Bioleaching of heavy metals from a contaminated soil using indigenous Penicillium chrysogenum strain F1

Journal of Hazardous Materials 233–234 (2012) 25–32

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Bioleaching of heavy metals from a contaminated soil using indigenous Penicillium chrysogenum strain F1 Xinhui Deng a,b , Liyuan Chai a,c,∗ , Zhihui Yang a,c , Chongjian Tang a,c , Haixia Tong a , Pingfu Yuan a a b c

College of Metallurgical Science and Engineering, Central South University, China College of Packing and Material Engineering, Hunan University of Technology, China Chinese National Engineering Research Center for Control & Treatment of Heavy Metal Pollution, China

h i g h l i g h t s     

The leaching efficiency of two-step bioleaching is better than one-step bioleaching using Penicillium chrysogenum strain F1. Carbonate fraction and Fe/Mn oxide fraction of Cu, Cd, Pb and Zn were easily removed. The residual metals were mainly bound in stable fractions after bioleaching. Heavy metals such as Cd, Cu, Pb and Zn have no influence on the metabolism of P. chrysogenum. The resistance method of P. chrysogenum to heavy metals was by adsorbing or preventing entry of metals into the cell.

a r t i c l e

i n f o

Article history: Received 27 January 2012 Received in revised form 1 May 2012 Accepted 22 June 2012 Available online 1 July 2012 Keywords: Penicillium chrysogenum Heavy metal Bioleaching

a b s t r a c t Bioleaching of heavy metals from contaminated soil using Penicillium chrysogenum strain F1 was investigated. Batch experiments were performed to compare leaching efficiencies of heavy metals between one-step and two-step processes and to determine the transformation of heavy metal fractions before and after bioleaching. The results showed that two-step process had higher leaching efficiencies of heavy metals than one-step process. When the mass ratio of soil to culture medium containing P. chrysogenum strain F1 was 5% (w/v), 50%, 35%, 9% and 40% of Cd, Cu, Pb and Zn were removed in one-step process, respectively. The two-step process had higher removals of 63% Cd, 56% Cu, 14% Pb and 54% Zn as compared with one-step process. The results of the sequential extraction showed that the metals remaining in the soil were mainly bonded in stable fractions after bioleaching. The results of TEM and SEM showed that during bioleaching process, although the mycelium of P. chrysogenum was broken into fragments, no damage was obviously observed on the surface of the living cell except for thinner cell wall, smaller vacuoles and concentrated cytoplasm. The result implied that P. chrysogenum strain F1 can be used to remove heavy metals from polluted soil. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Urbanization and industrialization are major causes of soil pollution. In Britain in 1993, 0.1 Mt of toxic waste and 0.26 Mt controlled waste were produced. Of the controlled waste, 22% came from industry. Solid wastes stocking, mining, atmospheric deposition and using of agrochemicals are resulting in the heavy metal pollution in soils. Some smelter slag has stocked without any management at Metallurgical Plant of China in the last decades. Soil pollution occurs when smelter slag is not disposed properly. It is estimated that 100,000 km2 of the cultivated land in China have been polluted, and 12 Mt of grain are contaminated by heavy metal

∗ Corresponding author. Tel.: +86 88836804; fax: +86 88836804. E-mail address: [email protected] (L. Chai). 0304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2012.06.054

every year. The pollution of soil by heavy metal is still a thoroughly unsolved environmental problem all over the world. Electrokinetics, solidification/stabilization and extraction techniques are available for the remediation of contaminated soils [1–5]. Electrokinetics process is particularly applicable to the finegrained soils of low hydraulic conductivity and large specific area, but it has the shortcoming of high cost and complicated operation. The effectiveness of electrokinetics may be declined by sorption of contaminants on soil particle surfaces and various effects induced by the hydrogen ions and hydroxide ions generated at the electrodes. Various enhancement techniques have been developed to improve the remediation of heavy metals, and it often couples with other remediation technologies [6–9]. Heavy metals are precipitated in the soil when solidification is applied, and their bioavailability and mobility are reduced. But the precipitates remaining in the soil may be solubilized under the change in the soil environment, thus solidification has the possibility of secondary

