Remediation of Metal Contaminated Soil by Organic Metabolitesfrom Fungi I—Production of Organic Acids

Zandra Arwidsson & Emma Johansson &

Thomas von Kronhelm & Bert Allard &

Patrick van Hees

Received: 9 January 2009 /Accepted: 30 March 2009 /Published online: 7 May 2009# Springer Science + Business Media B.V. 2009

Abstract Investigations were made on living strains offungi in a bioremediation process of three metal (lead)contaminated soils. Three saprotrophic fungi (Aspergillusniger, Penicillium bilaiae, and a Penicillium sp.) wereexposed to poor and rich nutrient conditions (no carbonavailability or 0.11 M D-glucose, respectively) andmetal stress (25 µM lead or contaminated soils) for5 days. Exudation of low molecular weight organicacids was investigated as a response to the metal andnutrient conditions. Main organic acids identified wereoxalic acid (A. niger) and citric acid (P. bilaiae).Exudation rates of oxalate decreased in response tolead exposure, while exudation rates of citrate were lessaffected. Total production under poor nutrient condi-tions was low, except for A. niger, for which nosignificant difference was found between the poor andrich control. Maximum exudation rates were 20 µmoloxalic acid g−1 biomass h−1 (A. niger) and 20 µmol

citric acid g−1 biomass h−1 (P. bilaiae), in the presenceof the contaminated soil, but only 5 µmol organic acidsg−1 biomass h−1, in total, for the Penicillium sp. Therewas a significant mobilization of metals from the soilsin the carbon rich treatments and maximum release ofPb was 12% from the soils after 5 days. This was notsufficient to bring down the remaining concentration tothe target level 300 mg kg−1 from initial levels of 3,800,1,600, and 370 mg kg−1in the three soils. Target levelsfor Ni, Zn, and Cu, were 120, 500, and 200 mg kg−1,respectively, and were prior to the bioremediationalready below these concentrations (except for Cu Soil1). However, maximum release of Ni, Zn, and Cu was28%, 35%, and 90%, respectively. The release of metalswas related to the production of chelating acids, but alsoto the pH-decrease. This illustrates the potential to usefungi exudates in bioremediation of contaminated soil.Nonetheless, the extent of the generation of organicacids is depending on several processes and mecha-nisms that need to be further investigated.

Keywords Bioremediation . Citric acid . Fungi .

Lead . Organic acids . Oxalic acid

1 Introduction

Contaminated soils represent severe environmentalproblems, both locally and globally. Persistent organicsubstances, like e.g. PCBs and PAHs, as well as

Water Air Soil Pollut (2010) 205:215–226DOI 10.1007/s11270-009-0067-z

Z. Arwidsson (*) : E. Johansson : B. Allard : P. van HeesMan-Technology-Environment Research Centre,School of Science and Technology, Örebro University,701 82 Örebro, Swedene-mail: zandra.arvidsson@sakab.se

Z. Arwidsson : T. von KronhelmSAKAB AB,692 85 Kumla, Sweden

P. van HeesEurofins Environment Sweden AB,Box 737, 531 17 Lidköping, Sweden

heavy metals, are of particular concern. The SwedishEnvironmental Protection Agency estimates that there areabout 83,000 contaminated areas in Sweden alone, manyof them with mixed contaminants, i.e., both organicsubstances and metals (SEPA 2007). Remediation withrespect to heavy metals can be achieved in variousways, e.g. through mobilization (soil washing) orimmobilization techniques. Persistent synthetic chelatingagents can in principle be used to enhance the release ofmetals from contaminated soil aggregates (Peters, 1999).However, many of these agents may have a negativeimpact on plant growth and mycorrhizal association, i.e.,rate of mycorrhizal infection and physiological inter-actions between the symbionts. Their presence in thesoil environment may also lead to stress symptoms ofthe soil-living fungi, due to their poor photo-, chemo-,and biodegradation. Additionally, there is also a risk ofgroundwater contamination when remediation is per-formed in situ using chemically stable chelating agents(Greman et al. 2001). There are presently severalbiodegradation procedures that are used for the remedi-ation of soils that are contaminated with organicsubstances, like e.g. petroleum products and PAHs(Laine and Jorgensen 1997; Jorgensen et al. 2000;Riffaldi et al. 2006). Unlike organic substances, metalscannot be biodegraded and bioremediation of metalcontaminated soil has not been applied in technicalscale. Nonetheless, microorganisms would have thepotential to alter the chemical state and thereby thedistribution and mobility of the metals in a soil matrix.

Saprotrophic fungi are known to interact with heavymetals. Various organic acids with a complexingcapacity, notably chelating agents, can be producedby the fungi in the presence of heavy metals. This canlead to immobilization of the metals that can be eitherincorporated within the cells or be bound to the fungalcell walls, but also to the formation of soluble metalcomplexes in solution (Gadd 2007). Several com-pounds produced by fungi and with high affinity for asingle element have been identified, e.g. siderophores(Braud et al. 2006; Illmer and Buttinger 2006; Gadd2007; Nair et al. 2007) and low molecular weightproteins like metallothioneins (Cervantes andGutierrez-Corona 1994; Gadd 2007). Oxalic and citricacid are examples of low molecular weight organicacids (LMWOAs) that can be produced and that havebeen extensively studied (Wasay et al. 1998; Karaffaand Kubicek 2003; Mulligan et al. 2004; Gadd2007; Ousmanova and Parker 2007). LMWOAs may

affect the metal distribution in two inter-linked ways,metal mobilization due to the formation of solublemetal complexes, and enhanced bioactivity in gen-eral through the mere production of biodegradablecompounds.

