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Fungal bioleaching of metals in preservative-treated wood
Process Biochemistry 42 (2007) 798–804 www.elsevier.com/locate/procbio
Fungal bioleaching of metals in preservative-treated wood Reyes Sierra-Alvarez * Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ 85721-0011, USA Received 18 September 2006; received in revised form 24 January 2007; accepted 25 January 2007
Abstract Twenty-four brown-rot and 10 white-rot fungi were screened to evaluate their applicability for detoxification of preservative-treated wood impregnated with copper and chromium (CC) salts. Brown-rot fungi generally showed higher tolerance towards copper inhibition than white-rot fungi. Additionally, brown-rot fungi were found to accumulate considerable quantities of oxalic acid (up to 44.3 mM) in liquid medium, while white-rot fungi generally accumulated only traces of this organic acid. Oxalic acid is a strong organic acid capable of complexing a variety of heavy metals. Four Antrodia vaillantii and two Poria placenta brown-rot strains that displayed both a high copper tolerance and a high oxalic acid production were selected for further study. The brown-rot fungi effectively decayed wood containing up to 4.4% CC causing corrected mass losses of up to 24.3% in 4 weeks. Fungal treatment was also found to promote extensive leaching of chromium (up to 52.4%), but only moderate leaching of copper (15.6% or less). These results indicate the potential of solid-state fermentation with copper-tolerant fungi for the remediation of preservative-treated wood. Improving the solubility of copper will be an important challenge for future research. # 2007 Elsevier Ltd. All rights reserved. Keywords: Preservative-treated timber; Copper; Chromium; Brown-rot fungi; White-rot fungi; Oxalic acid; Fungal bioleaching; Copper tolerance
1. Introduction Disposal of decommissioned timber treated with preservatives based on chromium (Cr), copper (Cu) and arsenic (As) is of increasing concern because of the potential public health and ecological risks associated with the release of the toxic inorganic compounds. In the United States and Canada alone, approximately 3–4 million m3 of chromated-copper arsenate (CCA) preservative-treated wood are currently being removed from service annually, and it is estimated that this amount will increase to 16 million m3 by 2020 [1]. Disposal of CCA-treated wood is also a growing problem in Europe. In France, as an example, about 25 million CCA-treated poles are currently in service and 500,000 of those poles (50,000 tons) are removed from service annually [2]. The total concentration of Cr, Cu and As in treated wood depends upon the intended use of the wood and typically varies from the thousands to the tens of thousands of milligram be kilogram by dry weight [3]. CCA and acid-chromated copper (CC) based preservatives are able to fix in the wood and resist leaching in aquatic environments [4]. CCA consists of a mixture of hexavalent Cr
* Tel.: +1 520 626 2896; fax: +1 520 621 6048. E-mail address: [email protected]. 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.01.019
(CrO3), divalent Cu (CuO) and pentavalent As (As2O5) oxides. The processes of fixation are complex and appear to involve reduction of Cr(VI) to Cr(III) and formation of sparingly soluble metal compounds, such as Cr(OH)3, CuCrO4 and CrAsO4, and metal complexes with lignin and carbohydrates [5,6]. Preservative components can penetrate the primary and secondary wall of woody cells [7]. There is an increasing need to develop techniques for the removal of heavy metals from decommissioned wood treated with preservatives containing Cu, Cr and/or As. Currently, preservative-treated wood is disposed of in approved landfills. However, there is a growing concern that decay of preservativetreated wood in microbially active landfills might result in contamination of soil and groundwater with toxic pollutants. The ability of some fungi [8–10] and bacteria [11–13] to degrade CCA wood, causing metal leaching, is well documented. Existing methods (e.g. extraction, incineration) are expensive and they burden the environment with organic solvents, salts, or polluting emissions to the atmosphere [2]. Fungal bioleaching is a promising alternative to present-day methods. This proposed biotechnological method is based on solid-state fermentation of the decommissioned wood by Cu-tolerant fungi. During fermentation, strongly acidic, complexing agents (i.e. polycarboxylic organic acids such as oxalic and citric acids) are produced, which can solubilize the heavy metals for subsequent
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leaching. In particular, oxalic acid, which is an important degradation product of numerous brown-rot fungi, has been reported to play a key role in the mobilization of metals by fungi from different matrices [14,15], including preservative-treated wood [16,17]. Fungal-mediated solubilization of a variety of metals has been demonstrated for metal ores, coal, kaolinite clay, quartz sand, fly ash and tannery sludges [14,18]. Leaching experiments with synthetic solutions containing organic acids similar to those produced by fungi have also confirmed the ability of these chelating agents to solubilize metals in complex matrices, including preservative-treated wood [11,18,19]. The objective of this study was to identify fungal strains which are good candidates for application in fungal bioleaching processes for the detoxification of timber preserved with Cu and Cr salts. Wood-degrading fungi were classified based on their copper tolerance and ability to accumulate oxalate in the extracellular medium, which are both features required for effective wood colonization and metal bioleaching. The best strains were tested for their effectiveness in the removal of Cu and Cr from CC-treated wood.
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dried overnight at 60 8C, their weight was determined and, subsequently, they were impregnated with a commercial aqueous solution containing hexavalent Cr (591 g l1 as CrO3) and divalent copper (238 g l1 as CuO) to a target retention of 1.4, 2.5 and 4.4% (based on wood dry weight (d.w.)). For treatment, the specimens were placed in a desiccator where they were degassed under vacuum (15–18 mmHg). Subsequently, the impregnation solution was allowed to flow in the desiccator till the specimens were submerged. After 2 h, the specimens were withdrawn, blotted lightly and weighted. Next, the treated specimens were fixed for 3 weeks (27 8C, 70% RH). After fixation, the initial weight of each block was determined and representative wood samples were analyzed for Cu and Cr. The average concentrations of Cu determined in the blocks treated to retentions of 1.4, 2.5 and 4.4% were 2.98 0.06, 4.62 0.16 and 7.81 0.32 g kg1 wood d.w., respectively, while the respective concentrations of Cr were 4.21 0.17, 7.79 0.26 and 15.32 0.15 g kg1 wood d.w. Sterile petri dishes containing agar-malt extract medium and benomyl (30 mg l1) were prepared. Benomyl, a specific inhibitor of ascomycetous fungi, was filtered sterilized (0.2 mm) to prevent thermal decomposition during autoclaving and it was subsequently added to the sterile agar-malt extract medium. Fungal cultures were inoculated on the solid medium and allowed to grow for 14 days. Subsequently, thin (2 mm thickness), sterile stainless steel screens were placed on the surface of the dishes to support and prevent water logging of the wood blocks. Non-inoculated controls were run in parallel to determine mass and metal losses by abiotic mechanisms. After 28 days, the specimens were removed and impregnated with distilled water (20 ml) under vacuum for 12 h. The aqueous extracts were analyzed for pH value and metal concentration. The experiments were performed in quadruplicate.
2. Materials and methods 2.5. Chemical leaching of metals from preservative-treated wood 2.1. Microorganisms and culture media Table 1 lists the designation and source of the brown-rot and white-rot fungal strains utilized in this study. Fungal cultures were maintained at 4 8C in agar-malt extract medium (per liter: 25 g of agar and 40 g malt extract). Assays in liquid medium utilized Kirk medium (pH 4.5) which consisted of glucose (10 g l1), sodium 2,2-dimethylsuccinic acid (10 mM), ammonium tartrate (0.2 g l1), thiamine (2 g l1) and BIII nutrient medium (10% v/v) [20]. All experiments were conducted in triplicate at a temperature of 27 8C and a relative humidity (RH) of 70%, unless otherwise indicated.
