxyhydroxide dueto its reducing capacity (Klarup 1997). Based on the se-quential fractionation study (Figure 1), the removal of62% As in the one-step process was not only from theeasily mobile fraction, which was only 19%, but alsofrom other fractions including the Fe and Mn oxides aswell as the residual fractions. Therefore, high As recov-ery suggested that there were dissolutions of As fromFe and Mn oxides and residual fractions by excretedoxalic acid. Removal of Fe also includes fractions otherthan the easily mobile fraction, which suggested that Feis available for reduction with the oxalic acid (Klarup1997). Manganese was removed at a very high rate(100%) in the one-step bioleaching process. Based on

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TABLE 4 Removal of Heavy Metals during the Bioleaching Processes at Various Tailings Concentration

TailingExperiment concentration (%) As (%) Fe (%) Mn (%) Pb (%) Zn (%)

One-step bioleaching 1 62.1 ± 4.5†a§ 58.4 ± 1.1 a 100.0 ± 6.3 a 56.1 ± 6.4 c 54.43 ± 1.3 a2 43.2 ± 2.9 b 36.2 ± 4.0 b 52.0 ± 3.6 c 33.0 ± 3.4 d 37.60 ± 2.2 b4 33.0 ± 0.75 c 27.0 ± 2.1 c 34.3 ± 3.8 d 17.3 ± 1.5 f 21.20 ± 1.9 d8 21.4 ± 1.9 d 13.9 ± 1.6 f 20.4 ± 2.3 e 2.3 ± 1.6 l 3.10 ± 0.3 g

Two-step bioleaching 1 32.0 ± 2.9 c 45.20 ± 3.5 b 58.4 ± 2.55 b 88.4 ± 6.7 a 31.30 ± 30 c2 13.9 ± 1.1 e 24.80 ± 4.1 e 33.4 ± 2.30 d 65.6 ± 9.0 b 15.70 ± 20 e4 9.2 ± 1.3 f 10.83 ± 2.2 g 21.5 ± 1.55 e 23.5 ± 3.4 e 10.15 ± 2.3 f8 3.4 ± 0.2 h 2.90 ± 1.4 j 6.6 ± 1.20 f 13.4 ± 1.5 g 3.20 ± 0.55 g

Control 1 1.06 ± 0.15 i 1.93 ± 0.11 j 0.20 ± 0.07 g 5.5 ± 0.17 k 0.11 ± 0.01 h2 3.0 ± 0.17 hi 3.9 ± 0.2 ij 0.95 ± 0.08 g 6.8 ± 0.30 j 0.46 ± 0.06 h4 3.7 ± 0.11 h 5.4 ± 0.12 hj 1.20 ± 0.14 g 8.8 ± 0.20 i 1.10 ± 0.10 h8 6.5 ± 0.57 g 6.0 ± 0.3 h 1.60 ± 0.03 g 11.0 ± 0.30 h 0.87 ± 0.05 h

†Mean of three replicates ± SD.§Different letters within a column of each experiment show significantly differences at the p < 0.05.

the fractionation study, Mn should be the most readilyextracted metal, since about 85% of the Mn was in thesoluble form. The results confirmed this expectation, as100% of Mn was removed in the one-step bioleachingprocess. The highest Zn removal (54%) was observedin the one-step bioleaching at 1% tailing concentration,which is an indication that oxalic acid was more able toform a soluble complex with Zn as compared to citricacid probably because the pH of the medium contain-ing oxalic acid was lower than the medium containingcitric acid. Zinc solubilization has been shown to be pHdependent (Mulligan and Kamali 2003). The removalefficiency of Zn was the lowest among the investigatedmetals in both bioleaching processes. The most effec-tive leaching agent for Zn among organic acids is tar-taric acid (Mulligan and Kamali 2003), which was notproduced at significant concentration in the currentstudy. In addition, data from the fractionation studyshow that Zn was mainly present in the residual form

(77%), which might explain why it was the least mobilemetal among the elements investigated in this study.However, the highest removal of zinc exceeded (54%)the expected mobile fractions (12%). The Lead removalwas low in the one-step bioleaching process despite thatit is present mainly in exchangeable and weakly com-plexed species forms (∼70%). The major leaching agentin the one-step bioleaching process is oxalic acid and itis not a good leaching agent for removing Pb becausethe acid will precipitate Pb as lead oxalate, which has alow solubility (K sp = 2:74 × 10−11).