The generation of organic compounds that may actas metal-chelating agents suitable for bioremediationprocedures is affected by several physical factors,such as temperature, humidity, and nutrient supply.Several studies have shown that the available nutrientsource is of great importance for the microbialproduction and exudation of organic acids (Dixon-Hardy et al. 1998; Gharieb and Gadd, 1999; Mandaland Banerjee 2005; Kim et al. 2006). Anotherdetermining factor for the efficiency is the rate ofdegradation of the exudates in the soil environment.LMWOAs constitute an easily accessible carbonsource for the soil microorganisms, and in organicsoils the turnover times can be less than 10 h (vanHees et al. 2003). However, the turnover time mightslow down when the exudates are associated with metals(Brynhildsen and Rosswall 1997). Processes like thesehave not been studied in detail in relation to the potentialuse of exudates for bioremediation purposes.

The overall aim of the present study is to assess thepossibility of using naturally occurring chelatingagents that are produced in situ by living strains offungi for bioremediation of heavy metal (lead)contaminated soils. The production of LMWOAswas investigated in the initial phase of the project.

2 Materials and Methods

2.1 Fungi

Three saprotrophic fungi were used in this study:Aspergillus niger, Penicillium bilaiae, and Penicilliumsp. The A. niger was provided from Department ofForest Mycology and Pathology, Swedish Universityof Agricultural Science and the two Penicilliumspecies from Department of Remediation Technology,SAKAB (in association with Department of NaturalScience, Orebro University). Characterization of thefungi was made at Swedish University of Agriculture(P Fransson).

The identity of these isolates was established byInternal Transcribed Spacer (ITS) sequence analysisusing standard techniques. DNA was extracted and

216 Water Air Soil Pollut (2010) 205:215–226

polymerase chain reactions (PCR) were carried outusing the primers ITS1 and ITS4 as described byWhite et al. (1990), with a final primer concentrationof 0.2 µM. Cycling parameters were 94°C for 5 min,then 35 cycles of 94°C for 30 s, 55°C for 30 s, and72°C for 30 s with a final extension at 72°C for7 min. PCR products were purified using theQIAquick PCR Purification Kit (Qiagen). Sequenceswere determined with a CEQTM 8000 GeneticAnalysis System (Beckman Coulter) using the DTCSQuick Start Kit (Beckman Coulter). The identificationof A. niger was unequivocal. For the two Peniciliumspecies (isolated from the same soil, sampled at amercury contaminated site, Gothenburg, Sweden)only one could be identified with the presenttechnique (P. bilaiae), while the other one could onlybe classified as being a Penicillium species.

Fungal cultures were inoculated on to 9-cm Petridishes containing a malt agar medium (pH 5.5±0.2)and incubated at 25ºC. Three 7-mm plugs were cutfrom the mycelial front of the fungal isolates after 5–8 days and transferred to 100-ml Erlenmeyer flaskscontaining 20 ml of a liquid growth medium(modified from Krantz-Rülcker et al., 1994). Thegrowth medium solution (M-GMS) had a pH of 5.9±0.06 and had the following composition: 0.16 mMC3H5(OH)2PO4Mg×2 H2O; 15 mM (NH4)2SO4;0.5 mM KNO3; 1.5 mM CaSO4×6 H2O; 47 mM3-N-Morpholino-propane sulphonic acid (MOPS-buffer); 110 mM D-glucose. The medium wasautoclaved at 115°C for 10 min. After autoclaving,1 ml of trace metals was added from a filtered stocksolution containing: 17 mM FeSO4×7 H2O; 5.8 mMZnSO4×7 H2O; 0.45 mM MnSO4×H2O; 0.40 mMCuSO4×5 H2O.

All glassware was washed with 10% HNO3 for24 h, followed by washing with distilled water, priorto the experiments, in order to remove traces ofmetals. Fungi were handled under sterile conditions,using a LAF-bench and autoclaving of all solutionsand glassware.

2.2 Soils

A sandy moraine soil (former shooting range, 100 kmwest of Stockholm) with a minor clay fraction (lessthan 5%) and organic fraction (less than 4%) was usedin the laboratory tests. A sample was taken from acontaminated area with high levels of lead (up to 4 g/kg)

and copper (up to 0.3 g/kg), as well as occasionallyorganic contaminants (hydrocarbons, up to 10%). Thesoil was fractionated in a conventional soil washingprocedure (with water). The fine suspended mobilefraction (less than 0.065 mm) was used in this study(Soil 1), as well as the course residual fraction (0.065–2 mm after sieving) (Soil 2). A similar but low-contaminated soil from the same area (industrial site,with somewhat elevated metal levels) was also selected(sieved, less than 2 mm) (Soil 3).