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pollution. Extraction technique is an economical and environment friendly process, which has the disadvantages of longer time, lower rate or less efficient than other remediation. Some measures must be taken to improve the rate of extraction. Bioleaching technology is one of the extraction techniques, which is an effective method to remove heavy metals from contaminated soil. In recent years, bioleaching has been widely used in extraction of metals from low-grade ore metallurgy, remediation of heavy metal polluted soil, removal of heavy metals from sludge, recovery of precious metals from spent batteries and extraction of Mn from electrolytic manganese slag [10–14]. Microorganisms being used for bioleaching are bacteria, such as Acidithiobacillus, Acetobacter, Acidophilum, Arthrobactor and Pseudomonas [15], and the fungi including Penicillium, Aspergillus and Fusarium [16,17]. But there are very few reports available on using P. chrysogenum to bioleach of heavy metals in polluted soil. The objectives of this study are to find out the most suitable method of bioleaching of heavy metals by using P. chrysogenum, to study the mechanisms of heavy metal leaching by P. chrysogenum, and to find out the resistant mechanism of heavy metals for P. chrysogenum. All these works were done in order to provide useful information to researchers and practitioners in this field, to promote the development of bioleaching technology. 2. Materials and methods 2.1. Soil The contaminated soil was collected from the sites under a slag heap at a large smelting industry in Zhuzhou, Hunan Province, Central-South China. This area is well known in China for its notorious heavy metal pollution. Surface (5–10 cm depth) soil samples were manually collected using a scoop and transported to the laboratory in airtight polythene bags. In the laboratory, they were stored at 4 ◦ C for further study. Soil samples were air dried, sieved through a 2 mm sieve and then mechanically mixed to ensure homogeneity and stored prior to experiment. 2.2. Soil characterization The total contents of heavy metals in soils were determined by subjecting to acid digestion mixture (HCl, HNO3 , HClO4 and HF) on an electric heating plate. The digestion solution was diluted with 1% (v/v) nitric acid for heavy metals analysis. Fractionation of heavy metals present in soil was carried out by sequential extraction as described by Tessier et al. [18]. The extractions were conducted in 50 mL polypropylene centrifuge tubes to minimize loss of soil. Between successive extractions, separation was done by centrifugation at 3500 × g for 20 min. The supernatant was filtered and analyzed for heavy metals by inductively coupled plasma-optical emission spectrometer (ICP-OES, Perkin-Elmer Optima 3000 V). Before carrying out next extraction, the residue was washed with 8 mL of distilled water and centrifuged for 20 min, this supernatant was discarded. Minimal volume of rinse water was used to avoid excessive solubilization of solid material. The details of extraction steps are summarized in Table 1. 2.3. Spore collection and inoculum preparation A fungi strain was isolated from the polluted soil under slag heap at a smelting industry in Zhuzhou, Hunan Province, Central-South China, which was identified as P. chrysogenum by sequencing 18S rDNA and ITS. The 18S rDNA sequence size was 1069, ITS4 and ITS5 sequences size were 559. Its similarity of gene sequence of 18S rDNA and ITS5 showed 100% with P. chrysogenum, which of ITS4

Table 1 Steps of heavy metal extract fractions. Step

Extract fraction

Extract reagent and method

Step1

Water-soluble fraction

Step2

Ion-exchange fraction

Step3

Carbonate fraction

Step4

Reducible fraction

Step5

Weak organic fraction

Step6

Strong organic fraction

Step7

Residual fraction

25 mL double-deionized water (pH 7.0), shaken at room temperature for 2h 25 mL 1 mol/L MgCl2 (pH 7.0), shaken at room temperature for 2 h 25 mL 1 mol/L NaAc (pH 5.0), shaken at room temperature for 5 h 50 mL 0.25 mol/L NH2 OH·HCl in 0.25 mol/L HCl, shaken at room temperature for 6 h. 50 mL 0.1 mol/L Na4 P2 O7 (pH 10), shaken at room temperature for 3 h (1) 5 mL 30% hydrogen peroxide and 3 mL HNO3 , shaken at 83 ± 3 ◦ C for 1.5 h, (2) 3 mL 30% hydrogen peroxide, stirred for 70 min, (3) 2.5 mL 3.2 mol/L NH4 Ac was added and diluted to 25 mL and standing for 10 h Acid digestion describe as the determination of total content of heavy metal in soil

showed 99% with P. chrysogenum. The strain was cultured in a modified Chashi liquid medium with a composition of 90 g/L glucose, 1 g/L K2 HPO4 , 3 g/L NaNO3 , 0.5 g/L MgSO4 , 0.5 g/L KCl and 0.01 g/L FeSO4 . The pH value of liquid medium was adjusted to 7.0 with 0.1 mol/L HCl. The spores for inoculation were collected from the modified Chashi agar medium, which were composed of glucose 30 g/L, K2 HPO4 1 g/L, NaNO3 3 g/L, MgSO4 0.5 g/L, KCl 0.5 g/L, FeSO4 0.01 g/L and 20 g/L agar at pH 7.0. The spores were counted using a Petroff-Hausser counting chamber where 1 mL of spore suspension (approximately 7.5 × 108 spores) was obtained. Then 1 mL of spore suspension was added to 49 mL medium in a 250 mL flask. Flasks containing modified Chashi liquid medium and soil were autoclaved at 115 ◦ C for 30 min to achieve sterilization. 2.4. Bioleaching experiments Bioleaching experiments were carried out in 250 mL autoclaved conical flasks. In the one-step bioleaching process, 2.5 g autoclaved soil and 1 mL of spore suspension were added into 49 mL culture medium. A control experiment was carried out in parallel without inoculation. All flasks were agitated in a rotary shaking incubator (120 rpm) at 25 ◦ C for 15 days. In the two-step bioleaching process, 1 mL of spore suspension was inoculated in 49 mL of modified Chashi liquid medium for 7 days (it is defined as the first step). After 7 days cultivation, 2.5 g autoclaved soil was added and bioleached in a shaking incubator (120 rpm) at 25 ◦ C for 8 days (it is defined the second step). All the experiments were run in triplicate. Before filtration, the samples were weighed and the water loss due to evaporation was replenished with distilled water every 3 days. During the bioleaching experiment, the variation of pH was measured at selected time by pH-meter (pHS-3B). After the bioleaching process, the mixtures of soil, mycelia and liquid medium were filtered, and then the mycelia adhering on the soil were sought out. The soil was washed with deionized water for three times. Thereafter, the soil was air-dried for 24 h. Five grams of the soil from assay were digested according acid digestion mixture (HCl, HNO3 , HClO4 and HF) as described in Section 2.2. 2.5. Observation of TEM and SEM The mycelium was collected for visual examination by a scanning electron microscope (SEM) and a transmission electron microscope (TEM) after 15 days incubation. Prior to observation

