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Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Bioremediation from wastewater and extracellular synthesis of copper nanoparticles by the fungus Trichoderma koningiopsis a
b
c
Marcia R. Salvadori , Rômulo A. Ando , Cláudio A. Oller Do Nascimento & Benedito Corrêa
a
a
Department of Microbiology, Biomedical Institute II, University of São Paulo, São Paulo, Brazil b
Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil c
Department of Chemical Engineering, Polytechnic, University of São Paulo, São Paulo, Brazil Published online: 26 Jun 2014.
To cite this article: Marcia R. Salvadori, Rômulo A. Ando, Cláudio A. Oller Do Nascimento & Benedito Corrêa (2014) Bioremediation from wastewater and extracellular synthesis of copper nanoparticles by the fungus Trichoderma koningiopsis, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:11, 1286-1295, DOI: 10.1080/10934529.2014.910067 To link to this article: http://dx.doi.org/10.1080/10934529.2014.910067
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Journal of Environmental Science and Health, Part A (2014) 49, 1286–1295 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.910067
Bioremediation from wastewater and extracellular synthesis of copper nanoparticles by the fungus Trichoderma koningiopsis ^ MARCIA R. SALVADORI1, ROMULO A. ANDO2, CLAUDIO A. OLLER DO NASCIMENTO3 and 1 ^ BENEDITO CORREA 1
Department of Microbiology, Biomedical Institute II, University of S~ ao Paulo, S~ ao Paulo, Brazil Department of Fundamental Chemistry, Institute of Chemistry, University of S~ ao Paulo, S~ ao Paulo, Brazil 3 Department of Chemical Engineering, Polytechnic, University of S~ ao Paulo, S~ ao Paulo, Brazil
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This is the first study describing the rapid extracellular production of copper nanoparticles by dead biomass of Trichoderma koningiopsis. The production and uptake of copper nanoparticles by dead biomass of Trichoderma koningiopsis were characterized by investigating physicochemical factors, equilibrium concentrations and biosorption kinetics, combined with scanning electron microscopy (SEM), energy dispersive X-ray (EDS), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). A successful route for the metallic copper nanoparticles synthesis was achieved, and followed a Langmuir isotherm where a high biosorption capacity was observed, 21.1 mg g¡1. The kinetic analysis showed that copper biosorption followed a pseudo– second-order model. The nanoparticles mainly exhibited a spherical shape, with an average size of 87.5 nm, and were synthesized extracellularly. The presence of proteins as stabilizing agents of the nanoparticles was demonstrated. The extracellular biosynthesis and uptake of copper nanoparticles using dead fungal biomass is a low-cost green processes, and bioremediation of impacted local. Keywords: Copper nanoparticles, dead biomass, extracellular synthesis, Trichoderma koningiopsis.
Introduction The possible use of biological materials for the synthesis of metal nanoparticles (NPs) has emerged as a more efficient and greener approach.[1] Because synthetic methods comprise complex physical and chemical processes that require the use of high temperatures and pressures, large amounts of energy are needed and toxic wastes are produced, posing environmental and human health risks.[2] Copper NPs can be used in printed electronics, are suitable substitutes of conductive and expensive noble metals such as gold and silver in chemical and metallurgical processes,[3,4] and have applications in gas sensing, catalytic processes, high temperature superconductors, solar cells, and wood treatment.[5–7] The filamentous fungi possess advantages over bacteria and algae such as, the fungal mycelial mesh that can withstand flow pressure and agitation and other conditions in the bioreactors or other chambers, ease of handling and culturing on a large scale, high tolerance towards metals, Address correspondence to Marcia R. Salvadori, Department of Microbiology, Biomedical Institute II, University of S~ ao Paulo, 05508000, S~ao Paulo, Brazil; E-mail:[email protected] Received December 16, 2013.
and a high wall-binding capacity, as well as intracellular metal uptake capacity.[8] Because metal ions are not biodegradable, they are usually removed from contaminated soil and water by chemical or physical treatment process. Typical treatment processes such as membrane separation, precipitation, ion exchange, reverse osmosis and electrolysis are often not feasible because of high costs, the need for continuous input of chemicals, and the production of a toxic sludge.[9] Recently, the bioremediation of metal ion pollution has emerged as an environmental friendly technology.[10] Some microorganisms are potent bioremediators that remove metals ions through biosorption mechanisms. Fungi are used in bioremediation processes since they are a versatile group that can adapt to and grow under various extreme conditions of pH, temperature and nutrient availability, as well as at high metal concentrations.[11] A literature review[2] has shown that few articles[12,13] on the biosynthesis of copper NPs by fungi have been published and none of those studies used the fungus Trichoderma koningiopsis (T. koningiopsis). To increase the scope of biosystems for the synthesis of metallic nanomaterials and bioremediation of wastewater, we explored for the first time the potential of the fungus T. koningiopsis for the removal and reduction of copper ions to copper NPs.