2.2. Fungal tolerance to copper The tolerance of fungal strains towards Cu(II) was determined by monitoring the growth response of the isolates when incubated on 4% malt-agar plates supplemented with various concentrations of Cu (0, 2, 4, 8, 16 and 32 mM as CuSO45H2O). The copper sulfate solution was filtered sterilized (0.2 mm pore size) and subsequently added to the autoclaved (121 8C, 20 min) agar-malt extract medium. Each Petri dish was filled with 25 ml medium under aseptic conditions. The medium was allowed to solidify and then it was inoculated with a mycelium plug (6 mm diameter) obtained from the leading edge of a 7–14day-old culture. Fungal growth was monitored periodically for 38 days by determining the diameter of the mycelium. The diameter was calculated as average of two measurements taken at perpendicular positions. The toxic threshold concentration is defined as the highest Cu(II) concentration at which fungal growth was still observed.
2.3. Accumulation of oxalic acid by fungal cultures Sterile Erlenmeyers (100 ml) containing Kirk medium (10 ml) were inoculated with one mycelium plug (diameter 6 mm) and then incubated under static conditions for 28 days. The culture medium was analyzed periodically for pH and oxalate content. The experiments were performed in duplicate.
2.4. Fungal bioleaching of metals from preservative-treated wood The assays utilized thin specimens (2 mm 40 mm 40 mm) of Scots pine (Pinus sylvestris L.) sapwood impregnated with Cu and Cr. Specimens were
Metal leaching by direct extraction with oxalic acid (150 mM, pH 1.2) and distilled water was evaluated. Wood meal samples (5 g o.d. weight) were degassed under vacuum (15–18 mmHg) for 10 min. Afterwards, the extraction solution (150 ml) was allowed to flow into the container holding the samples and the pressure was equilibrated to atmospheric conditions. The samples were shaken (150 rpm) for 5 h at 30 8C. The leachate was paper filtered and prepared for further analysis.
2.6. Chemical analyses Oxalic acid was determined by HPLC utilizing a Biorad Aminex HPX-87H column (300 mm 7.8 mm) at 210 nm (Alltech, Deerfield, IL, USA) at 40 8C [21]. The eluent was 5 mM H2SO4 at a flow rate of 0.5 ml min1. The lowest detection limit for oxalic acid in this system was 0.1 mg l1. To prevent analytical interferences by heavy metals, liquid samples were pretreated with the resin Chelex-100 (Sigma Aldrich, St. Louis, MO, USA). Samples (3.5 ml) were diluted with an equal volume of phosphate buffer (0.08 M, pH 6.5) and supplied with Chelex-100 (0.1 g). The mixture was shaken at 100 rpm for 1 h and subsequently passed through a membrane filter (0.45 mm). Cu and Cr concentrations in aqueous samples were determined by graphite oven atomic absorption spectroscopy (GF-AAS, Varian model SpectrAA 300, Springvale, Australia). Extraction and analysis of Cu and Cr in the treated timber was performed according to the protocol described in the English Standard Method BS 5666-3 [22]. Total losses of wood mass were calculated gravimetrically from the difference of the initial dry mass and the dry mass of the decayed wood samples. Dry mass was calculated indirectly by determining the moisture content (103 8C, overnight) in a wood sub-sample to avoid thermal modification of wood constituents.
2.7. Chemicals Copper sulfate (CuSO45H2O, 99.0% purity), potassium oxalate ((COOK)2H2O, 99.5%); nitric acid (65%, puriss. p.a. grade); sulfuric acid (95.0%, for trace analysis), sodium sulfate (Na2SO410H2O, 99.0%,) and hydrogen peroxide (30%, for trace analysis) were obtained from Sigma Aldrich.
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Table 1 Copper tolerance and oxalate accumulation by the wood-degrading fungi evaluated in this study Fungal strains
Source
Cu toxicity threshold (mM)
Oxalatea (mM)
Brown-rot isolates Antrodia vaillantii strain FPRL-14G A. vaillantii DFP-7919 A. vaillantii H525-Sp Poria placenta FPRL-280 A. vaillantii DFP-9679 A. vaillantii FP-90877R A. vaillantii CBS 330.29 Laetiporius sulphureus Plswrd P. placenta EMPA-45 P. placenta MAD-698R P. placenta MAD-575 Serpula incrassata Mon-12 A. vaillantii strain DFP-6878 Antrodia xhanta MAD 5096-35 Coniophora puteana CBS 230.87 Piptoporus betulinus Hevea94 Meruliporia incrassata MAD 563 Fomitopsis cajanderi CBS 378.82 Gloeophyllum trabeum MAD 617-R Gloeophyllum sepiarium MAD 537-T Meruliporia incrassata CBS 188.25 P. placenta CBS 384.8 Tyromyces stipticus ONO-2 Fistulina hepatica Dreyen 94
FPRL CSIRO WU FPRL CSIRO FPL CBS WU EMPA FPL FPL WU CSIRO FPL CBS WU FPL CBS FPL FPL WU CBS WU WU
32 32 * 32 32 32 * 32 * 32 32 * 16 16 16 16 8 8 8 8 8 4 4 2 2 2 2 2
44.3 2.8 29.6 2.8 17.3 1.2 14.1 0.4 7.2 0.1 7.2 0.2 6.6 1.2 0.3 0.0 13.0 0.2 6.5 0.5 9.3 0.5 NDb ND 13.3 0.1 9.0 0.1 2.6 0.0 2.3 0.1 42.2 1.5 0.8 0.1 1.8 0.0 0.1 0.0 9.0 0.3 ND ND
White-rot isolates Dichomitus squalens CSIC 631.11 Trametes versicolor CBS 114372 Polyporus hirsutus CSIC 619.9 Phellinus torulosus CSIC 630.8 Bjerkandera adusta strain Bos55 Phellinus pini CBS 162.65 Phellinus pini CSIC 628.13 Phellinus pomaceus CSIC 627.6 Trametes FR sp. CSIC 615.12 Peniophora pseudopini CBS 162.65
CIB WU CIB CIB WU CBS CIB CIB CIB CBS
8 8 8 8 4 4 4 4 4 2
15.7 1.7 ND ND ND ND ND ND ND 0.2 0.2 ND
The asterisk (*) indicates the most tolerant fungal strains which showed longitudinal mycelium growth in the presence of 32 mM Cu2+ before day 10. FPRL: Forest Products Research Laboratory (Princes Risborough, UK); CSIRO: Division of Forest Products, CSIRO (Clayton, Australia); WU: Division of Industrial Microbiology, Wageningen University (Wageningen, The Netherlands); FPL: Department of Agriculture, Forest Products Laboratory (Madison, WI, USA); CBS: Centraalbureau voor Schimmelcultures (Baarn, The Netherlands); EMPA: Swiss Federal Laboratories for Materials Testing and Research (St. Gallen, Zwitserland); CIB: Center for Biological Research, CIB-CSIC (Madrid, Spain; AE Gonza´lez). a Highest oxalate concentration detected in liquid cultures incubated for 28 days. b ND: not detected.
