Combined effects of Cu, Cd, Pb, and Zn on the growth and uptake of consortium of Cu-resistant Penicillium sp. A1 and Cd-resistant Fusarium sp. A19

Journal of Hazardous Materials 171 (2009) 761–766

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Combined effects of Cu, Cd, Pb, and Zn on the growth and uptake of consortium of Cu-resistant Penicillium sp. A1 and Cd-resistant Fusarium sp. A19 Rong Pan a , Lixiang Cao b , Renduo Zhang a,∗ a b

School of Environmental Science and Engineering, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, PR China School of Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, PR China

a r t i c l e

i n f o

Article history: Received 10 January 2009 Received in revised form 14 June 2009 Accepted 15 June 2009 Available online 21 June 2009 Keywords: Bioaccumulation Biosorption Fusarium Heavy metals Penicillium

a b s t r a c t Microorganisms can be important biosorbents for heavy metal remediation of contaminated soils and wastewaters. With different types and concentrations of heavy metals, strains display different resistance and removal abilities to the heavy metals. The objective of this study was to investigate the effects of single and multiple heavy metals on the growth and uptake of consortium of two types of fungal strains, Penicillium sp. A1 and Fusarium sp. A19. These fungal strains were tested to be tolerant to several heavy metals. A1, A19, and their combination (A1 + A19) were inoculated on potato dextrose agar (PDA), Czapek Dox agar (CDA), and potato dextrose broth (PDB) containing Cu2+ , Cd2+ , Pb2+ , and Zn2+ . Experimental results showed that the combined inoculation of A1 and A19 had profound effects on the growth of the two fungi in PDA and CDA under the treatments with Cu2+ and mixed Cd2+ + Zn2+ . The amount of metals through bioaccumulation by A1, A19, and A1 + A19 was significantly higher than that through biosorption by these fungi. The highest amount of Cd, Cu, and Zn accumulated by fungal biomass was obtained in the presence of Cd2+ + Cu2+ + Zn2+ in PDB. Compared with the individual A1 or A19 used in PDB, A1 + A19 accumulated higher amount of Cu and Pb in the presence of Cd2+ + Cu2+ + Pb2+ and higher amount of Pb in the presence of Cd2+ + Cu2+ + Zn2+ + Pb2+ . Our results indicated that there was no simple relationship between the metal biosorption by fungal biomass and the fungal metal tolerance. The biomass of A1 + A19 cultivated in PDB absorbed higher amount of metals than A1 or A19 in the presences of single metals and their combinations. The results suggested that the applicability of growing fungi tolerant to heavy metals provided a potential biotechnology for treatment of wastewaters with heavy metal pollutions. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Toxic heavy metals, especially copper (Cu), cadmium (Cd), lead (Pb), and zinc (Zn), are increasingly being released into the environment from industrial wastewaters and other human activities. Heavy metal contaminations bring potential health hazards to animals and human beings [1]. The adverse effects of heavy metals on the environment and their accretions through the food chain have promoted research to develop efficient and low costing techniques for wastewater treatment [2]. The treatment methods using microbial biomass are receiving much attention [3]. Microorganisms play an important role in the environmental fate of heavy metals, which are transformed to soluble and insoluble forms by various biological mechanisms. These mechanisms are part of natural biogeochemical cycles and potentially useful for both in situ and ex situ bioremediation processes for solid and

∗ Corresponding author. Tel.: +86 20 84110052; fax: +86 20 84110692. E-mail address: [email protected] (R. Zhang). 0304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.06.080

liquid wastes [4]. A wide variety of fungi, bacteria, and algae are now under study for possible applications as biosorbents for heavy metal remediation [5]. However, most biosorption studies focus on metabolically inactive dead biomass and research in using active and growing cells is still in infancy. Although several types of dead or pretreated microbial biomasses for high biosorptive potential have been reported, these biosorbents are not able to effectively remove metals from real industrial effluents attributable to presences of organic or inorganic ligands [6]. Under such situations, applications of active and growing cells should be a better choice because of their abilities of self-replenishment, continuous metabolic uptake of metals after physical adsorption. Nevertheless, metabolic uptake of metals may be limited because living cells are subject to extremes of pH, high metal concentrations, and multiple metals, which are common cases in industrial effluents. Therefore, in the isolation and selection of metal-resistant strains, it is crucial to study the factors affecting living cells of the strains. The positive interactions among constituent species may facilitate the survival of sensitive strains [6], which may lead to simultaneous removal of toxic metals, organic loads, and other inorganic impurities, as well as allow optimization through development of resistant species. The