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Fungal bioremediation of wastewater Materials and methods Growth and maintenance of the organism T. koningiopsis was isolated from the sediment collected in a copper waste pond at the Sossego mine, which belongs to the company Vale S.A. located in Cana~a dos Carajas, Par a, Brazilian Amazonia region (06 260 S latitude and 0 50 4 W longitude). T. koningiopsis was maintained and activated on Sabouraud Dextrose Agar (SDA) (Oxoid, England).[14]
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Minimum inhibitory concentration in agar medium Copper tolerance of the T. koningiopsis was determined as the minimum inhibitory concentration (MIC) by the spot plate method.[15] SDA plates containing different copper concentrations (50 to 2000 mg L¡1) were prepared and fungal inocula were spotted onto copper-containing and metal-free (control). The plates were incubated at 25 C for at least 5 days. The MIC is defined as the lowest concentration of the metal that inhibits visible growth of the isolate.
experiments were optimized using 45 mL of 100 mg L¡1 of Cu (II) test solution in plastic flask. Various concentrations of copper (II) were prepared by appropriate dilution of the copper (II) stock solution. The pH was adjusted with HCl or NaOH. The desired biomass dose was then added and the content of the flask was shaken. Next, the Cu (II) solution was separated from the biomass by vacuum filtration through a Millipore membrane. The metal concentration in the filtrate was determined by flame atomic absorption spectrophotometer. The efficiency (R) of metal removal was calculated using Eq. (1): R D .Ci -Ce /=Ci ¢100
(1)
where Ci and Ce are the initial and equilibrium metal concentrations, respectively. The metal uptake capacity, qe, was calculated using Eq. (2): qe D V.Ci -Ce /=M
(2)
where qe (mg g¡1) is the biosorption capacity of the biosorbent at any time, M(g) is the biomass dose, and V (L) is the volume of the solution.
Retention of copper NPs by the biosorbent Preparation of the adsorbate solutions All chemicals were of analytical grade and were used without further purification. The dilutions were prepared in double-deionized water (Milli-Q Millipore 18.2 MVcm¡1 resistivity). The copper stock solution was prepared by dissolving CuCl2.2H2O (Carlo Erba, Milan, Italy) in doubledeionized water. The working solutions were prepared by diluting this stock solution. Biomass preparation The fungal biomass was prepared in Sabouraud broth (Sb) (Oxoid, England), and incubated at 25 C for 5 days, under shaking at 150 rpm. After incubation, the pellets were harvested and washed with double-deionized water, corresponding to live biomass. For the preparation of dead biomass, an appropriate amount of live biomass was autoclaved. Dried biomass was obtained by drying the fungal mat at 50 C until it became crispy. The dried mat was ground to obtain uniformly sized particles.
Biosorption isotherm and kinetic models Two equilibrium models [16] were used to fit Cu (II) biosorption isotherm experimental data, as follows. The linearized Langmuir isotherm model can be described by Eq. (3): Ce =qe D 1=.qm ¢b/ C Ce =qm
(3)
where qm is the monolayer sorption capacity of the sorbent (mg g¡1), and b is the Langmuir sorption constant (L mg¡1). The linearized Freundlich isotherm model can be described by Eq. (4): Inqe D InKF C 1=n ¢InCe
(4)
where KF is a constant relating the biosorption capacity and the term written as “1/n” is related to the adsorption intensity of the adsorbent. The kinetics of Cu (II) biosorption was analyzed using pseudo–first-order, and pseudo–second-order models. The linear pseudo–first-order model [17] can be represented by Eq. (5):
Evaluation of the effects of physicochemical factors on the adsorption efficiency of copper NPs onto the biosorbent
log.qe ¡ qt / D logqe ¡ K1 =2:303 ¢t
The effect of pH (2–6), temperature (20–60 C), contact time (5–360 min), initial copper concentration (50–500 mg L¡1), agitation rate (50–250 rpm), and biosorbent dose (0.15–1.0 g) on the removal of copper was analyzed. These
where qe (mg g¡1) and qt (mg g¡1) are the amount of adsorbed metal on the sorbent at the equilibrium time and at any time t, respectively, and K1 (1 min¡1) is the rate constant of the pseudo–first-order adsorption process. The
(5)
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linear pseudo–second-order model by Eq. (6):
[18]
can be represented
t=qt D 1=K2 ¢qe C t=qe
(6)
where K2 (g mg¡1 min¡1) is the equilibrium rate constant of the pseudo–second-order model.