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Fig. 2. Change of pH value during bioleaching. Fig. 1. Different bioleaching methods of Penicillium chrysogenum strain F1.

under the electron microscope, the samples were washed three times with phosphate buffer (pH = 7.0) and prepared according to methods described by Chen et al. [19–21]. The morphology of P. chrysogenum was examined by a Hitachi S-4800 scanning electron microscope (SEM) and photographed in the TEM JEM-1230. 2.6. HPLC analysis The metabolites of P. chrysogenum, such as glucose acid, pyruvic acid, citric acid, oxalic acid, malic acid, succinic acid were determined with a HPLC system equipped with a Zorbax C18 column (250 mm × 4.6 mm) and a diode array detector (DAD) at 210 nm. The mobile phase was composed of 0.01 mol/L KH2 PO4 –H3 PO4 (pH 2.6) and 3% (v/v) methanol at a flow rate of 0.5 ␮L/min. While the glucose analysis was carried out on HPLC with carbohydrate column (250 mm × 4.6 mm) with differential refractive index detector (RID). The mobile phase was 80% acetonitrile and 20% (v/v) H2 O at a flow rate of 1 mL/min, and the operation was carried out at 30 ◦ C. An external standard method was used to quantify organic acids and glucose. 3. Results 3.1. Extraction of heavy metals Alternative bioleaching approaches (e.g. one-step bioleaching and two-step bioleaching) affected the metabolism of P. chrysogenum, and it could further affect the bioleaching efficiencies of heavy metals. Fig. 1 shows the metals (Cd, Cu, Pb and Zn) bioleaching efficiencies in the one-step and two-step bioleaching processes. The results indicated that the metals bioleaching efficiencies in two-step bioleaching were obviously higher than those in one-step bioleaching. For instance, in one-step bioleaching, 50% Cd, 35% Cu, 9% Pb and 40% Zn were leached from the polluted soil. While in two-step bioleaching, the highest metal extraction efficiencies of Cd, Cu, Pb and Zn were 63%, 56%, 14% and 54%, respectively. Only a small amount of Cu and Zn (Cu 1.8% and Zn 1.4%) were extracted in the control experiment. For both one-step and two-step bioleaching, the bioleaching efficiency of heavy metals from soil was in a decreasing order Cd > Zn ≈ Cu > Pb. Bioleaching efficiency is associated with the substrate concentration. In this study, the mass ratio of soil to culture medium containing P. chrysogenum strain F1 was 5% (w/v), indicating that 1 g

soil should add 20 mL culture medium. However, the mass ratio of soil to culture medium for Acidithiobacillus thiooxidans was 0.5–2% (w/v) as reported by Liu et al. [22–25], indicating that 1 g soil should add 50–100 mL culture medium. In addition, when P. chrysogenum was employed to remove heavy metals from soils, 50 mL medium can remove 11.0 mg of heavy metals (Cd, Cu, Pb and Zn). However, only 2.49 mg or 5.07 mg heavy metals (Zn, Cu, Pb) can be leached out when indigenous A. thiooxidans was used as reported by Liu et al. [22,23], The removal amount per unit medium for P. chrysogenum was higher than that for A. thiooxidans. Therefore, P. chrysogenum showed higher removal efficiencies of heavy metals as compared with A. thiooxidans in some cases. 3.2. pH variation P. chrysogenum produced organic acids through metabolism (e.g. the oxidation of glucose), which resulted in the change of pH value. Fig. 2 shows the pH variation during the one-step and the two-step bioleaching. It shows a reduction in pH at the beginning, but 10 days later, pH achieved an increase in the two bioleaching approaches. However, pH value decreased more dramatically in two-step process than in one-step process at the beginning. Seven days later, the pH attained about 2.9 in two-step process and about 4.6 in one-step. Then it increased to 3.8 in two-step process and 7.6 in one-step process after 20 days. 3.3. Fractions of heavy metal before and after bioleaching The mobility and bioavailability of the metals depend on the specific chemical fractions or binding forms in soils. In order to determine the suitability of decontaminated soil for land application, it is very necessary to examine the different fractions of heavy metals present in the soil and the changes in the fractions likely to occur after treatment. The bioleaching efficiency of heavy metals also depends on the specific chemical form of the metals present in the soil [26]. Heavy metals in exchangeable, carbonate and Fe/Mn oxide-bonded fraction are considered to be more mobile and bioavailable. The organic and residual fractions are considered to be more stable and non bio-available than metals in exchangeable, carbonate and Fe/Mn oxide-bonded fractions [22]. The heavy metal fractions before and after bioleaching are shown in Fig. 3. There was no denying that total content of all the heavy metal was decreased after bioleaching. The results