Total metal contents of the soils, Table 1, weredetermined by digestion with 7 M HNO3 in a microwaveoven. The organic content was estimated from the losson ignition (generally below 4%, not presented).

2.3 Leaching Experiments

Fungal biomass was produced using 20 ml of M-GMS in 100-ml Erlenmeyer flasks that were sealedwith glass wool to maintain sterile, but still aerobicconditions. The flasks were placed on a rotary shaker(100 rpm) at 20±1°C. After 8 days, the fungus wasrinsed with 20 ml of 0.1 M KNO3, after which thebiomass was transferred to another set of 100-mlErlenmeyer flasks containing 20 ml of experimentalsolution (and contaminated soil in some treatments,see below). The rinsing with KNO3 was done in orderto reduce the content of M-GMS in the experimentalsolutions. The flasks were once again sealed withglass wool and placed on a rotary shaker (100 rpm) at

Table 1 Metal concentrations (microwave digestion in 7 MHNO3) in the soils (>5 mg/kg, besides Na, Mg, K, and Ca;N=3, mean values with standard deviations)

Metal Soil 1 mg/kg Soil 2 mg/kg Soil 3 mg/kg

Al 23,000±940 5,400±690 6,000±790

Ti 1,700±50 520±190 830±39

V 61±1.9 16±2.2 20±2.7

Co 6.0±2.0 6.0±1.0

Cr 62±3.6 29±6.7 39±3.3

Mn 450±12 230±38 230±9.0

Fe 31,000±520 13,000±2,500 17,000±360

Ni 39±0.6 28±6.1 42±5.5

Cu 260±2.0 140±43 110±19

Zn 240±7.0 110±17 150±6.0

Ba 150±3.0 41±5.6 170±2.6

Pb 3,800±40 1,600±390 370±72

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20±1°C. After 5 days, the solutions were filteredthrough 0.45 µm filter (Milli-Q corp.) and stored at –20°C until analysis. The fungal biomass was dried at40°C until constant weight was obtained and the dryweights were recorded. The pH was measured in thesolutions at the start and at the end of the experiments.

In the 5-day experiments, poor nutrient solutionsconsisted of 100 µM KNO3 and 10 µM K2HPO4

(Poor-Control), whereas the rich nutrient solutionshad the same salt contents but also 0.11 M D-glucoseas a carbon source (Rich-Control). In the experimentswith metal stress, the solutions also contained 25 µMPb(NO3)2 (Poor-Pb2+and Rich-Pb2+ treatments). Thechosen lead concentration was considered to exertenough stress that the fungi would respond byproducing exudates, including LMWOAs. In thebioremediation experiments, 10 g of soil (1, 2, or 3)was added to the poor and rich nutrient solutions(Poor-Soil and Rich-Soil treatments). Triplicates ofblanks were also performed, i.e., the same experi-mental set-up as in the description above, but withoutthe presence of any fungi isolate.

2.4 Chemical Analysis of Organic Acids

LMWOAs were determined with a capillary electropho-resis system (Agilent 3D CE, Agilent Technologies)equipped with a diode array detector and hydrostaticinjection. Separations were carried out in a fused-silicacapillary with a total length of 72 cm and with an innerdiameter of 75 µm. Data evaluation were carried outwith the Agilent Chemstation software and the analysiswas performed as described by Dahlén et al. (2000).Eleven different LMWOAs was separated and analyzed:

Monocarboxylic: formic, acetic, propionic, butyricDicarboxylic: oxalic, malonic, succinic, fumaric,shikimicHydrocarboxylic: lactic, citric

Identification of the LMWOAs was performed byspiking with the known LMWOAs and by comparingmigration times, whereas quantification were achievedby using internal standards and calibration curves(solutions containing 2–20 µM pure LMWOAs).

2.5 Chemical Analysis of Metals

The metal concentrations in the fungal systems andsoils were analyzed by ICP-OES (Plasma 4000 DV,

Perkin Elmer). Altogether 12 elements were quantita-tively determined: Al, Ti, V, Co, Cr, Mn, Fe, Ni, Cu,Zn, Ba, and Pb. Data evaluation were carried out withthe Plasma 4000 DV software and quantification andidentification were achieved by using standard sol-utions (0.02–7 mg/l) with calibration curves for eachelement. All solutions were diluted with 1% or 5%HNO3 before analysis (depending on the matrix of thestandards for the different methods).

2.6 Calculation of Lead Speciation

Aqueous concentrations of lead species, includingcomplexes with oxalate, carbonate, phosphate, andhydroxide, were calculated using the chemical equi-librium program MINEQL+ (Environmental ResearchSoftware). The composition of the nutrient solutionand added metal salts were used as input values. Toreflect fungal uptake, total phosphate concentrationwas set to 5 µM based on capillary electrophoresisdeterminations. Total carbonate concentrations wereevaluated from the analytical runs and ranged from 0to 0.1 mM depending upon pH. The model was runover the observed pH interval (1.5–5.3) and measuredtotal oxalate and citrate concentrations. Saturationindices were calculated using modeled aqueousconcentrations. A number of solid phases wereconsidered: Pb3(PO4)2(s), PbHPO4(s), PbCO3(s), Pb(OH)2(s), as well as Pb-oxalate(s). All equilibriumand solubility constants were taken from theMINEQL+ database.