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Biosynthesis of copper NPs by T. koningiopsis Only dead biomass of T. koningiopsis, which showed a higher capacity to adsorb copper metal ions than live and dried biomass, was used. Biosynthesis of copper NPs by dead T. koningiopsis biomass was investigated at a concentration of 100 mg L¡1 copper (II) using the equilibrium model data. The size, shape and location of the copper NPs on the biosorbent were determined by analyzing ultrathin sections by TEM (JEOL-1010, Peabody, MA, USA). Small fragments of the biological material obtained before and after the formation of copper NPs were mounted on pin stubs, coated with gold under vacuum, and examined by SEM (JEOL 6460 LV) equipped with EDS. The XPS analysis was carried out at a pressure of less than 5 £ 10¡7 Pa using a commercial spectrometer (UNISPECS UHV System, Berlin, Germany). The Mg-Ka line was used (hn D 1253.6 eV) and the analyzer pass energy was set to 10 eV. The inelastic background of the C 1s, O 1s, N 1s and the Cu 2p3/2 electron core-level spectra was subtracted using Shirley’s method. The composition (at.%) of the near surface region was determined with an accuracy of §10% from the ratio of the relative peak areas corrected by Scofield’s sensitivity factors of the corresponding elements. The binding energy scale of the spectra was corrected using the C 1s hydrocarbon component of the fixed value of 285.0 eV. The spectra were fitted without placing constraints using multiple Voigt profiles. The width at half maximum (FWHM) varied between 1.2 and 2.1 eV and the accuracy of the peak positions was § 0.1 eV.
Results and discussion The effect of different copper concentrations (50–2000 mg L¡1) was tested in agar medium and the results showed that T. koningiopsis exhibited high tolerance to copper (1057 mg L¡1). It has previously been demonstrated that fungi are potential biosorbents of heavy metals,[19,20] and this fact was confirmed in the present study. Fungi offer a wide range of chemical groups that can attract and sequester metals present in the biomass. The cell wall of fungi consists of structural polysaccharides, proteins and lipids that contain metal-binding functional groups.[20]
Effects of physicochemical factors on biosorption The removal of copper by T. koningiopsis biomass is influenced by physicochemical factors such as biomass concentration, pH, temperature, contact time, rate of agitation, and metal ion concentration. The amount of biosorbent is an important parameter to determine the sorbent-sorbate equilibrium of the system.[21,22] Figure 1a shows that dead biomass was more efficient in removing copper compared to live and dried biomass. This finding indicates that dead biomass possesses a higher affinity for copper than live and dried biomass. The use of live biomass for the binding of metal ions depends on nutrient availability, environmental conditions and cell age.[23] In addition, live biomass may be subject to the toxic effect of heavy metals when present at high concentrations. Therefore, dead biomass is preferred to overcome these disadvantages.[24] The efficiency of ionization of functional groups present on the surface of the biomass cell wall depends on the pH of the solution.[25] The influence of pH on copper uptake is shown in Figure 1b. Maximum removal occurred at pH 5 for the three types of biomass. With respect to temperature, maximum removal of copper was observed at a temperature of 40 C for the three types of biomass (Fig. 1c). According to the theory of adsorption, adsorption decreases with increasing temperature, as molecules adsorbed earlier on a surface tend to desorb from the surface at higher temperatures.[26] The graph obtained for the three types of biomass showed sigmoid kinetics (Fig. 1d), characterizing an enzyme-catalyzed reaction. The results showed that this was a rapid process, with more than 90% of the NPs formed by dead biomass within 60 min of the reaction. Regarding agitation speed, optimum copper removal was observed at 150 rpm for the three types of biomass (Fig. 1e). At higher agitation speeds, vortex phenomena occur and the suspension is no longer homogenous, a fact impairing metal removal.[27] The initial metal concentration, provides an important driving force to overcome all mass transfer resistance of the metal between the aqueous and solid phases.[28] The percentage of copper adsorption decreased with increasing metal concentration (50–500 mg L¡1) for the three types of biomass (Fig. 1f). The same has been observed for the removal of zinc by fungi at concentrations ranging from 100–400 mg L¡1.[29]
Biosorption isotherm and kinetics models The experimental Cu (II) isotherm was tested using the Langmuir and Freundlich models. The Langmuir isotherms for Cu (II) biosorption obtained for the three types of T. koningiopsis biomass are shown in Figures 2a, 2b and 2c. Table 1 shows the isotherm constants, maximum loading capacity estimated by the Langmuir and Freundlich models, and regression coefficients. The Langmuir
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Amount of biosorbent (g)
c
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5 10 15 20 25 30 40 60 120 180 240 300 360
100 90 80 70 60 50 40 30 20 10 0
% removal of copper
% removal of copper
a
250
Agitation rate (rpm)
100 90 80 70 60 50 40 30 20 10 0
Live Dried Dead 50 100 150 200 300 400 500 Amount of copper (mg/L)
Fig. 1. Effects of physicochemical factors on live, dried and dead biomass of T. koningiopsis. (a) Amount of biosorbent; (b) pH; (c) temperature; (d) contact time; (e) agitation rate; and (f) initial copper concentration.
adsorption model was the best to fit the experimental biomass data. The dead biomass of T. koningiopsis (21.1 mg g¡1) had a similar or higher adsorption capacity than those described for other known biosorbents, such as, Pleurotus pulmonaris, Schizophyllum commune, Penicillium spp, Rhizopus arrhizus, Trichoderma viride, Pichia stiptis, Pycnoporus sanguineus with adsorption rates of 6.2, 1.52, 15.08, 19.0, 19.6, 15.85 and 2.76 mg g¡1 respectively.[14,30–34] Comparison with biosorbents of bacterial origin showed that the
Cu (II) adsorption rate of T. koningiopsis is similar to that of Bacillus subtilis IAM 1026 (20.8 mg g¡1).[35] In contrast the filamentous fungus T. koningiopsis exhibited a higher rate of metal ion adsorption than the algae Cladophora spp (14.28 mg g¡1).[36] The kinetic results suggested that Cu (II) adsorption by T. koningiopsis was best described using the pseudo–second-order kinetic model (Figs. 2d, 2e and 2f). This adsorption kinetics is typical of the adsorption of divalent metals onto biosorbents.[37] The constants and regression coefficients are shown in Table 2.