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applicability of growing cells shows the potential in bioremediation of industrial effluents. Simultaneous interactions among multiple heavy metals with microorganisms may result in either synergic, antagonistic, or additive effects. These mechanisms of interactions may be particularly complex and unique, depending on the combinations of heavy metals and microbial strains. The combined effects (e.g. growth stimulation or toxicity) of multiple metals in the same microbial consortium are usually different from the additive effects from the individual metals involved [7]. To better understand applications of biosorbents for heavy metal remediation, the objective of this study was to quantify the effects of different heavy metals on the growth and metal uptake of fungal consortia. In this study, four heavy metals of Cd2+ , Cu2+ , Pb2+ , and Zn2+ were selected because of their toxicities in contaminated soils and wastewaters. Two metal-tolerant fungi (Cu-resistant Penicillium sp. A1 and Cd-resistant Fusarium sp. A19), which can be easily isolated from heavy metal contaminated soils, were used for inoculations in various media with different treatments of the heavy metals. Finally we investigated the potential of the fungal consortia for bioremediation of multi-metal contaminated waters by bioaccumulation and biosorption. 2. Materials and methods 2.1. Strains and media

bioaccumulation of fungi was calculated as follows: q=

(C0 − C)V W

(1)

where q is the bioaccumulation amount of mycelia (mg g−1 ), C0 is the initial concentration of heavy metal used (mg l−1 ), C is the final concentration of heavy metal in PDB (mg l−1 ), V is the volume of the liquid medium (0.05 l in the experiments), and W is the biomass of mycelia (g). 2.3. Metal biosorption of biomass The biomasses of A1, A19, and A1 + A19 were grown on PDB and CDM with a pH adjusted to 6.4. The biosorption procedure was adopted from Zafar et al. [11] with minor modification. The fungi were cultured in filamentous form at 30 ◦ C for 2 d in flasks on a rotatory shaker (150 rpm). After being harvested with filteration through a 150 ␮M sieve, the biomass was thoroughly washed with distilled water for three times to remove residual of the growth media. Then 1 g (fresh weight) of the washed biomass was added into each of 25 ml heavy metal solution containing 100 mg l−1 of Cd2+ , Cu2+ , Zn2+ , and Pb2+ , respectively, in a conical flask. The flasks were shaken on a rotatory shaker (160 rpm) for 45 min at 25 ◦ C. The biomass was separated by the filteration, dried at 80 ◦ C, and weighted. The filtrate was digested in 67% HNO3 . The HNO3 solution was evaporated and the solids were redissolved in 0.1 M HCl.

Penicillium sp. strain A1 and Fusarium sp. strain A19 were isolated from a copper-mining soil in Shaoguan, Guangdong Province of China [8,9]. Penicillium sp. A1 was tested to be tolerant to 150 mM Cu2+ , 5 mM Cd2+ , 15 mM Ni2+ , 5 mM Cr6+ , 150 mM Zn2+ , and 35 mM Pb2+ , respectively. Fusarium sp. A19 was tolerant to 10 mM Cu2+ , 15 mM Cd2+ , 10 mM Ni2+ , 1 mM Cr6+ , 100 mM Zn2+ , and 20 mM Pb2+ , respectively. The A1 and A19 cultures were maintained on slants of potato dextrose agar (PDA, Huankai Ltd. Guangzhou, China) at 4 ◦ C. Czapek Dox medium (CDM) was prepared by dissolving 0.2 g NaNO3 , 0.001 g FeSO4 , 0.1 g K2 HPO4 , 3.0 g d-glucose, 0.05 g KCl, and 0.05 g MgSO4 • 7H2 O in 100 ml deionized water. Two percent agar was added into CDM to obtain the solid medium (Czapek Dox agar, CDA). The heavy metals used for the experiments included Cd2+ (CdCl2 ), Cu2+ (CuCl2 • 2H2 O), Zn2+ (ZnCl2 ), and Pb2+ (Pb(NO3 )2 ). 2.2. Growth and bioaccumulation of fungi Fungal growth was determined based on linear measurements of colony radii on the solid media, PDA and CDA. The measurements were carried out at an interval of 3 d throughout an incubation period about 12 d, during which the average radial growth rate (mm d−1 ) was estimated [10]. The fungal biomass in potato dextrose broth (PDB, Huankai Ltd. Guangzhou, China) at the end of the incubation period was harvested by filteration through a 150 ␮M sieve. The biomass was determined from the dry weight of mycelia for each fungal species. Applied concentrations of the heavy metals were as follows: Cd2+ 2 mM, Cu2+ 5 mM, Zn2+ 50 mM, and Pb2+ 20 mM. The concentration values were selected so that the tested fungi could grow well and form obvious colony. The fungi A1, A19, and A1 + A19 (1:1) were inoculated into PDB contained different combinations of the four heavy metals. The media without adding heavy metals were used as controls, represented by CK in the following sections. The fungi in different treatments were cultivated at 25 ◦ C for 7 d with shaking (160 rpm). Concentrations of the heavy metals in PDB were measured using an inductively coupled plasma optical emission spectrometry (ICP-OES) (PerkinElmer Optima, 5300DV). The metal