3. Results and discussion 3.1. Fungal tolerance to copper The tolerance of 24 brown-rot and 10 white-rot fungal isolates to inhibition by Cu was assayed in agar-malt plates supplemented with Cu(II) concentrations ranging from 0 to 32 mM (0–2033 mg l1). Brown-rot fungi generally showed a remarkable tolerance towards Cu inhibition (Table 1). The most tolerant strains, which belonged to the species A. vaillantii, P. placenta and Laetiporus sulphureus, were able to grow at the highest concentrations tested (32 mM Cu). A. vaillantii species appeared to show a outstanding ability to withstand the toxic effects of Cu, and six out of the seven strains tested were among the top performers. In contrast, none of the 10 white-rot fungi
strains were able to grow at Cu concentrations exceeding 8 mM (Table 1). Poor Cu tolerance was also observed in nearly half of all the brown-rot fungi assayed. Other reports confirm the high Cu tolerance of brown-rot fungi in the genera Antrodia P. Karst, Postia (Fr.) and Serpula Murrill [23–25]. Considerable variation in Cu tolerance appears to exist among isolates of a given species as was reported for A. vaillantii [23]. Fig. 1 shows the growth response determined in the Cu toxicity assays for four of the most tolerant brown-rot fungi, Antrodia vaillantii (Syn. Fibroporia vaillantii) strain DFP7919, A. vaillantii DFP-9679, A. vaillantii FRLP-14G and Poria placenta (Syn. Poria placenta) FRLP-280. The toxic threshold concentration determined for these strains was as high as 32 mM, indicating their high Cu tolerance. Nonetheless, Cu concentrations of 8 mM or higher caused partial inhibition
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Fig. 1. Growth response of the brown-rot fungi, Antrodia vaillantii strain DFP-7919 (A), Poria placenta FRLP-280 (B), A. vaillantii FRLP-14G (C), A. vaillantii DFP-9679 (D), cultured on malt-agar plates supplemented with various concentrations of CuSO45H2O (mM): (&) 0; (&) 2; (*) 4; (*) 8; (~) 16 and (4) 32.
of mycelium growth even in the most tolerant isolates. The response to the toxicant was distinct in the four brown-rot strains. Cu concentrations exceeding 4 mM delayed the onset of fungal growth by A. vaillantii strain DFP-7919 cultures, but the rate of fungal growth in the Cu-amended and Cu-free plates was very similar once that the mycelium diameter extended beyond 2 cm. A long lag phase was also observed in the growth of A. vaillantii DFP-9679, A. vaillantii FRLP-14G and P. placenta FRLP-280 at Cu concentrations above 16 mM, which was accompanied by a noticeable decrease in growth rate.
9679 and FRLP-14G. The maximum concentrations of oxalate determined in the culture media ranged from 7.2 to 44.3 mM. The temporal patterns of oxalic acid accumulation varied depending on the strain. The concentration of oxalate in the
3.2. Fungal accumulation of oxalic acid The ability of the selected wood-degrading fungi to accumulate oxalate in liquid culture was evaluated to make a preliminary assessment of their applicability in the remediation of decommissioned wood treated with CC preservatives. The maximum concentration of oxalate detected in the brown-rot fungal cultures varied widely (Table 1). The highest oxalate levels of 44.3 and 42.2 mM were detected in cultures of A. vaillantii DFP-7919 and Fomitopsis cajanderi CBS 378.82, respectively. The most effective oxalate producers reduced the pH to acidic values as low as 1.8–2.0. In contrast with the brown-rot cultures, only traces of oxalic acid were detected in the growth media of white-rot fungi, with the exception of Dichomitis squalens which accumulated up to 15.7 mM of the organic acid (Table 1). The activity of oxalate decarboxylase, an enzyme that converts oxalate into formic acid and carbon dioxide, is thought to account for the low oxalate levels generally detected in white-rot cultures [26,27]. Fig. 2 shows the concentration of oxalic acid and the pH value as a function of the incubation time for four brown-rot fungi that displayed high tolerance against Cu toxicity, P. placenta FRLP-280, and A. vaillantii strains DFP-7919, DFP-
Fig. 2. Concentrations of oxalic acid (A) and pH values (B) determined in liquid cultures of the brown-rot fungi Antrodia vaillantii strain DFP-7919 (&), Poria placenta FRLP-280 (*), A. vaillantii FRLP-14G (&) and A. vaillantii DFP-9679 (). The fungal isolates were grown on a basal medium (pH 4.5) containing glucose (10 g l1) as described in Section 2.
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cultures of A. vaillantii FRLP-14G and P. placenta FRLP-280 increased with time. In contrast, accumulation of oxalate in the A. vaillantii DFP-7919 culture reached a maximum after 16 days and decreased sharply thereafter. Oxalic acid is believed to play an important role in the initial stages of wood decay by brown-rot fungi [28] and to participate in the ligninolytic system of white-rot fungi [26]. Recent studies also indicate that some brown-rot fungi acquire biochemical energy by oxidizing glucose to oxalate [29]. It is well established that although most brown-rot fungi initially accumulate large quantities of oxalate, the compound is broken down in later stages of decay. Hydroxyl radicals have been proposed to facilitate the decomposition of oxalate in brown-rot fungi [28]. The enzyme oxalate decarboxylase has also been implicated in oxalate degradation by some brown-rot fungi [30]. The accumulation of oxalate by cultures of the brown-rot isolates, A. vaillantii strains DFP-7919, DFP-9679 and FRLP14G, and P. placenta FRLP-280, was accompanied by a decrease in the pH of the culture media to highly acidic values ranging from 3.4 to only 2.2 (Fig. 2B). The lowest pH values detected in each culture coincided with the highest oxalate concentration, which is in agreement with the strong acidic character of oxalic acid (pKA1 = 1.23, pKA2 = 4.19) [31]. Wood decay by brown-rot fungi generally leads to oxalate accumulation and reduction of wood pH values [28]. 3.3. Relation copper tolerance–oxalic acid production Fungal production of oxalic acid, a compound that can immobilize Cu by precipitation of copper oxalate, has been implicated in the mechanisms of Cu tolerance of brown-rot fungi [32–34]. However, few studies have evaluated the correlation between Cu tolerance and oxalic acid production. Recently, Clausen et al. [32] reported no statistical relationship between the amount of oxalic acid accumulated by the brownrot fungus Wolfiporia cocos growing in liquid culture or wood and its tolerance to Cu. Similarly, our results reveal a poor correlation between the observed peak oxalic acid concentrations and Cu tolerance. Although the most Cu-tolerant strains in our study were generally oxalate overproducers, very low levels of oxalic acid were detected in the cultures of several brown-rot fungi with outstanding Cu tolerance, e.g. L. sulphureus strain Plswrd. Also, some of the highest oxalate levels were detected in cultures of F. cajanderi in spite of the low Cu tolerance of this isolate. These findings suggest that factors other than oxalic acid accumulation alone must account for the ability of some fungi to grow in the presence of high levels of Cu. Intracellular chelation of metals in the cytosol by a range of ligands (glutathione, metallothioneins) is a key mechanism known to confer Cu tolerance to a variety of ascomycetous fungi [35]; however, very limited information is available to date about metal–peptide complexes in wood-rotting basidiomycetous fungi. Brown-rot fungi produce other metal-chelating organic acids aside from oxalate [27] which may play a role in imparting metal resistance. Cu tolerance and oxalate dynamics in artificial media may differ from those in the wood matrix. For that reason, the