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40
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40 20
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Fig. 2. Langmuir plots for live (a), dried (b) and dead (c) biomass of T. koningiopsis. Pseudo–second-order models for live (d), dried (e) and dead (f) biomass.
Table 1. Adsorption constants from simulations with Langmuir and Freundlich models. Langmuir model b Type of qm biomass (mg g¡1) (L mg¡1) Live Dried Dead
6.0 10.0 21.1
0.021 0.021 0.043
Table 2. Kinetic parameters for adsorption of copper. Pseudo–first-order
Freundlich model R2 0.989 0.984 0.984
KF (mg g¡1) 16 n 0.41 0.44 1.10
R2
0.41 0.815 0.47 0.817 0.57 0.898
Type of biomass Live Dried Dead
Pseudo–second-order
K1 (1 min¡1)
R2
K2 (g mg¡1 min¡1)
R2
4.83 £ 10¡3 4.37 £ 10¡3 2.53 £ 10¡3
0.395 0.469 0.139
18.90 £ 10¡3 15.26 £ 10¡3 19.35 £ 10¡3
0.981 0.986 0.987
Fungal bioremediation of wastewater
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Biosynthesis and characterization of copper NPs using dead biomass of T. koningiopsis Knowledge of the mechanisms underlying the formation of NPs by biological systems is important in order to develop more credible and reproducible processes for their biosynthesis. For this purpose a fraction of dead T. koningiopsis biomass was examined by TEM, which revealed the presence of NPs in the cell wall, but not in the cytoplasm or cytoplasmic membrane. No NPs were detected in the control. The ultrastructural changes observed included shrinking of the cytoplasm in the control and in biomass impregnated with copper, probably as a result of the autoclaving process (Figs. 3a and 3b). From the point of view of applications, this extracellular location offers a major advantage compared to an intracellular synthesis process. Because the NPs formed inside the biomass require an additional processing step for their release from the biomass by ultrasound treatment or by reaction with suitable detergents,[38] the extracellular location permits the fast and easy removal of large amounts of NPs and the reutilization of biomass in the production process. In this study,
1291 the copper NPs had an average diameter of 87.5 nm and a predominantly spherical shape, although, few aggregates of NPs with an average diameter of 328.27 nm (Fig. 3c) were also observed. The presence of copper NPs was confirmed by spot profile SEM-EDS measurements. Figures 4a and 4b show the SEM photomicrographs before and after the biosorption of Cu (II) by the fungal biomass. Surface modifications could be seen that were mainly due to an increase in irregularity after the binding of copper NPs onto the surface of dead biomass. The spot profile EDS spectra recorded in the region of the mycelium examined indicated signals from copper (Figs. 5a and 5b) to the fungus. Furthermore, the C, N and O peaks correspond to the presence of proteins as capping material on the surface of the copper NPs and other biopolymers present in the sample. These peaks are likely due to proteins secreted by the fungus. The XPS spectra Figure 6 shows C 1s, N 1s, O 1s and Cu 2p core level after biosynthesis of copper NPs by dead biomass of T. koningiopsis. As can be seen in the high resolution carbon spectra (C 1s), the components of higher binding energy were deconvoluted within four elements. The main component
Fig. 3. TEM micrographs of T. koningiopsis sections. (a) Control (without copper). (b) Section of the fungus showing the extracellular localization of copper nanoparticles (arrow). (c) Aggregated of nanoparticles (arrow).
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Fig. 4. Dead biomass of T. koningiopsis analyzed by SEM-EDS. (a) Control (without copper), and (b) biomass exposed to copper.
at 284.8 eV can be attributed to the hydrocarbon chains of the cellular phase, the peak at 286.5 eV to the a- carbon, the peak at 288.0 eV to the carbonyl groups and, finally, the peak at 289.0 eV to the carboxylic groups of the peptides/proteins bound to copper NPs.[39] The deconvoluted spectra of oxygen (O 1s), showed peaks at 531.4 eV, 532.7 eV and 533.2 eV and corresponded to the peaks found in the C 1s spectra. The nitrogen spectra (N 1s) had two components, the main component at 400.0 eV and a lower one at 402.0 eV. In the O 1s and N 1s spectra, major binding energies were
observed at 532.7 eV and 400.0 eV, respectively, confirming the presence of proteins involving copper NPs. These findings suggest the possibility that these agents act as capping agents.[39] The Cu 2p core level showed a sharp peak at 932.8 eV, corresponding to the Cu 2p3/2 level characteristic of Cu(0).[40–42] The presence of a CuO (Cu (II)) phase can be ruled out because of the lack of a signal at 933.7 eV. The presence of Cu2O can also be ruled out by the fact that no Cu 2p satellite peak appears with Cu2O.[43,44] These observations indicate the presence and binding of
Fig. 5. EDS spectra of dead biomass of T. koningiopsis. (a) Before exposure to copper solution, and (b) after exposure to the metal confirming the presence of copper.