Fig. 1. Radial growth rates of fungi A1, A19 and A1 + A19 on Czapek Dox agar (CDA) (I) and potato dextrose agar (PDA) (II) under different treatments of heavy metals. Different letters on the bars indicate that multiple comparison differences are significant under the same treatment of heavy metals (p < 0.05). The line on each bar is the standard deviation. CK represents the control.

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inoculations were conducted with a 5% least significant difference (LSD test, p < 0.05). 3. Results 3.1. Effects of heavy metals on the growth of test fungi

Fig. 2. Biomasses of fungi A1, A19 and A1 + A19 on potato dextrose broth (PDB) under different treatments of heavy metals. Different letters on the bars indicate that multiple comparison differences are significant under the same treatment of heavy metals (p < 0.05). The line on each bar is the standard deviation. CK represents the control.

Then the heavy metal concentrations in the filtrate were determined using ICP-OES. Each treatment was repeated for three times [12,13]. 2.4. Statistical analysis For all the data, including the radial growth rate, biomass, metal bioaccumulation and biosorption, 2 -tests were carried out with the mean of three replicates of each treatment using the statistical analysis software (SAS Institute, Inc., Cary, NC). Significance analyses of different inoculations under the same treatment with heavy metals and of different heavy metals under the same treatment with

Fig. 1I shows radial growth rates of fungi A1, A19, and A1 + A19 on CDA with the heavy metal treatments. The growth rate of A19 was significantly higher than that of A1 in the control (as indicated with CK in Fig. 1I). The growth rate of A19 with Cd2+ was higher than that of A1, whereas with Pb2+ and combined Cu2+ + Zn2+ , the growth rate of A19 was lower than that of A1. The radial growth rates of different fungal inoculations with other heavy metal treatments on CDA were not significantly different (p < 0.05) (Fig. 1I). The radial growth rates of A1 under the different treatments of heavy metals on CDA were in the following order: CK (a) >Cu2+ (b) >Zn2+ (c) >Cd2+ (d) >Cu2+ + Zn2+ (de) >Pb2+ , Cd2+ + Zn2+ (e) >Cd2+ + Cu2+ , Cd2+ + Cu2+ + Zn2+ (f). Here and below the different letters following the treatments indicate that the results are significantly different (p < 0.05). For A19, the radial growth rates under the different treatments were in the order of CK (a) >Cd2+ (b) >Cu2+ and Cd2+ + Zn2+ (c) >Zn2+ (cd) >Cd2+ + Cu2+ (de) >Pb2+ , Cu2+ + Zn2+ , and Cd2+ + Cu2+ + Zn2+ (e). For the combined inoculation of A1 + A19, the radial growth rates under the different treatments were in the following order: CK (a) >Cd2+ (b) >Cu2+ (c) >Zn2+ (d) >Cd2+ + Cu2+ (de) >Cd2+ + Zn2+ (def) >Pb2+ and Cu2+ + Zn2+ (ef) >Cd2+ + Cu2+ + Zn2+ (f). The results showed that the heavy metals had significant inhibitory effects on the fungal growth. Pb2+ had the most inhibition effect on the fungal growth than other heavy metals. Inhibitory effects of the combined heavy metals were more profound than those of the single heavy metals.