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Fig. 3. Fraction contents of heavy metals (Cd, Cu, Pb and Zn) before and after bioleaching (one-step/two-step), alphabet A, B, C, D, E, F and G represent water-soluble, ion-exchangeable, carbonate bound, ferric-manganese oxidation, weak organics bound, strong organics bound and residual fractions, respectively.

also indicated that the heavy metal fractions were changed after bioleaching. Fraction variation of cadmium in soil is shown in Fig. 3(a). Prior bioleaching, cadmium in soils was mainly present in the residual fraction (15.4 mg/kg) followed by ion-exchangeable fraction (9.8 mg/kg), Fe/Mn oxide-bonded fraction (8.2 mg/kg) and strong organic fraction (7.2 mg/kg) (Table 2). After one-step bioleaching the residual fraction was decreased largely. Meanwhile, there also showed a decrease of ion-exchangeable fraction, carbonate-bonded fraction, and strong organics fraction individually. Water-soluble fraction was increased slightly. There were different changes of heavy metal fractions after two-step bioleaching, and all the fractions except for water-soluble fraction were dramatically decreased. Changes in concentration of copper bonded to different fractions during bioleaching are shown in Fig. 3(b). Before bioleaching, copper was mainly bonded to Fe Mn oxides (651.1 mg/kg). Residual fraction of copper was 523.7 mg/kg. Strong organic fraction was 429.5 mg/kg. Carbonate-bonded fraction of copper accounted for 238 mg/kg (Table 2). Water-soluble, ion-exchangeable and weak organic fractions showed the lowest concentrations in soil,

the respective values being 1.1 mg/kg, 2.0 mg/kg, and 3.0 mg/kg. Water-soluble fraction, ion-exchangeable fraction and organic fraction were increased after the one-step bioleaching, however carbonate-bonded, Fe Mn oxide-bonded and residual fractions were decreased. During two-step bioleaching, carbonate-bonded fraction was completely solubilized, The contents of Fe Mn oxidebonded, strong organic and residual fractions were decreased too. On the contrary, two easier mobile fractions such as water-soluble fraction and ion-exchangeable fraction were increased. Fractionation variation of lead in soil during bioleaching is shown in Fig. 3(c). Prior to bioleaching, lead was mainly present in Fe Mn oxide-bonded fraction followed by residual fraction and strong organic fraction. There was no weak organic fraction and ion-exchangeable fraction of lead before bioleaching. Scarcely any water-soluble fraction of lead existed in the soil before bioleaching. Water-soluble fraction, weak organic fraction and the residual fraction were all increased both in the one-step bioleaching and two-step bioleaching. Especially, the residual fraction showed a large increase. A decrease of carbonate-bonded fraction, Fe Mn oxide-bonded fraction and strong organic fraction were observed similarly in the two methods. Ion-exchangeable

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Table 2 Fractionation of heavy metals present in soil. Sl. no.

Total content before bioleaching (mg/kg)

Water soluble fraction (mg/kg)

1 2 3 4

Cd (48.4) Cu (1848.6) Pb (1889.6) Zn (5682)

1.03 1.12 0.12 13.42

± ± ± ±

0.1 0.4 0.1 3.1

Ion-exchangeable fraction (mg/kg) 9.78 2.02 0 44.82

± ± ± ±

0.6 0.3 0 4.3

Carbonate fraction (mg/kg) 6.5 238.1 161.94 386.44

± ± ± ±

Fe Mn oxide fraction (mg/kg) 0.2 31 7.4 11

8.24 651.14 672 2027.2

± ± ± ±

0.4 59 63 122

Weak organic fraction (mg/kg) 0.24 2.96 0 3

± ± ± ±

0.04 0.8 0 0.1

Strong organic fraction (mg/kg) 7.2 429.54 501 1266.26

± ± ± ±

0.5 61 28 136

Residual fraction (mg/kg) 15.41 523.72 554.54 1940.86

± ± ± ±

0.2 52 32 142

Data represent average of quintuplicates and ± indicates standard deviation.

fraction was increased a small amount in the two-step bioleaching and no change was observed in the one-step bioleaching. Variation in fraction of zinc present in soil during bioleaching is showed in Fig. 3(d). Before bioleaching, zinc was mainly bonded to Fe Mn oxide-bonded fraction at a concentration of 2027.2 mg/kg, followed by residual fraction at 1940.9 mg/kg and strong organic fraction at 1266.3 mg/kg. Zinc in carbonate-bonded fraction was 386.4 mg/kg. Zinc concentrations in water-soluble fraction, ion-exchangeable fraction and weak organic fraction were 13.4 mg/kg, 44.8 mg/kg and 3 mg/kg, respectively (Table 2). The similar changes showed both in the one-step bioleaching and in the two-step bioleaching. For example, water soluble fraction and ion-exchangeable fraction were increased, while the other fractions including carbonate-bonded fraction, Fe Mn oxide-bonded fraction, organic fraction and residual fraction were decreased.

bioleaching. As a result, glucose acid, pyruvic acid, citric acid, oxalic acid, malic acid and succinic acid were all detectable. The retention time and concentrations of organic acids were listed in Table 4. The total organic acid concentrations were 472.6 mg/L in one-step bioleaching and 520.8 mg/L in two-step bioleaching after 15 days. A large amount of glucose acid, oxalic acid and pyruvic acid were generated both in the two-step bioleaching and in onestep bioleaching. Little citric acid and malic acid (<0.02 mg/L) were produced. Succinic acid in the two-step bioleaching process was more than in one-step bioleaching process. Meanwhile, almost all glucose was oxidized after bioleaching, with the residual concentration of 0.02 mg/L.