suitability of the strains selected from the screening for application in fungal bioleaching was further tested in assays with preservative-treated wood. 3.4. Fungal metal bioleaching Six brown-rot fungal isolates (A. vaillantii strains DFP7919, DFP-9679, FRLP-14G and H525-Sp, as well as P. placenta strains EMPA-45 and FPRL-280), which displayed both a high Cu tolerance and a high oxalic acid production, were evaluated for their ability to promote the leaching of heavy metals in CC-treated wood. Fig. 3A illustrates the losses of wood mass following solid-state fermentation experiments of wood blocks containing up to 4.4% CC by the selected isolates. Although the extent of wood decay decreased with increasing CC concentration, fungal treatment led to considerable mass losses even in the blocks containing the highest CC levels. P. placenta EMPA-45 was the most aggressive strain with fungal-mediated mass losses of 21.5% from the CCtreated wood (4.4% retention) after 28 days. Fungal decay was accompanied by a decrease in wood pH values from 4.8–5.8 to 2.7–3.2. Fig. 3B and C show the losses of Cr and Cu, respectively, determined after fungal treatment and subsequent leaching of the decayed wood blocks with water. The most effective isolates were A. vaillantii strains DFP-9679, H525-Sp and FRLP-14G, as well as P. placenta strain EMPA-45, which resulted in leaching of up to 61.2% of the Cr from treated wood (2.5% CC) and up to 52.4% Cr from wood treated at the highest retention (4.4% CC) in 28 days. Less than 0.4% of Cr was leached from sterile incubated wood, which indicates that the removal of Cr was achieved by bioleaching. In contrast with the extensive removal of Cr observed in our study, Cu solubilization was rather poor and did not exceed 15.6%. Removal of Cu in the sterile incubated wood ranged from 1.0 to 6.0%. Cu removal efficiencies attained during the treatment of preservative-treated wood by Cu-tolerant microorganisms, including oxalate-producing fungi, have been very variable ranging from 7 to 100% [16,17,36]. Kakitani et al. [37] has suggested that Cu extraction from preservative-treated wood by oxalate can be enhanced by increasing the pH of the extractant. Most metal oxalates are only sparingly soluble, but their solubility can be increased through complex formation. In the case of the Cu–oxalate system, formation of soluble Cu–oxalate complexes (Cu(C2O4)22) is favored at pH values above 2.0 [38]. Several studies have considered the chemical leaching of preservative-treated wood with oxalate or other chelating agents [19,36,37]. High concentrations of chemical extractants and stringent treatment conditions, e.g. mechanical or thermochemical pretreatments such as steam explosion, wood pulping or pyrolysis, have been often applied in those studies to promote metal mobilization [11,39–40]. The metal removal efficiencies attained in chemical extraction experiments appear to vary widely depending on the nature of the preservativetreated wood and extraction conditions applied. Our results showed that chemical extraction of CC-treated wood with oxalic acid attained Cu and Cr removal efficiencies comparable
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produced from degradation of carbohydrate substrates in wood. Enzymatic fungal attack disrupts the fiber structure, thereby increasing lignocellulose porosity and facilitating mass transport. This is expected to favor penetration of chelating agents into the wood cell-wall matrix, promoting dissolution of heavy metals. Improved mass diffusion will likely allow the application of bioleaching at milder treatment conditions as compared to chemical extraction (i.e., lower temperatures, extractant dose and chip size). The economic feasibility of chemical leaching and bioleaching processes for remediating heavy metal pollution will ultimately depend on the effectiveness of these processes, scale of the operation, etc. [41,42]. Taken together, the results of this study indicate the potential of biological treatment with Cu tolerant, oxalate-producing brown-rot fungi to decrease metal levels in decommissioned wood treated with Cu-based preservatives. Improving the solubility of Cu will be the main challenge for future research. Processes combining bioleaching with chemical extraction might offer the best opportunity to improve the removal of Cu from treated wood. Acknowledgements We would like to thank S. Kortekaas for providing skilled technical assistance and helpful discussion. This study was supported by the Nederlandse Maatschappij voor Energie en Milieu (NOVEM, grant no. 351650 6210). References
Fig. 3. Losses of wood mass (A), chromium (B) and copper (C) determined for Scots pine sapwood specimens treated with different concentrations of acidchromated copper preservative (in g CC preservative/100 g wood d.w.), 0 (solid bars), 1.4 (hatched bars), 2.5 (empty bars) and 4.4 (dotted bars) after 28 days of incubation with different Cu-tolerant brown-rot fungi: Antrodia vaillantii strain DFP-7919 (1), A. vaillantii DFP-9679 (2), A. vaillantii H525-Sp (3), A. vaillantii FRLP-14G (4), Poria placenta EMPA-45 (5) and P. placenta FPRL-280 (6). Reported metal and mass losses are corrected for losses determined in abiotic controls. Abiotic mass losses for the untreated and treated wood were equal or lower than 1.4 0.2% of the initial d.w. mass. Abiotic losses of Cr for the treated wood specimens were equal or lower than 0.4 0.2% of the initial Cr content. Abiotic losses of Cu for the wood containing 1.4, 2.5 and 4.4 g CC/100 g wood d.w. were 5.3 0.2, 6.0 0.6 and 3.2 1.3% of the initial Cu content, respectively.
to those of fungal bioleaching, 22.0 and 64.8%, respectively. However, the chemical extraction conditions were aggressive and included vacuum impregnation of wood meal followed by chemical extraction for 5 h with a pH 1.2 solution of highly concentrated oxalic acid (150 mM; liquid to solid ratio = 30/ 1 g g1). A key advantage of bioleaching when compared to chemical leaching is that fungal solid-state fermentation does not require the addition of costly leaching agents as these are
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[13] Daniel G, Nilsson T. Ultrastructural and TEM-EDAX studies on the degradation of CCA-treated Radiata pine by tunneling bacteria. International Research Group on Wood Preservation, Document IRG/WP/1260, Stockholm, Sweden; 1985. [14] Torma AE. Use of biotechnology in mining and metallurgy. Biotechnol Adv 1988;6:1–8. [15] Siegel SM, Siegel BZ, Clark KE. Bio-corrosion—solubilization and accumulation of metals by fungi. Water Air Soil Pollut 1983;19:229–36. [16] Kartal SN, Munir E, Kakitani T, Imamura Y. Bioremediation of CCAtreated wood by brown-rot fungi Fomitopsis palustris, Coniophora puteana, and Laetiporus sulphureus. J Wood Sci 2004;50:182–8. [17] Stephan I, Leithoff H, Peek RD. Microbial conversion of wood treated with salt preservatives. Mater Organism 1996;30:179–99. [18] Burgstaller W, Schinner F. Leaching of metals with fungi. J Biotechnol 1993;27:91–116. [19] Kartal SN, Kose C. Remediation of CCA-C treated wood using chelating agents. Holz Roh Werkst 2003;61:382–7. [20] Tien M, Kirk TK. Lignin peroxidase of Phanerochaete chrysosporium. Meth Enzym 1988;161:238–49. [21] Lapeyrie F, Chilvers GA, Bhem CA. Oxalic acid synthesis by the mycorrhizal fungus Paxillus involutus (Batsch. Ex Fr.) Fr. New Phytol 1987;106:139–46. [22] BSI. British Standard BS 5666-3:1991: methods of analysis of wood preservatives and treated timber. Quantitative analysis of preservatives and treated timber containing copper/chromium/arsenic formulations. The British Standards Institution. UK: London; 1991. [23] Collett O. Comparative tolerance of the brown-rot fungus Antrodia vaillantii (Dc-Fr) Ryv isolates to copper. Holzforsch 1992;46:293–8. [24] Collett O. Variation in copper tolerance among isolates of the brown-rot fungi Postia placenta (Fr) M-Lars and Lomb and Antrodia xantha (Fr) Ryv. Mater Organism 1992;27:263–71. [25] Hastrup ACS, Green F, Clausen CA, Jensen B. Tolerance of Serpula lacrymans to copper-based wood preservatives. Int Biodet Biodeg 2005;56:173–7. [26] Shimada M, Ma DB, Akamatsu Y, Hattori T. A proposed role of oxalicacid in wood decay systems of wood-rotting basidiomycetes. FEMS Microb Rev 1994;13:285–96. [27] Takao S. Organic acid production by basidiomycetes. I. Screening of acidproducing strains. Appl Microbiol 1965;13:732–7.