2.7 Statistical Analysis

All fungal experiments were performed in triplicatesand the results are presented as mean ± standarddeviation (SD). Statistical analysis was performedwith MINITAB 15 for WINDOWS (Minitab Inc).

A general linear model (GLM) multivariate analysiswas used to test for effects of nutrient treatments (poorand rich), lead treatments, and fungal species (A. niger,P. bilaiae, and Penicillium sp.) on exudation and metalmobilization (Ni, Cu, Zn, and Pb). The followingdependent variables were tested: exudation rate ofoxalate (μmol g−1 fungal dw h−1), exudation rate ofcitrate (μmol g−1 fungal dw h−1), exudation rates fortotal LMWOA (μmol g−1 fungal dw h−1), changes inpH in the experimental solutions, and metal concen-trations. The GLM post hoc test used for multiple

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comparisons of observed means was Tukey’s test at the5% level of significance. Pair wise comparisons weremade both between fungal species and within individualspecies.

3 Results and Discussion

The production of LMWOAs is estimated from theconcentrations measured in the leachates. Thus, anyfractions of the acids that were produced by the fungibut not present in the leachates (adsorbed on soilaggregates, cell walls, remaining inside cells etc) arenot determined.

3.1 Production of LMWOA

A. niger has been extensively studied for manydifferent applications and it is also the main organismused in the commercial production of citric acid(Karaffa and Kubicek 2003), whereas both A. niger(Sayer et al. 1999) and P. bilaiae (Takeda and Knight,2006) have been used in studies as phosphate-solubilizing fungi, due to their ability to acidify theirenvironment and/or producing organic acid anions.

Only eight of the 12 acids were quantified andexudation rates of LMWOAs after 5 days in thevarious systems are summarized in Table 2. Maximumexudation rates of LMWOAs in total, in the systemswithout soil, was in the order 30–60 μmol g−1 fungaldw h−1 (A. niger), 50–90 μmol g−1 fungal dw h−1

(P. bilaiae), and 1–2 μmol g−1 fungal dw h−1

(Penicillium sp.). Table 3 displays the concentrationsof lead, pH, and production (mM) of oxalic acid, citricacid, and total LMWOAs after 5 days. Both exudationrates and concentrations of LMWOAs are presented,since the latter is used in the subsequent leadspeciation calculations. There was no LMWOA pro-duction in the blank systems without soil. In the blanksystems containing soil, the soil microorganismsproduced very low levels of acids (<10 µM, in total)compared to the fungus.

3.1.1 A. niger

The production of LMWOAs was similar in the Rich-Control and Poor-Control systems, as well as in theRich-Pb2+system, with a final concentration of theorder 30–50 mM and pH of 1.5. The production was

significantly reduced in the Poor-Pb2+system. Thepresence of contaminated soil enhanced the produc-tion by a factor of 2–3 and the corresponding pH wasin the range 2–4, in the Rich-Soil systems (Table 3).The production was significantly lower in the Poor-Soil systems, and pH consequently higher. Theproduction in Poor-Soil 1 (highest Pb-level) was onlysome 2–3% of the production in Rich-Soil 1, whichmay indicate an inhibition in the nutrient poor systemin the absence of the additional carbon sourceprovided in the rich system. Dominating organicacids were oxalic and citric acid. There was also asignificant production of acetic acid in the Rich-Soilsystems (cf. Table 2).

Although the difference in total production of acidswas comparable between the Rich-Control and Poor-Control systems, there was a significantly higherexudation rate of oxalic acid in the Poor-Controlsystems. The fungal dry-weight was almost twice ashigh in the Rich-Control system, but the exudation ofoxalic acid did, however, on a biomass basis notincrease.

Ousmanova and Parker (2007) studied the exuda-tion of LMWOAs from different strain of Aspergillusand the subsequent pH variations over time. After8 days, A. niger 1 produced at most 7–9 mM oxalicacid and 1–3 mM citric acid. The pH in all solutionswas close to 4 at day 8, but in some solutions the pHwas a low as 2.5. This is comparable to the exudationof oxalic and citric acid in the Rich-Control in thisstudy, which reached concentrations of 29 mM and1 mM, respectively, and with a final pH of 1.5. Thevery low pH can be explained by the high concentra-tion of oxalic acid in the solutions, sine oxalic acid isa much stronger acid than citric acid. In another studywith heavy metal leaching from soil with A. niger(Wasay et al. 1998), the experiments were set-up as tofavor the exudation of citric acid rather than oxalicacid, which according to the authors would hinder themobilization of Pb. The optimum pH for heavy metalleaching with citrate ranged between pH 5 to 7.Nonetheless, the pH in the solutions was kept below4, since a pH above 5 would have led to the unwantedproduction of oxalic and gluconic acid. The citric acidyield (from sucrose) in this study was about 90%(200 mM). According to Karaffa and Kubicek (2003),there are some requirements to be met for highaccumulation of citric acid by A. niger, e.g., thehighest yield of citric acid are obtained on sucrose as