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Fungal bioremediation of wastewater
Fig. 6. XPS spectra of C 1s, N 1s, O 1s and Cu 2p core-level binding energies after the biosynthesis of copper NPs by T. koningiopsis.
proteins to copper NPs. In dead biomass, proteins are probably released from the cell during the autoclaving process and bind to the cell surface. This finding indicates that proteins are bound to the surface of spherical copper NPs, thereby acting as stabilizing agents. However, the type of protein involved in the interactions with copper NPs remains to be determined. This information may permit the development of more efficient processes for the biosynthesis of copper NPs.
Conclusion In the present work we explored for the first time the extracellular biosynthesis and removal of copper NPs from aqueous solution using dead biomass of the fungus T. koningiopsis. Using T. koningiopsis as the reducing agent, a suitable strategy was developed for the rapid, inexpensive, environmental friendly and easily scalable biosynthesis and removal of copper NPs. The biosynthesis
1294 mediated by the fungus provides a promising approach for scaling up industrial and technological of the synthesis of copper NPs and appears to be a good candidate for industrial application in mycoremediation of copper NPs from contaminated water systems.
Salvadori et al.
[13]
[14]
Acknowledgments The authors are very much thankful to the Laboratory of Photoelectron Spectroscopy (LEFE), Universidade Estadual Paulista “J ulio de Mesquita Filho” (UNESP), Araraquara, S~ ao Paulo, Brazil, by the performing the XPS analysis in this research work.
[15]
[16]
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[17]
Funding
[18]
This work was supported by the Funda¸c ~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP grant 2010/ 52305-1).
[19] [20]
References
[22]
[1] Bhattacharya, D.; Gupta, R.K. Nanotechnology and potential of microorganisms. Crit. Rev. Biotechnol. 2005, 25(4), 199–204. [2] Varshney, R.; Bhadauria, S.; Gaur, M.S. A review: Biological synthesis of silver and copper nanoparticles. Nano. Biomed. Eng. 2012, 4(2), 99–106. [3] Lee, Y.; Choi, J.; Lee, K.J.; Stott, N.E.; Kim, D. Large-scale synthesis of copper nanoparticles by chemically controlled reduction for application of inkjet-printed electronics. Nanotechnology 2008, 19(41), 415604–415611. [4] Song, X.; Sun, S.; Zhang, W.; Yin, Z. A method for the synthesis of spherical copper nanoparticles in the organic phase. J. Coll. Interf. Sci. 2004, 273(2), 463–469. [5] Li, Y.; Liang, J.; Tao, Z.; Chen, J. CuO particles and plates: Synthesis and gas-sensor application. Mater. Res. Bull. 2007, 43(9), 2380–2385. [6] Guo, Z.; Liang, X.; Pereira, T.; Scaffaro, R.; Hahn, H.T. CuO nanoparticle filled viny-ester resin nanocomposites: Fabrication, characterization and property analysis. Compos. Sci. Tech. 2007, 67, 2036–2044. [7] Evans, P., Matsunaga, H.; Kiguchi, M. Large-scale application of nanotechnology for wood protection. Nat. Nanotechnol. 2008, 3, 577. [8] Saha, S.; Sarkar, J.; Chattopadhyay, D.; Patra, S.; Chakraborty, A.; Acharya, K. Production of silver nanoparticles by a phytopathogenic fungus Bipolaris nodulosa and its antimicrobial activity. Dig. J. Nanomater. Bios. 2010, 5(4), 887–895. [9] Han, R.; Zhang, J.; Zou, W.; Xiao, H.; Shi, J.; Liu, H. Biosorption of copper(II) and lead(II) from aqueous solution by chaff in a fixed-bed column. J. Hazard. Mater. 2006, 133(3), 262–268. [10] Volesky, B. Detoxification of metal bearing effluents: biosorption for the next century. Hydrometallurgy 2001, 59(1), 203–216. [11] Anand, P.; Isar, J.; Saran, S.; Saxena, R.K. Bioaccumulation of copper by Trichoderma viride. Bioresour. Technol. 2006, 97(1), 1018–1025. [12] Salvadori, M.R.; Lepre, L.F.; Ando, R.A.; do Nascimento, C.A. O.; Corr^ea, B. Biosynthesis and uptake of copper nanoparticles by
