1 Introduction

Chromated copper arsenate (CCA) is a chemical compound mixture containing inorganic copper, chromium, and arsenic used for wood preservation since the 1940s. CCA-treated wood has been restricted in most European and Asian countries, and the use of CCA-treated wood will be limited to certain industrial and commercial applications in the USA by 2004. An increase in the amount of waste wood treated with CCA wood preservatives is expected within the next few decades due to CCA-treated wood being removed from service after an average service life of 20–40 years. The release of copper, chromium, and arsenic elements into the environment from CCA-treated wood removed and recycled from service, placed in landfills, and incinerated in commercial kilns has become an increasing concern due to their accumulation in forms leading to toxic effects on biotic life. As a result of public concern over CCA-treated waste wood, research on remediation of such waste has increased during the past decade. Remediation of CCA-treated waste wood prior to recycling, reusing, incineration, and disposal by landfilling will decrease concerns about environmental pollution and the safety of workers involved in such facilities (Kartal and Clausen 2001b, Kartal 2002, 2003, Kartal and Kose 2003).

Previous studies on the chemical and biological remediation of CCA-treated wood showed that certain isolates of fungi and bacteria can readily remediate wood that has been treated with CCA. Clausen and Smith (1998), Clausen (2000), Clausen et al. (2000, 2001), Kartal and Clausen (2001a, b) also showed that the removal of copper, chromium, and arsenic from CCA-treated waste wood increased significantly during oxalic acid extraction. In these studies, oxalic acid has played an important role in partial solubilization of the insoluble metal compounds of CCA wood preservative fixed in the wood. Oxalic acid can be produced in a biotechnological process, because microorganisms such as fungi are capable to secrete oxalic acid at several concentrations into the culture broth. For instance, Aspergillus niger, a filamentous fungus, produces not only oxalic acid but also citric acid and gluconic acids (Cameselle et al. 1998). Santoro et al. (1999) showed that A. niger produced oxalic acid concentration of 37 kg/m3 in the optimum substrate concentration. Pazouki et al. (2000) showed that maximum citric acid concentration of 12 kg/m3 was obtained with A. niger using molasses.

The purpose of the present study was to determine the ability of A. niger for removing heavy metals from treated waste wood as solid medium instead of aqueous solutions. The bioremediation process consisted of two stages. In the first stage, A. niger was cultivated in carbohydrates media in order to produce large quantities of oxalic acid. In the second stage, the fermentation medium and biomass were used as a leaching agent in order to remove heavy metals from CCA-treated wood.

2 Materials and methods

2.1 Wood source

Scots pine (Pinus sylvestris L.) poles were obtained from a preservative treatment plant in Adana, Turkey. The poles were previously treated with chromated copper arsenate Type C wood preservative (CCA-C) solution using a full-cell process at a retention of 21 kg/m3. Sapwood and heartwood portions of the treated poles were separated and the sapwood portions were then chipped with a commercial chipper to approximately 10–17 mm×0.20–0.35 mm×0.15–0.35 mm. The chips were screened and sorted to remove knots and over/under sized chips. The chips were conditioned at 23°C and 65% relative humidity (RH) for two weeks.

2.2 Analysis of preservative retention in the ground wood

The chips were ground to pass a US Standard 40-mesh screen (420 μm). The ground wood samples from the poles were then analyzed for copper, chromium, and arsenic content to determine the initial amounts of the elements in the samples using an ASOMA X-ray fluorescence analyzer (XRF) (ASOMA Instruments, Austin, TX, U.S.A.) according to American Wood Preservers’ Association (AWPA) A9-99 test method (AWPA 1999).

2.3 Microorganism and cultivation

The mutant strain Aspergillus niger was obtained from The Scientific and Technical Research Council of Turkey (TUBITAK). Spores from an established culture (6–7 days old) incubated on potato/glucose agar (PGA) plates at 28°C were used for the preparation of inocula. The cultures were routinely (every 7–10 days) transferred onto fresh PGA plates by streaking. Before A. niger cultures were used for inoculation of liquid growth medium, the fungus was subjected to three transfers on PGA plates.

2.4 Biomass production

Fungal biomass was cultivated in a liquid medium in shaking flasks. The growth medium had the following composition (Camaselle et al. 1998): Sucrose 150 kg/m3; NH4NO3 0.45 kg/m3; KH2PO4 kg/m3; MgSO4 7H2O 0.3 kg/m3; FeSO4 7H2O 0.1 kg/m3; ZnSO4 7H2O 0.25 kg/m3. The pH of the growth medium was adjusted to 6.0 before autoclaving. The medium was autoclaved for 20 min at 121°C. Spores and mycelium from the PGA spreadplate cultures were transferred to 500 ml Erlenmayer flasks containing 300 ml of growth medium. Cultivation of A. niger in the flasks inoculated with spores at a concentration of 106 spores/ml was carried out on a rotary shaker at 150 rpm. The culture temperature was maintained at 28°C.