[28] Goodell B. Brown-rot fungal degradation of wood: our evolving view. Wood Deterior Preserv 2003;845:97–118. [29] Munir E, Yoon JJ, Tokimatsu T, Hattori T, Shimada M. A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris. Proc Natl Acad Sci USA 2001;98:11126–30. [30] Micales JA. Oxalate decarboxylase in the brown-rot wood decay fungus Postia placenta. Mater Organism 1995;29:177–86. [31] Perrin DD. Ionisation constants of inorganic acids and bases in aqueous solution. Oxford, NY: Pergamon Press; 1982 (IUPAC Chemical Data Series no. 29). [32] Clausen CA, Green F, Woodward BM, Evans JW, DeGroot RC. Correlation between oxalic acid production and copper tolerance in Wolfiporia cocos. Int Biodet Biodeg 2000;46:69–76. [33] Murphy RJ, Levy JF. Production of copper oxalate by some copper tolerant fungi. Trans Br Mycol Soc 1983;81:165–8. [34] Sutter HP, Jones EBG, Walchli O. The mechanism of copper tolerance in Poria placenta (Fr) Cke and Poria vaillantii (Pers) Fr. Mater Organism 1983;18:241–62. [35] Gadd GM. Interactions of fungi with toxic metals. New Phytol 1993;124:25–60. [36] Clausen CA. CCA removal from treated wood using a dual remediation process. Waste Manag Res 2000;18:485–8. [37] Kakitani T, Hata T, Kajimoto T, Imamura Y. A novel extractant for removal of hazardous metals from preservative-treated wood waste. J Environ Qual 2006;35:912–7. [38] Marta L, Horovitz O, Zaharescu M. Analytical study of oxalates coprecipitation. Leonardo J Sci 2003;72–82. [39] Stephan I, Nimz H, Peek RD. Detoxification of salt-impregnated wood by organic acids in a pulping process. International Research Group on Wood Preservation, Document IRG/WP/93-50012, Stockholm, Sweden; 1993. [40] Kakitani T, Hata T, Kajimoto T, Imamura Y. Effect of pyrolysis on solvent extractability of toxic metals from chromated copper arsenate (CCA)treated wood. J Hazard Mater B 2004;109:53–7. [41] Gadd GM. Microbial influence on metal mobility and application for bioremediation. Geoderma 2004;122:109–19. [42] Sreekrishnan TR, Tyagi RD. A comparative study of the cost of leaching out heavy metals from sewage sludges. Process Biochem 1996;31:31–41.
Fungal bioleaching of metals in preservative-treated wood Reyes Sierra-Alvarez * Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ 85721-0011, USA Received 18 September 2006; received in revised form 24 January 2007; accepted 25 January 2007
Abstract Twenty-four brown-rot and 10 white-rot fungi were screened to evaluate their applicability for detoxification of preservative-treated wood impregnated with copper and chromium (CC) salts. Brown-rot fungi generally showed higher tolerance towards copper inhibition than white-rot fungi. Additionally, brown-rot fungi were found to accumulate considerable quantities of oxalic acid (up to 44.3 mM) in liquid medium, while white-rot fungi generally accumulated only traces of this organic acid. Oxalic acid is a strong organic acid capable of complexing a variety of heavy metals. Four Antrodia vaillantii and two Poria placenta brown-rot strains that displayed both a high copper tolerance and a high oxalic acid production were selected for further study. The brown-rot fungi effectively decayed wood containing up to 4.4% CC causing corrected mass losses of up to 24.3% in 4 weeks. Fungal treatment was also found to promote extensive leaching of chromium (up to 52.4%), but only moderate leaching of copper (15.6% or less). These results indicate the potential of solid-state fermentation with copper-tolerant fungi for the remediation of preservative-treated wood. Improving the solubility of copper will be an important challenge for future research. # 2007 Elsevier Ltd. All rights reserved. Keywords: Preservative-treated timber; Copper; Chromium; Brown-rot fungi; White-rot fungi; Oxalic acid; Fungal bioleaching; Copper tolerance
1. Introduction Disposal of decommissioned timber treated with preservatives based on chromium (Cr), copper (Cu) and arsenic (As) is of increasing concern because of the potential public health and ecological risks associated with the release of the toxic inorganic compounds. In the United States and Canada alone, approximately 3–4 million m3 of chromated-copper arsenate (CCA) preservative-treated wood are currently being removed from service annually, and it is estimated that this amount will increase to 16 million m3 by 2020 [1]. Disposal of CCA-treated wood is also a growing problem in Europe. In France, as an example, about 25 million CCA-treated poles are currently in service and 500,000 of those poles (50,000 tons) are removed from service annually [2]. The total concentration of Cr, Cu and As in treated wood depends upon the intended use of the wood and typically varies from the thousands to the tens of thousands of milligram be kilogram by dry weight [3]. CCA and acid-chromated copper (CC) based preservatives are able to fix in the wood and resist leaching in aquatic environments [4]. CCA consists of a mixture of hexavalent Cr
* Tel.: +1 520 626 2896; fax: +1 520 621 6048. E-mail address: [email protected]. 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.01.019
(CrO3), divalent Cu (CuO) and pentavalent As (As2O5) oxides. The processes of fixation are complex and appear to involve reduction of Cr(VI) to Cr(III) and formation of sparingly soluble metal compounds, such as Cr(OH)3, CuCrO4 and CrAsO4, and metal complexes with lignin and carbohydrates [5,6]. Preservative components can penetrate the primary and secondary wall of woody cells [7]. There is an increasing need to develop techniques for the removal of heavy metals from decommissioned wood treated with preservatives containing Cu, Cr and/or As. Currently, preservative-treated wood is disposed of in approved landfills. However, there is a growing concern that decay of preservativetreated wood in microbially active landfills might result in contamination of soil and groundwater with toxic pollutants. The ability of some fungi [8–10] and bacteria [11–13] to degrade CCA wood, causing metal leaching, is well documented. Existing methods (e.g. extraction, incineration) are expensive and they burden the environment with organic solvents, salts, or polluting emissions to the atmosphere [2]. Fungal bioleaching is a promising alternative to present-day methods. This proposed biotechnological method is based on solid-state fermentation of the decommissioned wood by Cu-tolerant fungi. During fermentation, strongly acidic, complexing agents (i.e. polycarboxylic organic acids such as oxalic and citric acids) are produced, which can solubilize the heavy metals for subsequent
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leaching. In particular, oxalic acid, which is an important degradation product of numerous brown-rot fungi, has been reported to play a key role in the mobilization of metals by fungi from different matrices [14,15], including preservative-treated wood [16,17]. Fungal-mediated solubilization of a variety of metals has been demonstrated for metal ores, coal, kaolinite clay, quartz sand, fly ash and tannery sludges [14,18]. Leaching experiments with synthetic solutions containing organic acids similar to those produced by fungi have also confirmed the ability of these chelating agents to solubilize metals in complex matrices, including preservative-treated wood [11,18,19]. The objective of this study was to identify fungal strains which are good candidates for application in fungal bioleaching processes for the detoxification of timber preserved with Cu and Cr salts. Wood-degrading fungi were classified based on their copper tolerance and ability to accumulate oxalate in the extracellular medium, which are both features required for effective wood colonization and metal bioleaching. The best strains were tested for their effectiveness in the removal of Cu and Cr from CC-treated wood.