Table 1 Metal bioaccumulation of mycelia (mg g−1 ) of the fungi cultivated in potato dextrose broth (PDB) under different treatments with heavy metals. Treatments

Heavy metals in mycelia

Cd2+ Cu2+ Pb2+ Zn2+ Cd2+ + Cu2+

Cd Cu Pb Zn Cd Cu Cd Zn Cd Pb Cu Zn Cu Pb Pb Zn Cd Cu Zn Cd Cu Pb Cu Pb Zn Cd Pb Zn Cd Cu Pb Zn

Cd2+ + Zn2+ Cd2+ + Pb2+ Cu2+ + Zn2+ Cu2+ + Pb2+ Zn2+ + Pb2+ Cd2+ + Cu2+ + Zn2+

Cd2+ + Cu2+ + Pb2+

Cu2+ + Zn2+ + Pb2+

Cd2+ + Zn2+ + Pb2+

Cd2+ + Cu2+ + Zn2+ + Pb2+

* **

The values are mean ± standard deviation (n = 3). Different letters indicate that the results are significantly different (p < 0.05).

Fungus A1 0.00 7.65 372.54 123.98 7.18 23.73 20.69 400.88 12.26 61.98 36.07 170.56 31.06 100.12 175.23 107.23 118.15 134.15 1013.01 11.30 25.33 178.28 44.30 517.57 353.17 39.03 892.63 624.53 9.68 3.98 227.42 149.71

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Fungus A19 0.00* c** 1.56 ab 32.00 a 37.10 c 2.01 b 10.25 a 6.18 a 9.29 a 2.10 a 6.72 c 11.34 a 39.81 a 17.58 a 55.65 a 37.42 a 12.12 a 23.17 a 32.94 a 39.09 b 0.54 b 1.55 b 1.20 b 0.61 b 61.14 b 22.97 b 9.44 a 37.33 a 95.53 a 0.99 b 1.48 a 66.88 ab 13.57 b

22.98 10.87 166.11 475.21 17.10 39.97 31.25 453.22 24.86 320.70 39.69 158.75 32.50 123.43 213.44 257.79 74.06 163.07 1394.00 38.39 38.41 347.66 71.17 703.47 584.66 35.66 544.60 270.58 30.28 22.96 120.35 372.85

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.46 a 2.85 a 48.10 b 14.53 a 1.71 a 3.55 a 4.65 a 23.88 a 7.33 a 13.42 a 7.72 a 35.78 a 13.01 a 9.69 a 61.60 a 68.13 a 9.12 a 20.00 a 53.84 a 6.91 a 8.86 ab 37.75 b 2.22 a 4.86 a 86.58 a 0.45 a 24.12 b 81.15 b 4.92 ab 6.48 a 17.64 b 26.59 a

Fungi A1 + A19 19.59 4.20 270.84 275.30 15.79 26.12 20.99 334.47 9.13 105.09 46.41 163.47 30.23 135.81 119.20 120.57 100.99 159.94 1094.32 45.69 60.29 540.62 28.31 232.40 302.75 38.47 693.75 321.86 31.09 39.84 401.42 331.01

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.72 b 1.48 b 78.56 ab 1.20 b 3.13 a 17.05 a 8.35 a 96.13 a 3.11 a 3.13 b 9.96 a 65.86 a 6.62 a 30.82 a 2.02 a 44.29 a 27.76 a 37.06 a 81.01 b 3.40 a 8.17 a 85.44 a 6.47 c 4.93 c 65.12 b 4.45 a 71.42 b 57.92 b 9.86 a 14.75 a 90.21 a 17.98 a

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Table 2 Metal biosorption (mg g−1 ) of the fungi cultivated in potato dextrose broth (PDB) under different treatments with heavy metals. Treatments 2+

Cd Cu2+ Pb2+ Zn2+ Cd2+ + Cu2+ Cd2+ + Zn2+ Cd2+ + Pb2+ Cu2+ + Zn2+ Cu2+ + Pb2+ Zn2+ + Pb2+ Cd2+ + Cu2+ + Zn2+