2.5 pH adjustment

During the first two days of the fermentation, pH of the growth medium was maintained at the optimal values of citric acid production (pH: 2.0–3.0). The pH was then shifted after 48 h and adjusted daily to 6 by addition of 5 N NaOH and 2 N HCl. An additional nitrogen and phosphorus source was added into the flasks in order to enhance the oxalic acid production after 48 h fermentation. Nitrogen and phosphorus sources used were NH4NO3 (2.5 kg/m3) and KH2PO4 (2.5 kg/m3), respectively (Cameselle et al. 1998, Santoro et al. 1999).

2.6 Oxalic acid assay

The concentration of oxalic acid produced during the fermentation in the flasks was determined by High Performance Liquid Chromatography (HPLC) (Hewlett Packard 1090).

2.7 Bioremediation of CCA-C treated wood

For the bioremediation process, CCA-C-treated particles were autoclaved for 20 min at 121°C. The particles of 10 g were placed in the flasks containing 300 ml of growth medium incubated with A. niger for 15 days as described above. The flasks were agitated for 6 h, 1, 3, 5, 7, and 10 days at 150 rpm on a rotary shaker at 28°C. Uninoculated growth medium (UGM) and deionized (DI) water extraction served as control. Two replicates of 10 g particles were removed at each time interval. The contents in the flasks were filtered through Whatman #4 filter papers using a vacuum pump, and rinsed three times with 300 ml of DI water at 20°C. Bioremediated chips were collected on cheesecloth covered screens, oven-dried at 60°C for 24 h and conditioned at 23°C and 65% RH for two weeks.

Bioremediated chips were ground to pass a US Standard 40-mesh screen (420 μm) and then analyzed for remaining copper, chromium, and arsenic content using an ASOMA X-ray fluorescence analyzer (XRF) (ASOMA Instruments, Austin, TX, U.S.A.) according to the AWPA A9–99 test method (AWPA 1999). The percent reduction of copper, chromium, and arsenic in the sawdust samples was calculated based on the initial amount of elements in the samples.

3 Results and discussion

Oxalic acid production into the medium began quickly after the second day of the fermentation. The oxalic acid secretion was then increased as the fermentation continued with controlled pH in order to maximize oxalic acid production. After 11 days, the production rate was constant and the oxalic acid concentration in the medium reached 13.4 kg/m3 at the end of the fermentation with a pH adjustment daily. The pH value plays an important role in the oxalic acid production with Aspergillus niger fermentation in sucrose medium. In addition to the pH effect, an enriched nitrogen and phosphorus medium is necessary in order to conduct the fermentation towards oxalic acid production (Camaselle et al. 1998). Camaselle et al. (1998) also showed that when the fermentation with A. niger was carried out only at a pH higher than 6, the main product was oxalic acid. Under controlled conditions, the oxalic acid production rate and productivity can be greatly increased with better pH control. In our study, the pH shifting technique (fermentation at acid pH in the first two days and then adjusting pH to 6) according to Camaselle et al. 1998 allowed to produce oxalic acid in the shaking flasks.

The results of the X-ray fluorescence spectroscopy analysis for CuO, CrO3, and As2O5 following extraction with deionized (DI) water and uninoculated growth medium (UGM), as well as the fermentation of CCA-treated chips by A. niger are given in Table 1. The results are expressed as mg of each component retained per gram of treated wood following exposure condition. The values represent the average of triplicate samples. CCA-treated chips contained 12.1 mg/g CuO, 31.4 mg/g CrO3, and 20.7 mg/g As2O5 before the bioremediation process. In the chips exposed to DI water and UGM extraction for 10 days, the As2O5 content remaining in the chips was 17.1 mg/g and 12.1 mg/g, respectively; however, A. niger fermentation caused significantly more As2O5 removal (0.6 mg/g).

Table 1 Tabelle 1 Amount of CCA components remaining in chips following exposure to deionized water, uninoculated growth medium and A. niger fermentation (mg/g)a Menge an CCA-Komponenten, die in Spänen verblieben nach Behandlung mit Wachstumsmedium ohne und mit Pilzbeimpfunga deionisiertem Wasser ausgesetzt waren
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The percentages of copper, chromium, and arsenic elements removed from treated chips following extraction with DI water and UGM, and A. niger fermentation are shown in Fig. 1 and Fig. 2, respectively. The total percentage of elements removed from chips with DI water extraction was considerably lower than of those with UGM extraction and A. niger fermentation. Extraction with DI water for 10 days removed about 20% copper, 13% chromium, and 18% arsenic from CCA-treated chips. The exposure of treated chips to an extraction with UGM for 10 days enhanced the removal of metals. This remediation process removed about 30%, 32%, and 42% of the initial concentrations of copper, chromium and arsenic, respectively in CCA-treated chips. These results suggested that the UGM containing mainly sucrose had a potential to absorb metals released from CCA-treated wood particles during extraction.