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dried overnight at 60 8C, their weight was determined and, subsequently, they were impregnated with a commercial aqueous solution containing hexavalent Cr (591 g l1 as CrO3) and divalent copper (238 g l1 as CuO) to a target retention of 1.4, 2.5 and 4.4% (based on wood dry weight (d.w.)). For treatment, the specimens were placed in a desiccator where they were degassed under vacuum (15–18 mmHg). Subsequently, the impregnation solution was allowed to flow in the desiccator till the specimens were submerged. After 2 h, the specimens were withdrawn, blotted lightly and weighted. Next, the treated specimens were fixed for 3 weeks (27 8C, 70% RH). After fixation, the initial weight of each block was determined and representative wood samples were analyzed for Cu and Cr. The average concentrations of Cu determined in the blocks treated to retentions of 1.4, 2.5 and 4.4% were 2.98 0.06, 4.62 0.16 and 7.81 0.32 g kg1 wood d.w., respectively, while the respective concentrations of Cr were 4.21 0.17, 7.79 0.26 and 15.32 0.15 g kg1 wood d.w. Sterile petri dishes containing agar-malt extract medium and benomyl (30 mg l1) were prepared. Benomyl, a specific inhibitor of ascomycetous fungi, was filtered sterilized (0.2 mm) to prevent thermal decomposition during autoclaving and it was subsequently added to the sterile agar-malt extract medium. Fungal cultures were inoculated on the solid medium and allowed to grow for 14 days. Subsequently, thin (2 mm thickness), sterile stainless steel screens were placed on the surface of the dishes to support and prevent water logging of the wood blocks. Non-inoculated controls were run in parallel to determine mass and metal losses by abiotic mechanisms. After 28 days, the specimens were removed and impregnated with distilled water (20 ml) under vacuum for 12 h. The aqueous extracts were analyzed for pH value and metal concentration. The experiments were performed in quadruplicate.
2. Materials and methods 2.5. Chemical leaching of metals from preservative-treated wood 2.1. Microorganisms and culture media Table 1 lists the designation and source of the brown-rot and white-rot fungal strains utilized in this study. Fungal cultures were maintained at 4 8C in agar-malt extract medium (per liter: 25 g of agar and 40 g malt extract). Assays in liquid medium utilized Kirk medium (pH 4.5) which consisted of glucose (10 g l1), sodium 2,2-dimethylsuccinic acid (10 mM), ammonium tartrate (0.2 g l1), thiamine (2 g l1) and BIII nutrient medium (10% v/v) [20]. All experiments were conducted in triplicate at a temperature of 27 8C and a relative humidity (RH) of 70%, unless otherwise indicated.
2.2. Fungal tolerance to copper The tolerance of fungal strains towards Cu(II) was determined by monitoring the growth response of the isolates when incubated on 4% malt-agar plates supplemented with various concentrations of Cu (0, 2, 4, 8, 16 and 32 mM as CuSO45H2O). The copper sulfate solution was filtered sterilized (0.2 mm pore size) and subsequently added to the autoclaved (121 8C, 20 min) agar-malt extract medium. Each Petri dish was filled with 25 ml medium under aseptic conditions. The medium was allowed to solidify and then it was inoculated with a mycelium plug (6 mm diameter) obtained from the leading edge of a 7–14day-old culture. Fungal growth was monitored periodically for 38 days by determining the diameter of the mycelium. The diameter was calculated as average of two measurements taken at perpendicular positions. The toxic threshold concentration is defined as the highest Cu(II) concentration at which fungal growth was still observed.
2.3. Accumulation of oxalic acid by fungal cultures Sterile Erlenmeyers (100 ml) containing Kirk medium (10 ml) were inoculated with one mycelium plug (diameter 6 mm) and then incubated under static conditions for 28 days. The culture medium was analyzed periodically for pH and oxalate content. The experiments were performed in duplicate.
2.4. Fungal bioleaching of metals from preservative-treated wood The assays utilized thin specimens (2 mm 40 mm 40 mm) of Scots pine (Pinus sylvestris L.) sapwood impregnated with Cu and Cr. Specimens were
Metal leaching by direct extraction with oxalic acid (150 mM, pH 1.2) and distilled water was evaluated. Wood meal samples (5 g o.d. weight) were degassed under vacuum (15–18 mmHg) for 10 min. Afterwards, the extraction solution (150 ml) was allowed to flow into the container holding the samples and the pressure was equilibrated to atmospheric conditions. The samples were shaken (150 rpm) for 5 h at 30 8C. The leachate was paper filtered and prepared for further analysis.
2.6. Chemical analyses Oxalic acid was determined by HPLC utilizing a Biorad Aminex HPX-87H column (300 mm 7.8 mm) at 210 nm (Alltech, Deerfield, IL, USA) at 40 8C [21]. The eluent was 5 mM H2SO4 at a flow rate of 0.5 ml min1. The lowest detection limit for oxalic acid in this system was 0.1 mg l1. To prevent analytical interferences by heavy metals, liquid samples were pretreated with the resin Chelex-100 (Sigma Aldrich, St. Louis, MO, USA). Samples (3.5 ml) were diluted with an equal volume of phosphate buffer (0.08 M, pH 6.5) and supplied with Chelex-100 (0.1 g). The mixture was shaken at 100 rpm for 1 h and subsequently passed through a membrane filter (0.45 mm). Cu and Cr concentrations in aqueous samples were determined by graphite oven atomic absorption spectroscopy (GF-AAS, Varian model SpectrAA 300, Springvale, Australia). Extraction and analysis of Cu and Cr in the treated timber was performed according to the protocol described in the English Standard Method BS 5666-3 [22]. Total losses of wood mass were calculated gravimetrically from the difference of the initial dry mass and the dry mass of the decayed wood samples. Dry mass was calculated indirectly by determining the moisture content (103 8C, overnight) in a wood sub-sample to avoid thermal modification of wood constituents.
2.7. Chemicals Copper sulfate (CuSO45H2O, 99.0% purity), potassium oxalate ((COOK)2H2O, 99.5%); nitric acid (65%, puriss. p.a. grade); sulfuric acid (95.0%, for trace analysis), sodium sulfate (Na2SO410H2O, 99.0%,) and hydrogen peroxide (30%, for trace analysis) were obtained from Sigma Aldrich.