Cd2+ + Cu2+ + Pb2+

Cu2+ + Zn2+ + Pb2+

Cd2+ + Zn2+ + Pb2+

Cd2+ + Cu2+ + Zn2+ + Pb2+

* **

Heavy metals in biomass Cd Cu Pb Zn Cd Cu Cd Zn Cd Pb Cu Zn Cu Pb Pb Zn Cd Cu Zn Cd Cu Pb Cu Pb Zn Cd Pb Zn Cd Cu Pb Zn

Fungus A1 3.59 3.09 13.94 2.22 0.00 3.12 0.00 0.83 0.00 10.67 1.62 0.22 0.36 5.19 3.39 0.42 0.00 0.53 0.00 0.00 1.90 2.27 1.18 1.41 0.00 0.00 3.01 0.00 0.00 1.08 1.37 0.00

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Fungus A19 *

**

0.54 b 0.02 b 0.48 b 0.11 b 0.00 b 0.37 c 0.00 b 0.07 c 0.00 b 0.10 a 0.50 b 0.05 b 0.41 b 0.53 b 0.68 c 0.00 c 0.00 a 0.47 b 0.00 b 0.00 a 0.02 b 0.27 b 0.25 b 0.20 b 0.00 b 0.00 a 0.18 b 0.00 c 0.00 a 0.16 b 0.10 b 0.00 b

5.86 4.37 13.05 3.79 0.54 3.68 1.47 1.93 2.33 7.78 3.53 1.16 3.66 6.10 6.69 1.68 0.00 0.00 0.00 0.00 0.00 1.16 0.00 0.00 0.00 0.00 2.14 1.79 0.00 0.00 3.00 2.66

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.91 ab 0.66 ab 0.44 b 0.13 a 0.03 a 0.00 b 0.10 a 0.11 b 0.15 a 0.48 b 0.07 a 0.01 a 0.44 a 0.56 a 0.46 b 0.08 b 0.00 a 0.00 b 0.00 b 0.00 a 0.00 c 0.44 c 0.00 c 0.00 c 0.00 b 0.00 a 0.29 c 0.30 b 0.00 a 0.00 c 1.62 b 1.61 a

Fungi A1 + A19 6.76 5.39 16.51 3.72 0.06 4.55 1.74 2.32 2.43 11.06 3.43 0.93 3.64 6.83 9.67 2.10 0.00 3.46 0.80 0.00 3.51 6.55 2.44 5.26 4.75 0.00 8.30 7.78 0.00 2.10 4.85 4.33

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.94 a 1.16 a 1.82 a 0.16 a 0.04 b 0.04 a 0.28 a 0.17 a 0.02 a 0.74 a 0.16 a 0.29 a 0.01 a 0.13 a 0.16 a 0.19 a 0.00 a 0.22 a 0.05 a 0.00 a 0.10 a 0.19 a 0.40 a 0.24 a 0.24 a 0.00 a 0.15 a 0.15 a 0.00 a 0.29 a 0.23 a 0.20 a

The values are mean ± standard deviation (n = 3). Different letters indicate that the results are significantly different (p < 0.05).

Table 3 Metal biosorption (mg g−1 ) of the fungi cultivated in Czapek Dox medium (CDM) under different treatments with heavy metals. Treatments

Heavy metals in biomass

Fungus A1

Cd2+ Cu2+ Pb2+ Zn2+ Cd2+ + Cu2+

Cd Cu Pb Zn Cd Cu Cd Zn Cd Pb Cu Zn Cu Pb Pb Zn Cd Cu Zn Cd Cu Pb Cu Pb Zn Cd Pb Zn Cd Cu Pb Zn

2.43 6.12 29.64 4.32 0.82 4.88 0.71 2.13 3.58 19.09 4.37 1.28 4.78 15.18 17.84 3.17 0.14 4.27 1.03 0.00 3.89 13.35 2.96 14.98 0.66 0.87 16.33 1.71 0.00 3.13 12.57 0.92

Cd2+ + Zn2+ Cd2+ + Pb2+ Cu2+ + Zn2+ Cu2+ + Pb2+ Zn2+ + Pb2+ Cd2+ + Cu2+ + Zn2+

Cd2+ + Cu2+ + Pb2+

Cu2+ + Zn2+ + Pb2+

Cd2+ + Zn2+ + Pb2+

Cd2+ + Cu2+ + Zn2+ + Pb2+

* **

The values are mean ± standard deviation (n = 3). Different letters indicate that the results are significantly different (p < 0.05).