[21]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
dead biomass of Hypocrea lixii isolated from the metal mine in the Brazilian Amazon region. Plos One 2013, 8(11), 1–8. Honary, S.; Barabadi, H.; Gharaei-Fathabad, E.; Naghib, F. Green synthesis of copper oxide nanoparticles using Penicillium aurantiogriseum, Penicillium citrinum and Penicillium waksmanii. Dig. J. Nanomater. Bios. 2012, 7(3), 999–1005. Kumar, B.N.; Seshadri, N.; Ramana, D.K.V.; Seshaiah, K.; Reddy, A.V.R. Equilibrium, thermodynamic and kinetic studies on Trichoderma viride biomass as biosorbent for the removal of Cu (II) from water. Separ. Sci. Technol. 2011, 46, 997–1004. Ahmad, I.; Ansari, M.I.; Aqil, F. Biosorption of Ni, Cr and Cd by metal tolerante Aspergillus niger and Penicillium sp using single and multi-metal solution. Ind. J. Exp. Biol. 2006, 44(1), 73–76. Volesky, B. Biosorption process simulation tools. Hydrometallurgy 2003, 71(1), 179–190. Lagergren, S. About the theory of so called adsorption of soluble substances. Kung. Sven. Veten. Hand. 1898, 24, 1–39. Ho, Y.S.; Mckay, G. Pseudo–second-order model for sorption process. Proc. Biochem. 1999, 34, 451–465. Volesky, B.; Holan, Z.R. Biosorption of heavy metals. Biotechnol. Prog. 1995, 11, 235–250. Veglio, F.; Beolchini, F. Removal of metals by biosorption: a review. Hydrometallurgy 1997, 44(3), 301–316. Hanif, M.A.; Nadeem, R.; Bhatti, H.N.; Ahmad, N.R.; Ansari, T. M. Nickel biosorption by Cassia fistula (Golden shower) biomass. J. Hazard. Mater. 2007, 139(2), 345–355. Sari, A.; Tuzen, M.; Uluuozlu, O.D.; Soylak, M. Biosorption of Pb (II) and Ni(II) from aqueous solution by lichen (Cladoni furcata) biomass. Biochem. Eng. J. 2007, 37(2), 151–158. Kapoor, A.; Viraraghavan, T. Fungal biosorption. An alternative treatment option for heavy metals bearing wastewater. A review. Bioresour. Technol. 1995, 53(3), 195–206. Butter, T.J.; Evison, L.M.; Hancoch, H.F.S.; Matis, K.A.; Philipson, A.; Sheikh, A.J.; Zouboulis, A.I. The removal of cadmium from dilute aqueous solution by biosorption and electrolysis at laboratory scale. Water Res. 1998, 32(2), 400–406. Ozer, A.; Ozer, D. Comparative study of the biosorption of Pb(II), Ni(II) and Cr(VI) ions onto S. cerevisiae: Determination of biosorption heats. J. Hazard. Mater. 2003, 100(3), 219–229. Iftikhar, A.R.; Bhatti, H.N.; Hanif, M.A.; Nadeem, R. Kinetic and thermodynamic aspects of Cu(II) and Cr(III) removal from aqueous solution using rose waste biomass. J. Hazard. Mater. 2009, 161(3), 941–947. Liu, Y.G.; Fan, T.; Zeng, G.M.; Li, X.; Tong, Q.; Ye, F.; Zhou, M.; Xu, W.H.; Huang, Y.E. Removal of cadmium and zinc ions from aqueous solution by living Aspergillus niger. Trans. Nonferrous. Met. Soc. China 2006, 16(3), 681–686. Xuejiang, W.; Siqing, X.; Ling, C.; Jianfu, Z.; Jeanmarc, C.; Renault, N.J. Biosorption of cadmium(II) and lead(II) ions from aqueous solution onto dried activated sluge. J. Environ. Sci. 2006, 18(5), 840–844. Faryal, R.; Lodhi, A.; Hameed, A. Isolation, characterization and biosorption of zinc by indigenous fungal strains Aspergillus fumigatus RH05 and Aspergillus flavus RH07. Pak. J. Bot. 2006, 38(4), 817–832. Veit, M.T.; Tavares, C.R.G.; Gomes-da-Costa, S.M.; Guedes, T. A. Adsorption isotherms of copper (II) for two species of dead fungi biomasses. Proc. Biochem. 2005, 40(10), 3303–3308. Du, A.; Cao, L..; Zhang, R.; Pan, R. Effects of a copper-resistant fungus on copper adsorption and chemical forms in soils. Water Air Soil Poll. 2009, 201(4), 99–107. Rome, L.; Gadd, D.M. Copper adsorption by Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum. Appl. Microbiol. Biotechnol. 1987, 26(1), 84–90.