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Table 1 Copper tolerance and oxalate accumulation by the wood-degrading fungi evaluated in this study Fungal strains
Source
Cu toxicity threshold (mM)
Oxalatea (mM)
Brown-rot isolates Antrodia vaillantii strain FPRL-14G A. vaillantii DFP-7919 A. vaillantii H525-Sp Poria placenta FPRL-280 A. vaillantii DFP-9679 A. vaillantii FP-90877R A. vaillantii CBS 330.29 Laetiporius sulphureus Plswrd P. placenta EMPA-45 P. placenta MAD-698R P. placenta MAD-575 Serpula incrassata Mon-12 A. vaillantii strain DFP-6878 Antrodia xhanta MAD 5096-35 Coniophora puteana CBS 230.87 Piptoporus betulinus Hevea94 Meruliporia incrassata MAD 563 Fomitopsis cajanderi CBS 378.82 Gloeophyllum trabeum MAD 617-R Gloeophyllum sepiarium MAD 537-T Meruliporia incrassata CBS 188.25 P. placenta CBS 384.8 Tyromyces stipticus ONO-2 Fistulina hepatica Dreyen 94
FPRL CSIRO WU FPRL CSIRO FPL CBS WU EMPA FPL FPL WU CSIRO FPL CBS WU FPL CBS FPL FPL WU CBS WU WU
32 32 * 32 32 32 * 32 * 32 32 * 16 16 16 16 8 8 8 8 8 4 4 2 2 2 2 2
44.3 2.8 29.6 2.8 17.3 1.2 14.1 0.4 7.2 0.1 7.2 0.2 6.6 1.2 0.3 0.0 13.0 0.2 6.5 0.5 9.3 0.5 NDb ND 13.3 0.1 9.0 0.1 2.6 0.0 2.3 0.1 42.2 1.5 0.8 0.1 1.8 0.0 0.1 0.0 9.0 0.3 ND ND
White-rot isolates Dichomitus squalens CSIC 631.11 Trametes versicolor CBS 114372 Polyporus hirsutus CSIC 619.9 Phellinus torulosus CSIC 630.8 Bjerkandera adusta strain Bos55 Phellinus pini CBS 162.65 Phellinus pini CSIC 628.13 Phellinus pomaceus CSIC 627.6 Trametes FR sp. CSIC 615.12 Peniophora pseudopini CBS 162.65
CIB WU CIB CIB WU CBS CIB CIB CIB CBS
8 8 8 8 4 4 4 4 4 2
15.7 1.7 ND ND ND ND ND ND ND 0.2 0.2 ND
The asterisk (*) indicates the most tolerant fungal strains which showed longitudinal mycelium growth in the presence of 32 mM Cu2+ before day 10. FPRL: Forest Products Research Laboratory (Princes Risborough, UK); CSIRO: Division of Forest Products, CSIRO (Clayton, Australia); WU: Division of Industrial Microbiology, Wageningen University (Wageningen, The Netherlands); FPL: Department of Agriculture, Forest Products Laboratory (Madison, WI, USA); CBS: Centraalbureau voor Schimmelcultures (Baarn, The Netherlands); EMPA: Swiss Federal Laboratories for Materials Testing and Research (St. Gallen, Zwitserland); CIB: Center for Biological Research, CIB-CSIC (Madrid, Spain; AE Gonza´lez). a Highest oxalate concentration detected in liquid cultures incubated for 28 days. b ND: not detected.
3. Results and discussion 3.1. Fungal tolerance to copper The tolerance of 24 brown-rot and 10 white-rot fungal isolates to inhibition by Cu was assayed in agar-malt plates supplemented with Cu(II) concentrations ranging from 0 to 32 mM (0–2033 mg l1). Brown-rot fungi generally showed a remarkable tolerance towards Cu inhibition (Table 1). The most tolerant strains, which belonged to the species A. vaillantii, P. placenta and Laetiporus sulphureus, were able to grow at the highest concentrations tested (32 mM Cu). A. vaillantii species appeared to show a outstanding ability to withstand the toxic effects of Cu, and six out of the seven strains tested were among the top performers. In contrast, none of the 10 white-rot fungi
strains were able to grow at Cu concentrations exceeding 8 mM (Table 1). Poor Cu tolerance was also observed in nearly half of all the brown-rot fungi assayed. Other reports confirm the high Cu tolerance of brown-rot fungi in the genera Antrodia P. Karst, Postia (Fr.) and Serpula Murrill [23–25]. Considerable variation in Cu tolerance appears to exist among isolates of a given species as was reported for A. vaillantii [23]. Fig. 1 shows the growth response determined in the Cu toxicity assays for four of the most tolerant brown-rot fungi, Antrodia vaillantii (Syn. Fibroporia vaillantii) strain DFP7919, A. vaillantii DFP-9679, A. vaillantii FRLP-14G and Poria placenta (Syn. Poria placenta) FRLP-280. The toxic threshold concentration determined for these strains was as high as 32 mM, indicating their high Cu tolerance. Nonetheless, Cu concentrations of 8 mM or higher caused partial inhibition
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Fig. 1. Growth response of the brown-rot fungi, Antrodia vaillantii strain DFP-7919 (A), Poria placenta FRLP-280 (B), A. vaillantii FRLP-14G (C), A. vaillantii DFP-9679 (D), cultured on malt-agar plates supplemented with various concentrations of CuSO45H2O (mM): (&) 0; (&) 2; (*) 4; (*) 8; (~) 16 and (4) 32.
of mycelium growth even in the most tolerant isolates. The response to the toxicant was distinct in the four brown-rot strains. Cu concentrations exceeding 4 mM delayed the onset of fungal growth by A. vaillantii strain DFP-7919 cultures, but the rate of fungal growth in the Cu-amended and Cu-free plates was very similar once that the mycelium diameter extended beyond 2 cm. A long lag phase was also observed in the growth of A. vaillantii DFP-9679, A. vaillantii FRLP-14G and P. placenta FRLP-280 at Cu concentrations above 16 mM, which was accompanied by a noticeable decrease in growth rate.
9679 and FRLP-14G. The maximum concentrations of oxalate determined in the culture media ranged from 7.2 to 44.3 mM. The temporal patterns of oxalic acid accumulation varied depending on the strain. The concentration of oxalate in the
3.2. Fungal accumulation of oxalic acid The ability of the selected wood-degrading fungi to accumulate oxalate in liquid culture was evaluated to make a preliminary assessment of their applicability in the remediation of decommissioned wood treated with CC preservatives. The maximum concentration of oxalate detected in the brown-rot fungal cultures varied widely (Table 1). The highest oxalate levels of 44.3 and 42.2 mM were detected in cultures of A. vaillantii DFP-7919 and Fomitopsis cajanderi CBS 378.82, respectively. The most effective oxalate producers reduced the pH to acidic values as low as 1.8–2.0. In contrast with the brown-rot cultures, only traces of oxalic acid were detected in the growth media of white-rot fungi, with the exception of Dichomitis squalens which accumulated up to 15.7 mM of the organic acid (Table 1). The activity of oxalate decarboxylase, an enzyme that converts oxalate into formic acid and carbon dioxide, is thought to account for the low oxalate levels generally detected in white-rot cultures [26,27]. Fig. 2 shows the concentration of oxalic acid and the pH value as a function of the incubation time for four brown-rot fungi that displayed high tolerance against Cu toxicity, P. placenta FRLP-280, and A. vaillantii strains DFP-7919, DFP-
Fig. 2. Concentrations of oxalic acid (A) and pH values (B) determined in liquid cultures of the brown-rot fungi Antrodia vaillantii strain DFP-7919 (&), Poria placenta FRLP-280 (*), A. vaillantii FRLP-14G (&) and A. vaillantii DFP-9679 (). The fungal isolates were grown on a basal medium (pH 4.5) containing glucose (10 g l1) as described in Section 2.