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.09* b** 0.95 a 3.61 a 0.81 ab 0.03 a 0.04 a 0.53 a 0.42 a 0.47 a 3.53 a 0.12 a 0.12 a 0.35 a 0.08 a 1.90 a 0.07 a 0.09 b 0.11 a 0.34 b 0.00 b 0.05 a 0.57 a 0.26 a 0.94 a 0.01 b 0.12 ab 1.70 a 0.34 b 0.00 b 0.42 a 0.11 a 0.05 c

Fungus A19 5.92 5.57 12.37 0.23 0.00 2.85 0.16 0.93 3.86 13.47 1.70 0.00 1.60 11.55 14.22 1.14 0.00 1.64 0.35 0.00 0.94 10.22 0.00 9.49 8.51 0.00 14.90 14.04 0.00 0.48 8.40 7.70

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.64 a 0.69 a 0.73 b 0.14 b 0.00 b 0.52 b 0.05 ab 0.03 b 0.38 a 0.54 a 0.49 b 0.00 b 0.39 b 0.77 b 0.47 b 0.11 b 0.00 b 0.22 b 0.15 c 0.00 b 0.48 b 0.96 b 0.00 b 0.76 b 0.70 a 0.00 b 4.79 a 4.61 a 0.00 b 0.08 b 0.04 c 0.08 b

Fungi A1 + A19 5.46 6.61 29.28 7.75 0.00 2.08 0.00 1.17 0.29 14.08 0.80 0.00 1.07 12.33 13.13 4.14 1.68 4.43 2.99 1.53 4.39 10.88 3.72 10.18 9.63 1.77 11.60 11.04 0.79 3.33 10.11 9.57

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.83 a 0.50 a 1.41 a 1.99 a 0.00 b 0.37 b 0.00 b 0.02 b 0.32 b 1.19 a 0.39 b 0.00 b 0.43 b 1.12 b 0.89 b 0.48 a 0.11 a 0.19 a 0.26 a 0.24 a 0.19 a 0.31 b 0.38 a 0.24 b 0.24 a 0.74 a 0.82 a 0.80 a 0.27 a 0.11 a 0.28 b 0.29 a

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On PDA, the radial growth rates of different inoculations under the treatments with heavy metals containing Pb2+ were low and not remarkably different (Fig. 1II). As shown in Fig. 1II, the growth rates of A1 + A19 were lower than those of A1 and A19 in the control. A19 grew more quickly than A1 in the presences of Cd2+ and mixed Cd2+ + Cu2+ , whereas A1 grew more quickly than A19 in the presences of Pb2+ and Zn2+ . In other treatments with the heavy metals, the differences among the radial growth rates of different fungal inoculations were not significant. The growth rates of A1 + A19 were similar to those of A19 in the presences of Cd2+ and Cd2+ + Cu2+ , while the growth rate of A1 + A19 was similar to that of A1 in the presence of Zn2+ . The growth rate of A1 + A19 under the Cd2+ + Zn2+ treatment was higher than those of A1 and A19. The radial growth rates of A1, A19, and A1 + A19 under the treatments of heavy metals on PDA showed different orders from those on CDA. For A1, the order of radial growth rates under the heavy metal treatments was as follows: CK (a) >Cu2+ (b) >Zn2+ (c) >Cu2+ + Zn2+ (cd) >Pb2+ and Cd2+ (d) >Cd2+ + Zn2+ (de) >Cd2+ + Cu2+ + Zn2+ (ef). For A19, the order of radial growth rates was as follows: CK (a) >Cd2+ and Cu2+ (b) >Cd2+ + Cu2+ (c) >Zn2+ (d) >Cu2+ + Zn2+ (de) >Cd2+ + Zn2+ (def) >Pb2+ (defg). For A1 + A19, the radial growth rates were in the following order: Cu2+ (a) >CK (ab) >Cd2+ (b) >Zn2+ , Cd2+ + Cu2+ , Cd2+ + Zn2+ , and Cu2+ + Zn2+ (bc) >Pb2+ (cd). Under the treatments with other mixed heavy metals, the radial growth rates were very low and not significantly different. The biomasses in the liquid medium (PDB) under the heavy metal treatments showed similar results to those in the solid medium (CDA) (Fig. 2). However, in PDB, the fungal biomasses of A1, A19, and A1 + A19 reached the highest values in the presence of Cu2+ compared with other treatments. The fungal biomass cultivated in PDB in the presence of single Pb2+ was lower than those cultivated in the media containing multimetals.