3.4. Morphology of strain F1

4.1. Bioleaching efficiency of heavy metals and liquid medium pH value

After bioleaching, the granular soil was converted into mud adhering to the surface of the mycelium and little soil minerals were precipitated under the culture medium. From the SEM images (Fig. 4(a) and (b)), the mycelium was broken into fragments which were mixed with spores and granular soil. The impact of heavy metals on the microorganism was evaluated by TEM images of living P. chrysogenum cell sections before (Fig. 4(c)) and after bioleaching (Fig. 4(d)). By comparison, no significant damage was observed on the surface of the living cell during bioleaching. After bioleaching, the cell was surrounded by extracellular materials and filled with concentrated cytoplasm. Some of the cell walls either became thinner or began to be lysed. Moreover, a part of vacuoles got smaller. 3.5. Mass balances of heavy metals The soils after bioleaching were analyzed to evaluate a mass balance for Cd, Zn, Pb and Cu. The content of Cd, Zn, Pb and Cu in the soils and in the leachate was determined after bioleaching. The total content of heavy metals in the soils and in the leachate after bioleaching was then compared to the initial content of Cd, Zn, Pb and Cu in the soils before bioleaching. The results showed that in the one-step bioleaching, 0.63 mg/kg Cd, 68.4 mg/kg Cu, 9.45 mg/kg Pb and 204.55 mg/kg Zn were lost. Approximately, the loss percentages of Cd, Cu, Pb and Zn accounted for 1.3%, 3.8%, 0.5%, and 3.6%, respectively. 0.82 mg/kg (1.7%) Cd, 72.1 mg/kg (3.9%) Cu, 13.23 mg/kg (0.7%) Pb and 215.92 mg/kg (3.8%) Zn were not accounted for in the two-step bioleaching. It could be concluded that most of missing Cd, Zn, Pb and Cu were likely adsorbed in fungal biomass [27] (Table 3). 3.6. Oxidation of glucose in the medium and production of organic acid Heavy metal and pH value can affect the regulation of enzyme. In this study, glucose acid, pyruvic acid, citric acid, oxalic acid, malic acid and succinic acid were selected to be determined after

4. Discussion

Heavy metal bioleaching efficiency in the control experiment can be negligible. The two-step bioleaching process was more efficient than the one-step bioleaching process to remove heavy metals from the contaminated soils. The results were attributed to the organic acids production and the decline of the pH value. In the one-step leaching process, pH value decreased with the production of organic acids during the growth of P. chrysogenum, but it did not decrease so much as compared with the two-step leaching process. In addition, the pH value began to increase after 7 days during one-step leaching process and even was higher at the end than that at the beginning. In contrast, the pH value did not change so much during the two-step bioleaching process. According to the pH value, an amount of organic acids has been produced by P. chrysogenum before adding into soil. Once the soil was added into pre-cultured medium containing P. chrysogenum, the heavy metals were solubilized rapidly and chelated tightly by the organic acids already produced in the two-step bioleaching. Thus higher bioleaching efficiencies of heavy metals were obtained in the two-step bioleaching. Hence, we may conclude that heavy metals in soil were extracted mainly due to the solubilization and chelation of metabolites by P. chrysogenum [28]. Comparatively, Cd and Zn were more easily removed from soil than Cu and Pb at low pH [29]. This is related to the interaction between the heavy metal and the soil surface, the chemical form of heavy metals and the characteristics of heavy metals themselves [30]. Since Cd concentration was sizeable in water-soluble, exchangeable, carbonate and Fe/Mn oxide-bonded fractions (Fig. 4(a)), these fractions of cadmium were more easily to be bioleached than other fractions. However, the higher bioleaching efficiency of Zn is due to the low affinity interaction between Zn and the soil surface [31,32]. Thus the higher affinity interaction with Cu/Pb and soil surface and their stable chemical forms in the soil led to their lower bioleaching efficiencies. In addition, there are Cl− and C2 O4 2− in the leaching liquid, insoluble salts are formed when Cl− , C2 O4 2− and Pb are in the same liquid. It is may be one factor of lower bioleaching efficiency of Pb.

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Table 3 Mass balances between before and after bioleaching. Initial heavy metal content (mg/kg)

Cd (48.4) Cu (1848.6) Pb (1889.6) Zn (5682)

Bioleaching amount (mg/kg)

Heavy metal content after bioleaching (mg/kg)

Difference between before and after bioleaching (mg/kg)

One-step bioleaching

One-step bioleaching

One-step bioleaching

Two-step bioleaching

0.63 68.4 9.45 204.55

0.82 72.1 13.23 215.92

24.2 647 170.1 2272.8

± ± ± ±

0.01 0.3 0.1 0.2

Two-step bioleaching 30.5 1035.2 264.5 3068.3

± ± ± ±

0.2 0.3 0.2 0.1

23.6 1133.2 1710.1 3204.7

± ± ± ±

0.01 0.3 0.1 0.2

Two-step bioleaching 17.1 741.3 1611.9 2397.8

± ± ± ±

0.2 0.3 0.2 0.1

Data represent average of triplicates and ± indicates standard deviation.