Fig. 1 Abb. 1
figure 1

Percentage removal of elements from CCA-treated wood by DI water extraction and UGM-extraction

Prozentuale Entfernung von Elementen aus CCA-behandeltem Holz durch DI Wasserextraktion und UGM-Extraktion

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Fig. 2 Abb. 2
figure 2

Percentage removal of elements from CCA-treated wood by A. niger fermentation

Prozentuale Entfernung von Elementen aus CCA-behandeltem Holz durch A. niger Fermentation

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However, the leaching rate and total percentage of arsenic released from treated chips following the A. niger fermentation were considerably higher than the extractions with DI water and UGM. The total percentage of arsenic removed from CCA-treated chips by A. niger fermentation for 10 days was about 97%, whereas about 18% and 42% of arsenic were released from the chips with DI water and UGM extraction for 10 days, respectively. In other words, the percentage arsenic removed from the chips by A. niger fermentation was about 5.4 and 2.3 times greater than that from DI water and UGM-extracted chips, respectively. In addition, the A. niger fermentation for 10 days removed about 49% of the copper and 55% of the chromium from CCA-treated chips. Compared to UGM-extracted chips, the copper and chromium removal was about 1.6 and 1.7 times higher in the bioremediated chips by A. niger. Figure 3 shows the percentage of copper, chromium, and arsenic following DI-water and UGM extraction, and the A. niger fermentation of CCA-treated chips for 10 days.

Fig. 3 Abb. 3
figure 3

Percentage of copper, chromium, and arsenic released following DI-water and UGM extraction, and A. niger fermentation of CCA-treated chips for 10 days

Prozentsatz von freigesetztem Kupfer, Chrom und Arsen durch DI-Wasser und UGM-Extraktion sowie A. niger 10-tägige Fermentation von CCA-behandelten Spänen

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Kartal and Clausen (2001a, b) showed that 0.8% oxalic acid extraction for 18 h removed 23% copper, 65% chromium, and 74% arsenic from CCA-treated wood particles. In addition, Clausen (2000) found that 1% oxalic acid extraction of CCA-treated wafers for 24 h resulted in about 25% copper, 45% chromium, and 90% arsenic removal. A study by Clausen et al. (2001) showed that 53% copper and 60% arsenic were removed from CCA-treated wood particles under the same extraction conditions. In studies by Kartal (2002) and Kartal and Kose (2003), 1% oxalic acid extraction for 24 h removed about 61% copper, 41% chromium, and 75% arsenic from CCA-treated wood. These results suggested that an oxalic acid extraction was relatively ineffective in the removal of chromium and copper from CCA-treated wood while a considerable amount of arsenic was removed due to the chelating ability of oxalic acid on this element. In our recent study, the first fermentation step was stopped at a final oxalic acid concentration of 13.4 kg/m3 in the flasks. In the bioremediation process of the study as a second step, an arsenic removal of 97% was achieved due to oxalic acid produced in the flasks. On the other hand, a passive metal removal or biosorption of heavy metals on fungi may have occurred, because of the functional groups present on the fungal cell surface (Kapoor and Viraraghavan 1997). Although oxalic acid is effective in the removal of heavy metals from CCA-treated wood due to its chelating and reducing capacity (Kartal and Clausen 2001b, Kartal 2002, Kartal and Kose 2003), biosorption of heavy metals from fermentation medium by fungus biomass can play an important role in the bioremediation of treated wood. Kapoor and Viraraghan (1997), Kapoor et al. (1999), and Goyal et al. (2002) also showed that A. niger has heavy metal biosorption sites in its structure and is capable of removing heavy metals from aqueous solutions. A. niger’s ability to produce high amounts of oxalic acid during fermentation under controlled conditions based on pH and effectively removing metals from CCA-treated wood can be used for remediation as an alternative to chemical extraction.

4 Conclusions

Aspergillus niger is able to produce a quite high concentration of oxalic acid using sucrose as a carbon source. Oxalic acid, a chelating and reducing agent, is one of the strongest organic acids. Because it is readily oxidized, it is useful as a reducing agent for bleaching and ink removal. On the other hand, A. niger biomass has an ability to absorb heavy metals from aqueous solutions. This study evaluated the removal of copper, chromium, and arsenic from CCA-treated wood using the A. niger fermentation. Oxalic acid produced by the fungus during fermentation was used for the removal of metal elements via bioleaching. A. niger fermentation removed 97% of arsenic found in the CCA-treated wood. Compared to arsenic, less copper and chromium were removed by an A. niger fermentation. We also observed that uninoculated growth medium containing sucrose can be used as a biosorbent for the removal of heavy metals. The ability of A. niger to remove heavy metals from CCA-treated wood can be considered as a potential alternative to oxalic acid extraction for the acid extraction of treated wood. However, further research on extraction via dual remediation process using different acids or microorganisms is needed to improve the removal efficiency of other CCA components.