Water Air Soil Pollut (2010) 205:215–226 219

Table 2 Production of organic acids after 5 days, expressed as nmol g−1 h−1 (N=3, mean values with standard deviations)

System Formic Acetic Oxalic Malonic Succinic Fumaric Citric Shikimic

A. niger

Poor

Control 36,000±7,900 7,900±10,000 1,800±100

Pb2+ 4,100±2,600 310±61

Soil 1 670±160 570±45

Soil 2 3,300±1,200 6,500±1,800 440±150

Soil 3 930±520 1,700±340

Rich

Control 17,000±4,200 530±320

Pb2+ 10,000±2,800 1,400±1,300 1,300±1,200 740±530

Soil 1 8,200±620 6,600±2,700 10,000±4,400

Soil 2 7,200±2,300 24,000±8,200 3,700±3,800

Soil 3 6,400±1,400 18,000±10,000 7,300±4,000

P. bilaiae

Poor

Control 1,700±560 150±29 1,700±670

Pb2+ 170±300 390±320 360±77

Soil 1 5.2±87 1,300±1,600 1,300±750

Soil 2 300±53 30±11 650±330

Soil 3 170±52

Rich

Control 37,000±5,100 1,800±54 2,900±1,300 4,200±460 9,500±10,000 19,000±3,500

Pb2+ 3,800±2,900 310±190 330±300 560±540 110±200 16,000±8,700

Soil 1 91±120 640±390 870±650

Soil 2 1,200±370 70±76 2,800±1,800 18,000±6,600

Soil 3 2,700±550 5.2±14 4,300±980 23,000±3,200

Penicillium sp.

Poor

Control 54±68 250±140 47±82 60±62 340±140

Pb2+ 92±98 200±110 160±140 510±200

Soil 1 40±12 1,600±170

Soil 2 670±69 2,100±890

Soil 3 200±61 120±12 38±37 2,500±540

Rich

Control 35±36 26±58 28±10 430±94 98±28 26±58 690±330

Pb2+ 100±120 180±45 39±23 270±120 74±47 23±10 210±69

Soil 1 280±100 170±73 270±260

Soil 2 650±500 1,400±1,400 460±120 2,700±960

Soil 3 1,400±890 290±190 71±74 1,000±330

Medium 100 µM KNO3, 10 µM K2HPO4; [Pb]s=3,780±40 mg/kg (Soil 1), 1,550±390 mg/kg (Soil 2), 369±72 mg/kg (Soil 3)

Rich with 20 g/l D-glucose, Poor without glucose, Pb2+ with 25 µM Pb(NO3)2, Control without Pb, Blank not detected or below 1%of DOC

220 Water Air Soil Pollut (2010) 205:215–226

a carbon source, the carbon source concentration mustbe higher than 50 g/l, and the pH of the media mustbe below 3. In this study, the available carbon sourcewas glucose and only at a concentration of 20 g/l.Furthermore, the initial pH in the growth media was

close to pH 6 and according to the previous studies,the requirements for high citric acid exudation werenot met. This could explain why A. niger in mostsystems exuded significantly more oxalic acid thancitric acid. However, it must be stressed that the high

System [Pb]aq Citric Oxalic Organic acid pHmg/l mM mM mM

A. niger

Poor

Control 0 1.6±0.2 33.8±8.9 43.2±19.5 1.5±0.0

Pb2+ 5.2 0.32±0.06 4.3±2.8 4.3±2.8 2.1±0.4

Soil 1 0.68±0.05 1.5±0.2 6.4±0.1

Soil 2 17.3±6.5 0.53±0.17 7.8±2.2 12.2±3.8 4.4±0.2

Soil 3 2.0±0.4 1.1±0.6 3.1±1.0 8.2±0.2

Rich

Control 0 1.1±0.2 29.1±4.5 30.2±4.7 1.5±0.0

Pb2+ 5.2 3.3±3.0 24.7±6.8 33.7±14.7 1.5±0.1

Soil 1 54±13 24.8±10.7 15.8±6.4 60.3±18.6 4.3±0.1

Soil 2 70±70 8.9±9.2 56.4±19.7 82.6±34.4 2.3±0.4

Soil 3 5.8±2.3 17.8±9.6 43.6±25.1 78.3±38.0 3.5±0.4

P. bilaiae

Poor

Control 0 0.62±0.17 0.64±0.14 1.36±0.38 5.5±0.1

Pb2+ 5.2 0.26±0.06 0.28±0.23 0.71±0.54 6.6±0.5

Soil 1 0.78±0.45 0.75±0.93 1.54±1.44 6.9±0.0

Soil 2 0.72±0.01 0.43±0.22 0.020±0.008 0.63±0.26 7.7±0.1

Soil 3 0.09±0.03 0.09±0.03 8.0±0.1

Rich

Control 0 10.5±1.6 1.0±0.1 42.2±10.6 2.4±0.1

Pb2+ 5.2 9.4±4.8 0.18±0.11 12.6±7.3 2.5±0.0

Soil 1 12±12 0.5±0.4 0.38±0.23 0.96±0.70 5.3±0.9

Soil 2 167±72 11.7±4.4 1.9±1.2 14.4±5.8 3.5±0.2

Soil 3 13.5±0.5 15.4±2.1 2.8±0.7 20.0±3.2 4.0±0.1

Penicillium sp.