3.2. Bioaccumulation of heavy metals of test fungal mycelia The fungus A19 accumulated more Cd in PDB containing Cd2+ , Cd2+ + Cu2+ , and Cd2+ + Cu2+ + Pb2+ than A1 (Table 1). Strain A19 also accumulated more Pb than A1 in the treatment of Cd2+ + Pb2+ . More Zn was accumulated by A19 than A1 under the conditions containing Cd2+ + Cu2+ + Zn2+ , Cu2+ + Zn2+ + Pb2+ , and Cd2+ + Cu2+ + Zn2+ + Pb2+ . However, A1 accumulated more Pb than A19 in PDB containing Pb2+ and Cd2+ + Zn2+ + Pb2+ , and more Zn in the medium containing Cd2+ + Zn2+ + Pb2+ . The combination A1 + A19 accumulated more Pb than A1 and A19 in PDB containing Cd2+ + Cu2+ + Zn2+ + Pb2+ , and more Cu and Pb in the presence of Cd2+ + Cu2+ + Pb2+ . However, less Cu and Pb was accumulated by A1 + A19 in the presence of Cu2+ + Zn2+ + Pb2+ . The accumulated amount ranged from 100 to 600 mg g−1 for Zn and Pb, and was lower than 100 mg g−1 for Cu and Cd with inoculations of A1, A19, and A1 + A19. The highest accumulated values of Cd, Cu, and Zn were obtained in PDB containing Cd2+ + Cu2+ + Zn2+ , and the highest amount of Pb2+ was in the presence of Cd2+ + Zn2+ + Pb2+ .

3.3. Metal biosorption by fungal biomass Biomasses of A1 + A19 cultivated in PDB absorbed higher amount of different metals than those of A1 or A19 in the presences of single metals and the metal combinations (Table 2). However, the metal concentrations absorbed by A1 + A19 cultivated in CDM were not significantly higher than those of A1 or A19 (Table 3). The metal concentrations biosorbed by the fungal biomasses cultivated in CDM were higher than those in PDB.