4.2. Change of heavy metal fraction The change of heavy metal fraction reflects the heavy metal mobility and bioavailability change. The sequential chemical extraction procedures can help in assessing the potential mobility and solubility of metals in the contaminated soils approximately.

The heavy metal fraction is mainly bonded to the stable fraction after bioleaching, resulting in the minimizing of heavy metal mobility and bioavailability [33]. Moreover, the heavy metal fraction is changed from difficultly extracted fraction to easily extracted fraction after bioleaching, which is favorable for the next extraction [34].

Fig. 4. Micrograph of strain F1 taken by transmission electron microscope (a means the interaction between mycelium and soil in the one-step bioleaching; b means the interaction between mycelium and soil in the two-step bioleaching; c means the control; d means the bioleaching mycelium; magnifications are at (a, b) 100,000×, (c, d) 3000×; arrows show some of the changes of the cells before and after bioleaching).

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Table 4 Production of organic acids and oxidation of glucose after 15 day bioleaching. Item

Retention time (min)

Equation of quantification

Correlation coefficient

Content in one-step bioleaching (mg/L)

Content in two-step bioleaching (mg/L)

Glucose acid Pyruvic acid Citric acid Oxalic acid Malic acid Succinic acid Glucose

3.512 5.272 9.149 3.981 8.688 10.810 6.532

Height = 13.051*Amt + 14.387 Height = 0.548*Amt + 0.331 Height = 80.050*Amt + 1.354 Height = 0.246*Amt − 3.518 Height = 0.013*Amt − 0.025 Height = 0.015*Amt − 0.034 Area = 103,691.153*Amt + 84,142.8

0.997 0.998 0.999 0.998 0.999 0.999 0.999

230.2 120.7 <0.01 121.7 <0.01 <0.01 0.03

102.2 191.6 0.02 156.4 0.03 70.6 0.02

Bioleaching efficiency of cadmium in two-step bioleaching was better than in one-step bioleaching. This is because that cadmium was exposed to a more acid environment for two-step bioleaching than that for one-step bioleaching (Fig. 2). Ion-exchangeable fraction, carbonate and Fe Mn oxide-bonded fractions, strong organic fraction and residual fraction of cadmium were solubilized largely. Cadmium remaining in soils was mainly bonded to the organic fraction and residual fraction. Heavy metals bonded to organic fraction and residual fraction is inert and the complexation is stronger. The results implied that Cd was not only decreased from the total content but also decreased from the mobility and bioavailability after bioleaching using P. chrysogenum. From the whole, bioleaching efficiency of Cu in two-step bioleaching was clearly better than in one-step bioleaching. Especially, carbonate-bonded fraction was completely solubilized. Fe Mn oxide-bonded fraction also showed a high solubility. Strong organic fraction and residual fraction were partly solubilized. After bioleaching, strong organic fraction and residual fraction of copper were still a large proportion, indicating that copper is more preferred to be associated with stable fraction [35]. For Cu, the influence of bioleaching was mainly on the carbonate and Fe Mn oxide-bonded fractions. At the same time, water-soluble and ionexchangeable fractions of Cu showed an increase, which may be helpful to be extracted for next step. After bioleaching, lead remaining in soils is mainly in the residual fraction, but carbonate and Fe Mn oxide-bonded fractions and strong organic fraction were mainly solubilized during bioleaching. Residual fraction content of lead was increased largely after bioleaching. The present results are different from those reported by Chen et al. [36]. This may be attributed to the bioleaching environmental factors, organic acids and heavy metal itself. Residual fraction and strong organic fraction were the main fractions of zinc. Carbonate and Fe Mn oxide-bonded fractions were mostly solubilized during bioleaching, resulting in the increasing of water-soluble and ion-exchangeable fractions. Most of Zn in the residual fraction and strong organic fraction was removed. It attributed to the low levels of pH at the end of bioleaching. It could lead to the removal of metal from these fractions when the soil remained in highly acidic environment for two weeks [37]. 4.3. Resistance mechanism of P. chrysogenum to heavy metals In general, microbial resistance to heavy metals includes a variety of strategies in the environment. These strategies are either to prevent entry of the metal into the cell or to actively pump the metal out of the cell. The resistance to heavy metals can be divided into two classes: metal dependent and metal independent [38]. Under high metal stress, it may become necessary for resistant microorganisms to use specifically directed metal-resistance mechanisms, including ATP-dependent efflux pumps and intracellular sequestration, which may be more effective for detoxifying by increasing the resistance ability of cell membranes for metal penetration under high bio-available metal conditions. In this study, strain F1 survived well and metabolized normally during bioleaching, and it is