Poor

Control 0 0.09±0.02 0.01±0.02 0.18±0.09 4.5±0.8

Pb2+ 5.2 0.15±0.06 0.04±0.04 0.26±0.15 6.5±0.5

Soil 1 0.38±0.04 0.009±0.003 0.39±0.05 6.8±0.1

Soil 2 0.51±0.21 0.66±0.23 7.7±0.2

Soil 3 0.60±0.13 0.69±0.16 8.9±0.1

Rich

Control 0 0.013±0.001 0.015±0.007 0.68±0.28 3.0±0.0

Pb2+ 5.2 0.012±0.005 0.02±0.01 0.45±0.21 3.2±0.1

Soil 1 0.55±0.31 0.13±0.13 0.08±0.04 0.35±0.21 6.5±0.2

Soil 2 5.3±2.2 0.22±0.06 0.68±0.65 2.5±1.4 5.3±0.0

Soil 3 0.2±0.2 0.49±0.16 0.14±0.09 1.3±0.7 6.6±0.6

Table 3 Lead concentrations,organic acid concentrations,and pH after 5 days (N=3,mean values with standarddeviations)

Medium 100 µM KNO3,10 µM K2HPO4; [Pb]s=3,780±40 mg/kg (Soil 1),1,550±390 mg/kg (Soil 2),369±72 mg/kg (Soil 3)

Rich with 20 g/l D-glucose,Poor without glucose, Pb2+

with 25 µM Pb(NO3)2,Control without Pb, Blank<0.1 mg/l (Pb), <10 μM(organic acids)

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exudation of oxalic acid from this A. niger speciesalso might be a natural variation of exudate produc-tion within the same strain.

3.1.2 P. bilaiae

The production of LMWOAs was highest in the Rich-Control system, with final concentrations reachingapproximately 30–50 mM and pH of 2.4 (Table 3).The production was significantly reduced in the Poor-Control and Poor-Pb2+systems, only some 2–3% ofthe production in the rich systems. The presence ofcontaminated soil greatly reduced the productionof acids, in contrast to the A. niger response, withcorresponding pH in the range 3.5–4. In all Poor-Soilsystems, as well as in Rich-Soil 1 (highest Pb-level),the final concentration was only 1–2 mM or below,and corresponding pH 7–8 (5.3 in Rich-Soil 1). Thedominating organic acid was citric acid, with con-tributions of oxalic acid, as well as acetic and formicacid (Rich-Soil systems). Most of the other acidswere also produced in the systems without soil (cf.Table 2).

Takeda and Knight (2006) investigated the pH-buffering capacity on the rock phosphate (RP)solubilization by P. bilaiae. The highest productionof oxalic and citric acid was found in the pH-bufferedmedia (pH 7.0), with concentrations reaching 13 and12 mM, respectively (final pH 4.9). This can becompared to the production of oxalic and citric acid inthe Rich-Control system in this study, that were 1 and11 mM, respectively, with a corresponding pH of 2.4.The concentration of oxalic acid was not as high as in thestudy by Takeda andKnight (2006). Despite this, the pHin the solution was much lower. This is most likely aneffect of the much weaker pH-buffering capacity in theexperimental systems in this study.

3.1.3 Penicillium sp.

The production of LMWOAs was similar in allsystems—Control, Pb2+, Rich, and Poor, with orwithout soils. Final concentrations were generally inthe range 0.2–1 mM, with corresponding pH in therange 6.5–8.9 (for the soil systems, 5.3 in the Rich-Soil 2 system). Thus, there was no pronounced effectof the presence of lead, or an additional carbonsource, on the total production, but on the distributionbetween the various acids that were identified. Citric

acid dominated in the Poor-Soil system with oxalicacid at the same level in the Rich-Soil systems.Dominating acid in the Rich-Soil 1 and 2 systems wasidentified as shikimic acid. All of the other acids wereobserved at low concentration levels (Table 2).

3.2 Metal Mobilization from Soils

The target levels in the soils after remediation are120 mg kg−1 Ni, 200 mg kg−1 Cu, 500 mg kg−1Zn,and 300 mg kg−1 Pb. Consequently, prior to thebioremediation, Pb is the dominating contaminant inSoil 1–3, as well as copper in Soil 1 (Table 1). Soil 3represents the background level at the location,however with metal concentrations slightly abovethe levels in pristine soil from the same area. Metalreleases (Ni, Cu, Zn, and Pb) from the soils after5 days are given in Table 4. Mobilization of metalsfrom the blank systems without any fungi were verylow (<0.1%).