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4. Discussion Microbial biomass can remove and concentrate a variety of metal ions from aqueous solutions. The uptake of heavy metals by microorganisms can be divided into two categories. One is known as biosorption, a metabolism-independent binding, consisting of negatively charged free groups in several biopolymers that form the fungal cell wall. Another is an energy-dependent metal influx known as bioaccumulation [2]. In this study, although A1 could tolerate higher Cu2+ than A19, the Cu concentrations absorbed by A1 were not always higher than those by A19 in the different treatments containing Cu2+ . Similarly, A19 could tolerate higher Cd2+ than A1, but A19 did not always absorb more Cd than A1 in the treatments containing Cd2+ . The lack of correlation between the metal tolerance and metal uptake by fungal biomass might be attributable to the different mechanisms between the resistance and biosorption. With the increase of metal types, the metal concentrations biosorbed by the fungal biomass decreased, which might be due to the competition for biosorping sites from other metals. The biomasses of A1 + A19 cultivated in PDB absorbed more metals than A1 or A19 under the different treatments of heavy metals. Nevertheless, the metal concentrations absorbed by the biomass of A1 + A19 cultivated in CDM were not significantly higher than those by A1 or A19. The metal concentrations biosorbed by the fungal biomasses cultivated in CDM were higher than those in PDB. Biosorption occurred as a consequence of physical binding to cell surfaces rather than of an active process. The autolysed mycelia in CDM increased the specific surface area of the autolysed cell wall and released intracellular binding sites after denaturation of the biomass. Therefore, additional negatively charged binding sites were provided in addition to those present on the surface of the fungal cells [2]. The media used could affect the absorption capability of the biomass. The metal concentrations through bioaccumulation by fungi A1, A19, and A1 + A19 were significantly higher than those through biosorption by these fungi. However, the fungal biomass was influenced by the toxicity of metals. The fungal biomass cultivated in PDB in the presence of single Pb2+ was lower than those cultivated in the media containing multi-metals. This result may be explained with the biological toxicity of Pb2+ to mycelia, which decreased in the presences of other heavy metals [14]. The highest fungal biomass in PDB in the presence of single Cu2+ may result from the detoxification of Penicillium A1 tolerant to Cu2+ [15]. An active detoxification process was possible when the fungus was cultivated on PDA. The fungi with the three types of inoculations grew faster on PDA containing heavy metals than on CDA. Therefore, the media used could affect the fungal tolerance to heavy metals. The interactions among Zn2+ , Cd2+ , and Pb2+ may affect the uptake capability of biosorbent [16]. However, the highest amount of Cd, Cu, and Zn accumulated by fungal biomass was obtained in the presence of Cd2+ + Cu2+ + Zn2+ . The results indicated that some heavy metals might introduce antagonism effects by interacting with fungi [17]. The fungal combination of A1 + A19 accumulated the highest concentrations of Cu and Pb in PDB containing Cd2+ + Cu2+ + Pb2+ , but the lowest in the presence of combined Cu2+ + Zn2+ + Pb2+ . It is difficult to explain the interactions of combined toxicities [18]. Nevertheless, compared with the toxicity of individual metals, it was suggested that the interactions of mixed heavy metals reduced toxicity for fungi [2]. Heavy metals may compete for binding on and transporting into the strains. Meanwhile, strains may act differently depending on the types and concentrations of heavy metals [16]. The metal concentrations accumulated by Fusarium sp. A19 were higher than those by Penicillium sp. A1. This result may be related to different detoxification mechanisms of the two fungi. Since toxicities of heavy metals to microorganism are influenced by a number of

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environmental factors (e.g., pH, biomedium composition, biomass concentration, presence of other heavy metals) [7,19], the resistant strains display different growth and removal abilities to heavy metal ions [20,21]. The fungi A1 + A19 accumulated more Pb than A1 and A19 in PDB containing Cd2+ + Cu2+ + Zn2+ + Pb2+ , and more Cu and Pb in the presence of Cd2+ + Cu2+ + Pb2+ . However, A1 + A19 accumulated less Cu and Pb than A1 and A19 in the presence of Cu2+ + Zn2+ + Pb2+ . The different resistances to metals between A1 and A19 suggest different detoxification mechanisms of the two fungi. Therefore, the co-inoculation of A1 + A19 resulted in higher resistance to multi-metals than single A1 or A19. The combination of A1 + A19 could accumulate higher amount of the metals on their biomasses than single A1 or A19. The combined fungal effects reflected the interactions of symbiosis or antagonism for competition to adsorb heavy metals between the fungal isolates [22]. The interactions should help microorganisms to better withstand adverse conditions often encountered in industrial wastewaters [6]. In sum, in bioremediation applications of heavy metals, it should be a better option to use the active biomass because growing cells have the abilities of self-replenishment, continuous metabolic uptake of metals after physical adsorption. Moreover, growing cells have unlimited capacities to cleave organic–metallic complexes, degrade organic compounds, and take up other inorganic ions such as ammonium, nitrate, and phosphate. Furthermore, dissolved and fine-dispersed metallic elements can also be removed via immobilization. For employing harvested biomass (dead or pretreated), metals are not taken into the cells, but adsorbed on the cell surfaces. Since only a small fraction of bioaccumulation capacity is exploited, biosorption alone may not sufficient for effective metal remediation [6]. In view of these facts, the applicability of growing fungi tolerant to heavy metals should provide potential biotechnology for treatment of wastewaters and soils with heavy metal pollutions. Acknowledgements This work was partly supported by grants from the Natural Science Foundation of Guangdong Province (no. 06202438) and the Chinese National Natural Science Foundation (nos. 30600011, 50779080). References [1] B. Volesky, Z. Holan, Biosorption of heavy metals, Biotechnol. Progr. 11 (1995) 235–250.

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