proved that filamentous fungi had strong resistance to heavy metals [39]. The heavy metals resistance strategies for P. chrysogenum’s were either by adsorbing or by preventing entry of the metal into the cell. 4.4. Production of organic acid Information on P. chrysogenum’s metabolism under heavy metal stress has not been reported. Glucose acid, pyruvic acid, citric acid, oxalic acid, malic acid and succinic acid were all detected in the culture medium. It can be speculated that hexose monophosphate pathway may be P. chrysogenum metabolic pathway during bioleaching. It indicated that heavy metals such as Cd, Cu, Pb and Zn have no influence on the metabolism of P. chrysogenum. This result was different from Penicillium simplicissimum reported by Amiri et al. [40]. Little citric acid and lots of pyruvic acid existed in the culture medium. The result illustrates that an extremely small amount of pyruvic acid may entered in tricarboxylic acid cycle during the metabolism of glucose after bioleaching for 15 days. At the same time, almost all glucose was consumed. So the concentration (90 g/L) was economical and suitable for glucose as carbon source. 5. Conclusion P. chrysogenum strain F1 cultivated in modified Chashi liquid medium is highly effective for removal of heavy metals in polluted soil at 5% (w/v) addition rate of soil to culture medium containing P. chrysogenum strain F1. When P. chrysogenum strain F1 is used for bioleaching, the two-step bioleaching is better than one-step bioleaching. P. chrysogenum strain F1 has strong resistance to Cu, Pb, Zn and Cd, and it can exist intactly at 5% (w/v) addition rate of soil to culture medium. The changing of heavy metals fractions after bioleaching can decrease their bioavailability and toxicity. The result implied that P. chrysogenum strain F1 can be used to remove heavy metals from polluted soil. Acknowledgments This work was financially supported by the National Funds for Distinguished Young Scientist (50925417), the National Natural Science Foundation of China (51074191) and the National Key Technology Research and Development Program (2012BAC09B04). We express our thanks to all colleagues in our group for their assistances in field work. References [1] R. Franz, L. Cornelia, Phytoextraction of cadmium and phytostabilisation with Mugwort, Water Air Soil Poll. 216 (2011) 93–103. [2] V.U. Valentina, Bioremediation of toxic heavy metals using acidothermophilic autotrophes, Bioresource Technol. 97 (2006) 1237–1242. [3] K.J. Jacek, P.S. Zofia, K. Piotr, Mycorrhizal fungi and ectomycorrhiza associated bacteria isolated from an industrial desert soil protect pine seedlings against Cd(II) impact, Ecotoxicology 16 (2007) 449–456. [4] A. Burt, W. Maher, A. Roach, F. Krikowa, P. Honkoop, B. Bayne, The accumulation of Zn, Se, Cd and Pb and physiological condition of Anadara trapezia transplanted

32

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15] [16] [17] [18]

[19]

[20]

[21]

X. Deng et al. / Journal of Hazardous Materials 233–234 (2012) 25–32 to a contamination gradient in Lake Macquarie, New South Wales, Australia, Mar. Environ. Res. 64 (2007) 54–78. L.Y. Chai, S.H. Huang, Z.H. Yang, B. Peng, Y. Huang, Y.H. Chen, Cr (VI) remediation by indigenous bacteria in soils contaminated by chromium-containing slag, J. Hazard. Mater. 167 (2009) 516–522. A. Giannis, E. Gidarakos, A. Skouta, Application of sodium dodecyl sulfate and humic acid as surfactants on electrokinetic remediation of cadmium contaminated soil, Desalination 211 (2007) 249–260. K. Maturi, K.R. Reddy, Cosolvent-enhanced desorption and transport of heavy metals and organic contaminants in soils during electrokinetic remediation, Water Air Soil Poll. 189 (2008) 199–211. D.M. Zhou, C.F. Deng, L. Cang, A.N. Alshawabkeh, Electrokinetic remediation of a Cu Zn contaminated red soil by controlling the voltage and conditioning catholyte pH, Chemosphere 61 (2005) 519–527. E. Gidarakos, A. Giannis, Chelate agents enhanced electrokinetic remediation for removal cadmium and zinc by conditioning catholyte pH, Water Air Soil Poll. 172 (2006) 295–312. P. Delvasto, A. Valverde, A. Ballester, J.A. Munoz, F. Gonzalez, M.L. Blazquez, J.M. Igual, C.B. Garcia, Diversity and activity of phosphate bioleaching bacteria from a high phosphorus iron ore, Hydrometallurgy 92 (2008) 124–129. R.K. Naresh, R. Nagendran, Changes in nutrient profile of soil subjected to bioleaching for removal of heavy metals using Acidithiobacillus thiooxidans, J. Hazard. Mater. 156 (2008) 102–107. C. Löer, A. Zehnsdorf, P. Hoffmann, H. Seidel, Remediation of heavy metal polluted sediment by suspension and solid-bed leaching: estimate of metal removal efficiency, Chemosphere 66 (2007) 1699–1705. L. Zhao, N.W. Zhu, X.H. Wang, Comparison of bio-dissolution of spent Ni Cd batteries by sewage sludge using ferrous ions and elemental sulfur as substrate, Chemosphere 70 (2008) 974–981. B.P. Xin, B. Chen, N. Duan, C.B. Zhou, Extraction of manganese from electrolytic manganese residue by bioleaching, Bioresource Technol. 102 (2011) 1683–1687. C.N. Mulligan, R.G. Cloutier, Bioremediation of metal contamination, Environ. Monitor. Assessment 84 (2003) 45–60. M. Valix, F. Usai, R. Malik, Fungal bioleaching of low grade laterite ores, Miner. Eng. 14 (2001) 197–203. W. Burgstaller, F. Schinner, Leaching of metals with fungi, J. Biotechnol. 27 (1993) 91–116. A. Tessier, P.G.C. Campbell, M. Bissom, Sequential extraction procedure for the speciation of particulate trace metals, Anal. Chem. 51 (1979) 844–851. X.C. Chen, J.Y. Shi, Y.X. Chen, X.H. Xu, S.Y. Xu, Y.P. Wang, Tolerance and biosorption of copper and zinc by Pseudomonas putida CZ1 isolated from metal polluted soil, Can. J. Microbiol. 52 (2006) 308–316. X.C. Chen, J.Y. Shi, Y.X. Chen, Immobilization of heavy metals by Pseudomonas putida CZ1/goethite composites from solution, Colloids Surf. B 61 (2008) 170–175. C. Chen, J.L. Wang, Investigating the interaction mechanism between zinc and Saccharomyces cerevisiae using combined SEM–EDX and XAFS, Appl. Microbiol. Biotechnol. 79 (2008) 293–299.