3.2.1 A. niger

The A. niger systems that produced the highest levelsof LMWOAs in the presence of contaminated soilwere Rich-Soil 1, 2, and 3 (Table 3), leading to arelease of lead of the order 3–5% of the total content(Table 4). Evidently, even though the total productionof acids was high, the release of Pb was low. Thisstrongly indicates that the mobilization of Pb fromthese soils is governed by other factors than merely ahigh production of acids. Rich-Soil 2 and 3 systemshad the highest production of oxalic acid (40–60 mM)and consequently the lowest pH (2–4). According toseveral previous studies (Wasay et al. 1998; Mulliganet al. 2004; Ousmanova and Parker 2007), citric acidseems to be the most favorable fungi exudate, in orderto reach a satisfactory release of metals from soil.Wasay et al. (1998) investigated the metal desorptionfrom soils with commercially available citric acid andstated that it required citric acid concentrationsranging between 30–160 mM in order to removeheavy metals from soil. The citric acid productionfrom A. niger in this study was in the order 10–30 mM. Thus, the poor mobilization of Pb from thesoils seems to be related to the high production ofoxalic acid, together with the low production of citricacid, in the systems. The release of Cu, Ni, and Zn,from Soil 1 (fine soil wash residue), was of the order

222 Water Air Soil Pollut (2010) 205:215–226

3–5%. However, the release from Soil 2 (coarse soilwash residue) and Soil 3 (background) was as high as15–36% of the total metal content for these threeelements and 90% for Cu from Soil 2 (highest level ofoxalic acid together with the lowest pH).

3.2.2 P. bilaiae

The P. bilaiae systems that produced the highest acidlevels in the presence of contaminated soils were

Rich-Soil 2 and 3 (Table 3), leading to a release oflead of the order 7–12% (less than 1% for Soil 1). Therelease of Cu, Ni, and Zn was in the range 9–37%from Soil 2 and 3 (but only 1–2% from Soil 1). Thedominating acid in these systems were citric acid andat the same level as in the systems with A. niger. Evenso, the release of Cu in Rich-Soil 2 and 3 wassignificantly higher in the systems with A. niger thanwith P. bilaiae. This is probably due to much higheroxalic acid concentrations in the systems with A.niger (15–30 times higher), consequently leading to alower soil pH, in which the release of Cu is promoted.

3.2.3 Penicillium sp.

The highest acid production by Penicillium sp. wasobserved in the Rich-Soil 2 system and thecorresponding releases of Ni, Cu, and Zn were inthe 2–10% range. The release of all the four metalswas below 1% in all other systems, with the exceptionof Cu (1–2%, Poor-Soil 2 and 3).

3.3 Lead Speciation

Lead releases and lead speciation in solution werecalculated for the A. niger and P. bilaiae Rich-Soilsystems, Table 5, considering the total analyticallevels of citrate, oxalate, and lead, as well as pH, inthe water phase. Complexes with oxalate are clearlydominating in all three of the A. niger-soil systemsand calculations indicate the presence of both 1:1 and1:2-complexes, particularly in the two soils withhighest pH (3.5 and 4.3; Table 5). Oxalate complexesare also dominating in all three P. bilaiae systems,including Soil 1 with the lowest level of organicacids. The 1:1 citrate complex constitutes a majorfraction despite the low citric acid concentration, butreflecting the high pH (5.3) and related dissociation ofthe acid (higher concentration of the complexinganion). Hydrolysis, as well as formation of carbonateor phosphate complexes, are not significant consider-ing the total phosphate and carbonate levels, and thelow pH (below 4.3, except for P. bilaiae Rich-Soil 2).However, at the highest pH the formation of Pb-oxalate (s) cannot be ruled out.

Precipitation of Pb-oxalates would be expected insolutions with, e.g. total oxalate concentrations above0.1 mM, total lead concentrations above 25 μM andpH above 4.5, not considering the presence of citrate

Table 4 Metal releases from the soils after 5 days (N=3, meanvalues with standard deviations)

System Ni Cu Zn Pb% % % %

A. niger

Poor

Soil 1

Soil 2 5.3±1.5 14.8±6.7 7.1±2.6 1.2±0.9

Soil 3 0.2±0.3

Rich

Soil 1 4.0±0.4 2.9±1.2 4.8±0.9 2.8±0.7

Soil 2 28.4±5.4 90.1±18 34.5±2.4 4.9±9.7

Soil 3 14.7±2.3 34.2±9.1 35.6±3.8 3.2±0.2

P. bilaiae

Poor

Soil 1 0.2±0.0

Soil 2 1.1±0.1

Soil 3 0.7±0.1

Rich

Soil 1 2.3±0.0 0.8±0.8 0.9±1.5 0.7±1.0

Soil 2 19.9±1.6 16.7±4.6 21.7±1.6 11.7±6.3

Soil 3 13.9±2.4 9.3±4.9 36.6±7.8 7.3±0.9

Penicillium sp.