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Fungal bioremediation of wastewater [33] Yilmazer, P.; Saracoglu, N. Bioaccumulation and biosorption of copper (II) and chromium (III) from aqueous solutions by Pichia stiptis yeast. J. Chem. Technol. Biot. 2009, 84, 604–610. [34] Yahaya, Y.A.; Matdom, M.; Bhatia, S. Biosorption of copper (II) onto immobilized cells of Pycnoporus sanguineus from aqueous solution: Equilibrium and Kinetic studies. J. Hazard. Mater. 2008, 161(1), 189–195. [35] Nakajima, A.; Yasuda, M.; Yokoyama, H.; Ohya-Nishiguchi, H.; Kamada, H. Copper sorption by chemically treated Micrococcus luteus cells. World. J. Microb. Biot. 2001, 17(4), 343–347. € [36] Elmacy, A.; Yonar, T.; Ozengin, N. Biosorption characteristics of copper (II), chromium (III), nickel (II) and lead (II) from aqueous solutions by Chara sp and Cladophora sp. Water. Environ. Res. 2007, 79, 1000–1005. [37] Reddad, Z.; Gerent, C.; Andres, Y.; LeCloirec, P. Adsorption of several metal ions onto a low-cost biosorbents: kinetic and equilibrium studies. Environ. Sci. Technol. 2002, 36(9), 2067– 2073. [38] Bhainsa, K.C.; D’Souza, S.F. Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Coll. Surf. B 2006, 47(1), 160–164.
1295 [39] Bansal, V.; Ahamad, A.; Sastry, M. Fungus-mediated biotransformation of amorphous silica in rice husk to nanocrystalline silica. J. Am. Chem. Soc. 2006, 128(43), 14059–14066. [40] Naumkin, A.V.; Kraut-Vass, A.; Gaarenstroom, S.W.; Powell, C. J. NIST X-ray Photoelectron Spectroscopy Database: NIST Standard Reference Database 20, v. 4.1. National Institute of Standards and Technology: Gaithersburg, MD, 2012. Available at http:// www.srdata.nist.gov/XPS/ (accessed Dec 2013). [41] Briggs, D.; Seah, M.P. Practical Surface Analysis, Auger and X-ray Photoelectron Spectroscopy. John Wiley & Sons: Chichester, UK, 1990. [42] Jeong, S.; Woo, K.; Kim, D.; Lim, S.; Kim, J.S.; Shin, H.; Xia, Y.; Moon, J. Controlling the thickness of the surface oxide layer on Cu nanoparticles for the fabrication of conductive structures by ink-jet printing. Adv. Funct. Mater. 2008, 18(5), 679–686. [43] Zhang, J.; Wang, Y.; Cheng, P.; Yao, Y.L. Effect of pulsing parameters on laser ablative cleaning of copper oxides. J. Appl. Phys. 2006, 99(1), 1–11. [44] Ghodselahi, T.; Vesaghi, M.A.; Shafielkhani, A.; Bachizadeh, A.; Lameii, M. XPS study of the Cu@Cu2O core-shell nanoparticle. Appl. Surf. Sci. 2008, 255(5), 2730–2734.
Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Bioremediation from wastewater and extracellular synthesis of copper nanoparticles by the fungus Trichoderma koningiopsis a
b
c
Marcia R. Salvadori , Rômulo A. Ando , Cláudio A. Oller Do Nascimento & Benedito Corrêa
a
a
Department of Microbiology, Biomedical Institute II, University of São Paulo, São Paulo, Brazil b
Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil c
Department of Chemical Engineering, Polytechnic, University of São Paulo, São Paulo, Brazil Published online: 26 Jun 2014.
To cite this article: Marcia R. Salvadori, Rômulo A. Ando, Cláudio A. Oller Do Nascimento & Benedito Corrêa (2014) Bioremediation from wastewater and extracellular synthesis of copper nanoparticles by the fungus Trichoderma koningiopsis, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:11, 1286-1295, DOI: 10.1080/10934529.2014.910067 To link to this article: http://dx.doi.org/10.1080/10934529.2014.910067
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Journal of Environmental Science and Health, Part A (2014) 49, 1286–1295 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.910067
Bioremediation from wastewater and extracellular synthesis of copper nanoparticles by the fungus Trichoderma koningiopsis ^ MARCIA R. SALVADORI1, ROMULO A. ANDO2, CLAUDIO A. OLLER DO NASCIMENTO3 and 1 ^ BENEDITO CORREA 1
Department of Microbiology, Biomedical Institute II, University of S~ ao Paulo, S~ ao Paulo, Brazil Department of Fundamental Chemistry, Institute of Chemistry, University of S~ ao Paulo, S~ ao Paulo, Brazil 3 Department of Chemical Engineering, Polytechnic, University of S~ ao Paulo, S~ ao Paulo, Brazil
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2
This is the first study describing the rapid extracellular production of copper nanoparticles by dead biomass of Trichoderma koningiopsis. The production and uptake of copper nanoparticles by dead biomass of Trichoderma koningiopsis were characterized by investigating physicochemical factors, equilibrium concentrations and biosorption kinetics, combined with scanning electron microscopy (SEM), energy dispersive X-ray (EDS), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). A successful route for the metallic copper nanoparticles synthesis was achieved, and followed a Langmuir isotherm where a high biosorption capacity was observed, 21.1 mg g¡1. The kinetic analysis showed that copper biosorption followed a pseudo– second-order model. The nanoparticles mainly exhibited a spherical shape, with an average size of 87.5 nm, and were synthesized extracellularly. The presence of proteins as stabilizing agents of the nanoparticles was demonstrated. The extracellular biosynthesis and uptake of copper nanoparticles using dead fungal biomass is a low-cost green processes, and bioremediation of impacted local. Keywords: Copper nanoparticles, dead biomass, extracellular synthesis, Trichoderma koningiopsis.