[22] Y.G. Liu, M. Zhou, G.M. Zeng, X. Wang, X. Li, T. Fan, W.H. Xu, Bioleaching of heavy metals from mine tailings by indigenous sulfur-oxidizing bacteria: effects of substrate concentration, Bioresource Technol. 99 (2008) 4124–4129. [23] Y.G. Liu, M. Zhou, G.M. Zeng, X. Wang, X. Li, T. Fan, Effect of solids concentration on removal of heavy metals from mine tailings via bioleaching, J. Hazard. Mater. 141 (2007) 202–208. [24] S.Y. Chen, J.G. Lin, Effect of substrate concentration on bioleaching of metal contaminated sediment, J. Hazard. Mater. 82 (2001) 77–89. [25] D. Mishra, D.J. Kim, D.E. Ralph, J.G. Ahn, Y.H. Rhee, Bioleaching of metals from spent lithium ion secondary batteries using Acidithiobacillus ferrooxidans, Waste Manage. 28 (2008) 333–338. [26] A. Pathak, M.G. Dastidar, T.R. Sreekrishnan, Bioleaching of heavy metals from sewage sludge: a review, J. Environ. Manage. 90 (2009) 2343–2353. [27] C.N. Mulligan, M. Kamali, B.F. Gibbs, Bioleaching of heavy metals from a lowgrade mining ore using Aspergillus niger, J. Hazard. Mater. 110 (2004) 77–84. [28] K. Bosecker, Bioleaching: metal solubilization by microorganisms, FEMS Microbiol. Rev. 20 (1997) 591–604. [29] C.N. Mulligan, R.N. Yong, B.F. Gibbs, Remediation technologies of metal contaminated soils and groundwater: an evaluation, Eng. Geol. 60 (2001) 193–207. [30] S.R. Gummuluru, R.R. Krishnamurti, Solid solution speciation and phytoavailability of copper and zinc in soils, Environ. Sci. Technol. 36 (2002) 2645–2651. [31] S.R. Burckhard, A.P.M.K.B. Schwab, The effects of organic acids on the leaching of heavy metals from mine tailings, J. Hazard. Mater. 41 (1995) 135–145. [32] Y.J. Wang, D.M. Zhou, Basic energy parameters of Cu/Zn at the interface of soil/water and their environmental chemical behaviors, Doctoral Dissertation, Nanjing Institute of Soil Science, Chinese Academy of Sciences, 2008, pp. 67–78. [33] W.H. Zhang, H. Huang, F.F. Tan, H. Wang, R. l. Qiu, Influence of EDTA washing on the species and mobility of heavy metals residual in soils, J. Hazard. Mater. 173 (2010) 369–376. [34] A. Giannisa, D. Pentarib, J.Y. Wang, E. Gidarakos, Application of sequential extraction analysis to electrokinetic remediation of cadmium, nickel and zinc from contaminated soils, J. Hazard. Mater. 184 (2010) 547–554. [35] L. Lazzari, L. Sperni, P. Bertin, B. Pavoni, Correlation between inorganic (heavy metals) and organic (PCBs and PAHs) micropollutant concentrations during sewage sludge composting process, Chemosphere 41 (2000) 427–435. [36] Y.X. Chen, Y.M. Hua, S.H. Zhang, G.M. Tian, Transformations of heavy metal forms during sewage sludge bioleaching, J. Hazard. Mater. B 123 (2005) 196–202. [37] R.N. Kumar, R. Nagendran, Fractionation behavior of heavy metals in soil during bioleaching with Acidithiobacillus thiooxidans, J. Hazard. Mater. 169 (2009) 1119–1126. [38] T.M. Roane, I.L. Pepper, Microbial remediation of soils co-contaminated with 2,4-dichlorophenoxyacetic acid and cadmium, in: Proceedings of the 12th Annual Conference on Hazardous Research, Kansas City, USA, 1997, pp. 343–356. [39] S.A. Sarabjeet, G. Dinesh, Microbial and plant derived biomass for removal of heavy metals from wastewater, Bioresource Technol. 98 (2007) 2243–2257. [40] F. Amiri, S. Yaghmaei, S.M. Mousavi, Bioleaching of tungsten-rich spent hydrocracking catalyst using Penicillium simplicissimum, Bioresource Technol. 102 (2011) 1567–1573.