In the two-step bioleaching process, the removalyield of As, Fe, Mn, and Zn were lower than the one-step bioleaching process. As previously described, themajor leaching agent in the two-step bioleaching pro-cess is citric acid. The lower As, Fe, Mn, and Zn removalin the two-step bioleaching process may be due to lesscapability of citric acid to release metals firmly boundin crystal lattices, such as Fe oxides and the solid matrix

TABLE 5 Comparison of Removal of Heavy Metals during Bioleaching by A. fumigatus in the Present Study and That of Other FungalBioleaching Reported Literature

Material Organism As Fe Mn Pb Zn References

Black shale Aspergillus niger — — — — 49 Anjum et al. 2010Contaminated soil Aspergillus niger — — — 30 19 Ren et al. 2009Incinerator fly ash Aspergillus niger — 40 100 70 100 Wu and Ting, 2006Incinerator fly ash Aspergillus niger — <10 32 52 66 Bosshard, Bachofen, and Brandl, 1996Industrial waste Penicillium sp. — — — — 93 Schinner and Burgstaller 1989Low-grade oxidized

mining oresAspergillus niger — 7 — — 46 Mulligan Kamali, and Gibbs 2004

Gold mine tailings Aspergillus fumigatus 62 58 100 56 54 Present study

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compared to oxalic acid. Oxalic acid was reported tobe 5 times more effective than citric acid in remov-ing Fe, and is capable of complexing and reducing Fe(Mulligan, Kamali, and Gibbs 2004). For Pb, the high-est removal efficiency was achieved in the two-step bi-oleaching process among the metal ions and this mightbe an indication of the formation of soluble com-plexes with citric acid. Wasay, Barrington, and Toku-naga (1998) studied leaching of lead from clay loam,loam, and sandy clay loam soils by A. niger with 85%,83%, and 58%, respectively. They showed that at thebeginning, Pb removal was slow as oxalic acid was pro-duced, when citric acid production started, lead leach-ing reached a peak after 7 days.

As a comparison, the one-step bioleaching generallygave higher metal extraction efficiencies than the two-step bioleaching at all tailing concentrations, with theexception of Pb. The higher removal of As, Fe, Mn, andZn in the one-step process may be due to the higheramount of organic acids (oxalic acid and gluconic acid)produced by the fungi. This also could be due to accu-mulation by the fungal pellets, which contribute to themetals dissolution by altering the equilibrium betweensolid and dissolved metals via sorption (Wu and Ting2006).

Chemical Leaching Experiment

Figure 3 shows the chemical leaching of mine tail-ings using oxalic (216 mM), gluconic (21.1 mM), citric(8.5 mM), and a mixture of these acids (216 mM oxalic,21.1 mM gluconic, and 8.5 mM citric acids). Oxalic

FIGURE 3 Removal of heavy metals during chemical leachingusing various organic acids.

acid (at 216 mM concentration) showed high removalefficiency for Mn (88%), As (51%), Fe (48%), and Zn(44%) and low removal efficiency for Pb (33%). Citricacid (at 8.5 mM concentration) showed relatively highleaching yield (65%) for Pb. Chemical leaching usinggluconic acid (at 21 mM concentration) was less effec-tive in removing metals from the tailings, which maybe due to the high pH of the acid. The pH of glu-conic acid (at 21 mM concentration) was 6.0, whereasthe pHs of the other acids were less than 2.5. The per-centage removals of As, Fe, Mn, and Zn using oxalicacid were not statistically different to the removals bythe mixture of acids. However, similar trends were notobserved for citric and gluconic acids. These results fur-ther confirmed that oxalic acid was the major leachingagent for As, Fe, Mn, and Zn removal from the tailingsin bioleaching. The lower removal of Pb by the mix-ture of acid is due to the precipitation of Pb as leadoxalate as discussed earlier. In the chemical leachingexperiment using a mixture of the three acids, 92% ofMn, 55% of As, 52% of Fe, 46% of Zn, and 36% of Pbwas removed. In comparison to chemical leaching, themetal removal in one-step bioleaching reached 100%,62%, 58%, 56%, and 54% for Mn, As, Fe, Pb, and Zn,respectively. Generally, chemical leaching using com-mercial organic acid was less effective in the removal ofheavy metals from mine tailings compared to bioleach-ing by A. fumigatus. This result is in agreement with theresults of Aung and Ting (2005), which showed thatbioleaching of spent catalyst by Aspergillus niger gave2.7–20% higher metal removal efficiency than chemi-cal leaching with commercial organic acids at the sameconcentration.

CONCLUSIONSThe major organic acids produced by A. fumigatus

isolated from the mine tailings were oxalic and citricacids in the one- and two-step bioleaching processes, re-spectively. The one-step bioleaching process was moreefficient in removing As, Fe, Mn, and Zn but removalof Pb was more efficient in the two-step bioleachingprocess. Oxalic acid was a good leaching agent for As,Fe, Mn, and Zn, whereas citric acid was more efficientremover for Pb. Our study shows the potential of in-digenous A. fumigatus to remove As, Fe, Mn, Zn, andPb using bioleaching.

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