Poor

Soil 1 0.3±0.0

Soil 2 1.5±0.2

Soil 3 2.5±0.7

Rich

Soil 1 0.2±0.0

Soil 2 1.9±0.2 3.5±0.4 10.8±9.9 0.4±0.4

Soil 3 0.4±0.0 0.5±0.4 0.1±0.1

Medium 100 µM KNO3, 10 µM K2HPO4; [Pb]s=3,780±40 mg/kg (Soil 1), 1,550±390 mg/kg (Soil 2), 369±72 mg/kg(Soil 3)

Rich with 20 g/l D-glucose, Poor without glucose, Blank below0.1%

Water Air Soil Pollut (2010) 205:215–226 223

and solid surfaces from the soil, or precipitating iron(III)hydroxides. The overall distribution of leadbetween the various chemical states that may existcan, however, not be accurately modeled. Thus, apartial precipitation of lead as Pb-oxalate would beexpected in the systems with highest pH, along withadsorption and exchange processes on the soilsurfaces as well as on the biomass over the wholepH range. It is not possible to distinguish and quantifythese processes from the present experiments.

4 Conclusions

After 5 days, and in Rich-Soil systems, the maximumconcentration of LMWOAs was in the range 60–80 mM with corresponding pH in the range 2–4 (A.niger). Dominant acids were oxalic and citric acid. Inthe same systems together with P. bilaiae, theconcentrations of LMWOAs were in the order 15–20 mM with corresponding pH of 3.5–4 (with theexception for Soil 1). For both fungi, the productionin the soils was reduced by approximately one orderof magnitude in the absence of the carbon source andthe dominating acids were oxalic and citric acids(some 70–90% of the total acid production). The high

exudation of mainly oxalate, but also citrate, fromthese two species is reflected in the large pH drop inthe solutions over time. The production by Penicilliumsp. gave concentrations below 1 mM and pH generallyabove 6.5 (in the soil systems) in the presence as wellas absence of the carbon source.

Mobilization of metals was accomplished in allsoils under nutrient rich conditions, with the highestmobilization in Soil 2 and 3 in the systems with A.niger and P. bilaiae. At most, 12% Pb, 28% Ni, 35%Zn, and 90% Cu was extracted from the soils. Oxalateproduction seemed to have a negative impact on therelease of Pb, whereas higher production of citric acid(approximately 10 mM), together with a low pH inthe soil slurries, gave the highest mobilizations of leadfrom the soils. For the other metals (Ni, Zn, Cu), citricand additionally high production of oxalic acid(approximately 50 mM) seemed to have a positiveimpact on desorption from the soils, together with lowpH in the soil slurries.

These studies have illustrated the potential of theselected fungi to exudate chelating agents and therebymobilize metals from contaminated soil. The extent ofthe generation of organic acids by fungi is dependingon several factors like nutrient availability and soilcomposition. However, the mechanisms behind the

Table 5 Summary of lead releases and lead speciation in solution after 5 days (N=3, mean values)

System Pba pH ∑(Citr + Ox)b [Pb]tot Pb2+ PbOx PbOx2 PbCitr% mM (%) mM % % % %

A. niger

Rich

Pb2+ – 1.5 27.9 (83) 0.025 53 47

Soil 1 2.8 4.3 40.6 (67) 0.26 45 55

Soil 2 4.9 2.3 65.3 (79) 0.34 5 86 9

Soil 3 3.2 3.5 61.0 (78) 0.028 52 48

P. bilaiae

Rich

Pb2+ – 2.5 9.6 (25) 0.025 92 8

Soil 1 0.7 5.3 0.9 (94) 0.058 13 47 1 39

Soil 2 11.7 3.5 13.5 (94) 0.80 15 79 4 2

Soil 3 7.3 4.0 18.2 (91) 0.065 4 78 9 9

Only complexes with citric (Citr) and oxalic (Ox) acids (and hydrolysis) are considered. Medium 100 µM KNO3, 10 µM K2HPO4;[Pb]s=3,780±40 mg/kg (Soil 1), 1,550±390 mg/kg (Soil 2), 369±72 mg/kg (Soil 3)

Rich with 20 g/l D-glucose, Pb2+ with 25 µM Pb(NO3)2a Lead release from the soilsb Sum of the concentrations of citric and oxalic acids and (within parenthesis) their fraction of the total concentration of organic acids

224 Water Air Soil Pollut (2010) 205:215–226

mobilization of metals from different soils needs to befurther investigated before any of these fungi can beused in large scale bioremediation. Nonetheless, thisstudy has shown that the release of metals from thesoil would be governed by several processes andmechanisms:

1. Generation and release of organic acids that mayform soluble complexes with the metals associatedwith the soil

2. Decrease of pH (and increase of the surfacecharges of the solid phases) due to generation ofacids

3. The composition of the metal-bearing phase in thesoil—the chemical composition of the metal species(elemental, discrete minerals or compounds likeoxides/hydroxides, silicates, etc, as well as metalsbound to surface sites, exchangeable etc.)

4. Secondary reactions—interactions of releasedmetals with solid surfaces, adsorption of metalions, and organic complexes

5. Formation or dissolution of new metal-carryingsolid phases, notably iron hydroxides

These aspects will be further studied in the secondphase of this project.

Acknowledgement Financial support was obtained from theFoundation for Knowledge and Competence Development aswell as Sakab-Kumla Environmental Foundation. The classifi-cation of the fungi was made by P Fransson at the Departmentof Forest Mycology and Pathology, Swedish University ofAgricultural Sciences, which is gratefully acknowledged.

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