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cultures of A. vaillantii FRLP-14G and P. placenta FRLP-280 increased with time. In contrast, accumulation of oxalate in the A. vaillantii DFP-7919 culture reached a maximum after 16 days and decreased sharply thereafter. Oxalic acid is believed to play an important role in the initial stages of wood decay by brown-rot fungi [28] and to participate in the ligninolytic system of white-rot fungi [26]. Recent studies also indicate that some brown-rot fungi acquire biochemical energy by oxidizing glucose to oxalate [29]. It is well established that although most brown-rot fungi initially accumulate large quantities of oxalate, the compound is broken down in later stages of decay. Hydroxyl radicals have been proposed to facilitate the decomposition of oxalate in brown-rot fungi [28]. The enzyme oxalate decarboxylase has also been implicated in oxalate degradation by some brown-rot fungi [30]. The accumulation of oxalate by cultures of the brown-rot isolates, A. vaillantii strains DFP-7919, DFP-9679 and FRLP14G, and P. placenta FRLP-280, was accompanied by a decrease in the pH of the culture media to highly acidic values ranging from 3.4 to only 2.2 (Fig. 2B). The lowest pH values detected in each culture coincided with the highest oxalate concentration, which is in agreement with the strong acidic character of oxalic acid (pKA1 = 1.23, pKA2 = 4.19) [31]. Wood decay by brown-rot fungi generally leads to oxalate accumulation and reduction of wood pH values [28]. 3.3. Relation copper tolerance–oxalic acid production Fungal production of oxalic acid, a compound that can immobilize Cu by precipitation of copper oxalate, has been implicated in the mechanisms of Cu tolerance of brown-rot fungi [32–34]. However, few studies have evaluated the correlation between Cu tolerance and oxalic acid production. Recently, Clausen et al. [32] reported no statistical relationship between the amount of oxalic acid accumulated by the brownrot fungus Wolfiporia cocos growing in liquid culture or wood and its tolerance to Cu. Similarly, our results reveal a poor correlation between the observed peak oxalic acid concentrations and Cu tolerance. Although the most Cu-tolerant strains in our study were generally oxalate overproducers, very low levels of oxalic acid were detected in the cultures of several brown-rot fungi with outstanding Cu tolerance, e.g. L. sulphureus strain Plswrd. Also, some of the highest oxalate levels were detected in cultures of F. cajanderi in spite of the low Cu tolerance of this isolate. These findings suggest that factors other than oxalic acid accumulation alone must account for the ability of some fungi to grow in the presence of high levels of Cu. Intracellular chelation of metals in the cytosol by a range of ligands (glutathione, metallothioneins) is a key mechanism known to confer Cu tolerance to a variety of ascomycetous fungi [35]; however, very limited information is available to date about metal–peptide complexes in wood-rotting basidiomycetous fungi. Brown-rot fungi produce other metal-chelating organic acids aside from oxalate [27] which may play a role in imparting metal resistance. Cu tolerance and oxalate dynamics in artificial media may differ from those in the wood matrix. For that reason, the
suitability of the strains selected from the screening for application in fungal bioleaching was further tested in assays with preservative-treated wood. 3.4. Fungal metal bioleaching Six brown-rot fungal isolates (A. vaillantii strains DFP7919, DFP-9679, FRLP-14G and H525-Sp, as well as P. placenta strains EMPA-45 and FPRL-280), which displayed both a high Cu tolerance and a high oxalic acid production, were evaluated for their ability to promote the leaching of heavy metals in CC-treated wood. Fig. 3A illustrates the losses of wood mass following solid-state fermentation experiments of wood blocks containing up to 4.4% CC by the selected isolates. Although the extent of wood decay decreased with increasing CC concentration, fungal treatment led to considerable mass losses even in the blocks containing the highest CC levels. P. placenta EMPA-45 was the most aggressive strain with fungal-mediated mass losses of 21.5% from the CCtreated wood (4.4% retention) after 28 days. Fungal decay was accompanied by a decrease in wood pH values from 4.8–5.8 to 2.7–3.2. Fig. 3B and C show the losses of Cr and Cu, respectively, determined after fungal treatment and subsequent leaching of the decayed wood blocks with water. The most effective isolates were A. vaillantii strains DFP-9679, H525-Sp and FRLP-14G, as well as P. placenta strain EMPA-45, which resulted in leaching of up to 61.2% of the Cr from treated wood (2.5% CC) and up to 52.4% Cr from wood treated at the highest retention (4.4% CC) in 28 days. Less than 0.4% of Cr was leached from sterile incubated wood, which indicates that the removal of Cr was achieved by bioleaching. In contrast with the extensive removal of Cr observed in our study, Cu solubilization was rather poor and did not exceed 15.6%. Removal of Cu in the sterile incubated wood ranged from 1.0 to 6.0%. Cu removal efficiencies attained during the treatment of preservative-treated wood by Cu-tolerant microorganisms, including oxalate-producing fungi, have been very variable ranging from 7 to 100% [16,17,36]. Kakitani et al. [37] has suggested that Cu extraction from preservative-treated wood by oxalate can be enhanced by increasing the pH of the extractant. Most metal oxalates are only sparingly soluble, but their solubility can be increased through complex formation. In the case of the Cu–oxalate system, formation of soluble Cu–oxalate complexes (Cu(C2O4)22) is favored at pH values above 2.0 [38]. Several studies have considered the chemical leaching of preservative-treated wood with oxalate or other chelating agents [19,36,37]. High concentrations of chemical extractants and stringent treatment conditions, e.g. mechanical or thermochemical pretreatments such as steam explosion, wood pulping or pyrolysis, have been often applied in those studies to promote metal mobilization [11,39–40]. The metal removal efficiencies attained in chemical extraction experiments appear to vary widely depending on the nature of the preservativetreated wood and extraction conditions applied. Our results showed that chemical extraction of CC-treated wood with oxalic acid attained Cu and Cr removal efficiencies comparable
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produced from degradation of carbohydrate substrates in wood. Enzymatic fungal attack disrupts the fiber structure, thereby increasing lignocellulose porosity and facilitating mass transport. This is expected to favor penetration of chelating agents into the wood cell-wall matrix, promoting dissolution of heavy metals. Improved mass diffusion will likely allow the application of bioleaching at milder treatment conditions as compared to chemical extraction (i.e., lower temperatures, extractant dose and chip size). The economic feasibility of chemical leaching and bioleaching processes for remediating heavy metal pollution will ultimately depend on the effectiveness of these processes, scale of the operation, etc. [41,42]. Taken together, the results of this study indicate the potential of biological treatment with Cu tolerant, oxalate-producing brown-rot fungi to decrease metal levels in decommissioned wood treated with Cu-based preservatives. Improving the solubility of Cu will be the main challenge for future research. Processes combining bioleaching with chemical extraction might offer the best opportunity to improve the removal of Cu from treated wood. Acknowledgements We would like to thank S. Kortekaas for providing skilled technical assistance and helpful discussion. This study was supported by the Nederlandse Maatschappij voor Energie en Milieu (NOVEM, grant no. 351650 6210). References
Fig. 3. Losses of wood mass (A), chromium (B) and copper (C) determined for Scots pine sapwood specimens treated with different concentrations of acidchromated copper preservative (in g CC preservative/100 g wood d.w.), 0 (solid bars), 1.4 (hatched bars), 2.5 (empty bars) and 4.4 (dotted bars) after 28 days of incubation with different Cu-tolerant brown-rot fungi: Antrodia vaillantii strain DFP-7919 (1), A. vaillantii DFP-9679 (2), A. vaillantii H525-Sp (3), A. vaillantii FRLP-14G (4), Poria placenta EMPA-45 (5) and P. placenta FPRL-280 (6). Reported metal and mass losses are corrected for losses determined in abiotic controls. Abiotic mass losses for the untreated and treated wood were equal or lower than 1.4 0.2% of the initial d.w. mass. Abiotic losses of Cr for the treated wood specimens were equal or lower than 0.4 0.2% of the initial Cr content. Abiotic losses of Cu for the wood containing 1.4, 2.5 and 4.4 g CC/100 g wood d.w. were 5.3 0.2, 6.0 0.6 and 3.2 1.3% of the initial Cu content, respectively.
to those of fungal bioleaching, 22.0 and 64.8%, respectively. However, the chemical extraction conditions were aggressive and included vacuum impregnation of wood meal followed by chemical extraction for 5 h with a pH 1.2 solution of highly concentrated oxalic acid (150 mM; liquid to solid ratio = 30/ 1 g g1). A key advantage of bioleaching when compared to chemical leaching is that fungal solid-state fermentation does not require the addition of costly leaching agents as these are
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