Introduction The possible use of biological materials for the synthesis of metal nanoparticles (NPs) has emerged as a more efficient and greener approach.[1] Because synthetic methods comprise complex physical and chemical processes that require the use of high temperatures and pressures, large amounts of energy are needed and toxic wastes are produced, posing environmental and human health risks.[2] Copper NPs can be used in printed electronics, are suitable substitutes of conductive and expensive noble metals such as gold and silver in chemical and metallurgical processes,[3,4] and have applications in gas sensing, catalytic processes, high temperature superconductors, solar cells, and wood treatment.[5–7] The filamentous fungi possess advantages over bacteria and algae such as, the fungal mycelial mesh that can withstand flow pressure and agitation and other conditions in the bioreactors or other chambers, ease of handling and culturing on a large scale, high tolerance towards metals, Address correspondence to Marcia R. Salvadori, Department of Microbiology, Biomedical Institute II, University of S~ ao Paulo, 05508000, S~ao Paulo, Brazil; E-mail:[email protected] Received December 16, 2013.
and a high wall-binding capacity, as well as intracellular metal uptake capacity.[8] Because metal ions are not biodegradable, they are usually removed from contaminated soil and water by chemical or physical treatment process. Typical treatment processes such as membrane separation, precipitation, ion exchange, reverse osmosis and electrolysis are often not feasible because of high costs, the need for continuous input of chemicals, and the production of a toxic sludge.[9] Recently, the bioremediation of metal ion pollution has emerged as an environmental friendly technology.[10] Some microorganisms are potent bioremediators that remove metals ions through biosorption mechanisms. Fungi are used in bioremediation processes since they are a versatile group that can adapt to and grow under various extreme conditions of pH, temperature and nutrient availability, as well as at high metal concentrations.[11] A literature review[2] has shown that few articles[12,13] on the biosynthesis of copper NPs by fungi have been published and none of those studies used the fungus Trichoderma koningiopsis (T. koningiopsis). To increase the scope of biosystems for the synthesis of metallic nanomaterials and bioremediation of wastewater, we explored for the first time the potential of the fungus T. koningiopsis for the removal and reduction of copper ions to copper NPs.
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Fungal bioremediation of wastewater Materials and methods Growth and maintenance of the organism T. koningiopsis was isolated from the sediment collected in a copper waste pond at the Sossego mine, which belongs to the company Vale S.A. located in Cana~a dos Carajas, Par a, Brazilian Amazonia region (06 260 S latitude and 0 50 4 W longitude). T. koningiopsis was maintained and activated on Sabouraud Dextrose Agar (SDA) (Oxoid, England).[14]
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Minimum inhibitory concentration in agar medium Copper tolerance of the T. koningiopsis was determined as the minimum inhibitory concentration (MIC) by the spot plate method.[15] SDA plates containing different copper concentrations (50 to 2000 mg L¡1) were prepared and fungal inocula were spotted onto copper-containing and metal-free (control). The plates were incubated at 25 C for at least 5 days. The MIC is defined as the lowest concentration of the metal that inhibits visible growth of the isolate.
experiments were optimized using 45 mL of 100 mg L¡1 of Cu (II) test solution in plastic flask. Various concentrations of copper (II) were prepared by appropriate dilution of the copper (II) stock solution. The pH was adjusted with HCl or NaOH. The desired biomass dose was then added and the content of the flask was shaken. Next, the Cu (II) solution was separated from the biomass by vacuum filtration through a Millipore membrane. The metal concentration in the filtrate was determined by flame atomic absorption spectrophotometer. The efficiency (R) of metal removal was calculated using Eq. (1): R D .Ci -Ce /=Ci ¢100
(1)
where Ci and Ce are the initial and equilibrium metal concentrations, respectively. The metal uptake capacity, qe, was calculated using Eq. (2): qe D V.Ci -Ce /=M
(2)
where qe (mg g¡1) is the biosorption capacity of the biosorbent at any time, M(g) is the biomass dose, and V (L) is the volume of the solution.
Retention of copper NPs by the biosorbent Preparation of the adsorbate solutions All chemicals were of analytical grade and were used without further purification. The dilutions were prepared in double-deionized water (Milli-Q Millipore 18.2 MVcm¡1 resistivity). The copper stock solution was prepared by dissolving CuCl2.2H2O (Carlo Erba, Milan, Italy) in doubledeionized water. The working solutions were prepared by diluting this stock solution. Biomass preparation The fungal biomass was prepared in Sabouraud broth (Sb) (Oxoid, England), and incubated at 25 C for 5 days, under shaking at 150 rpm. After incubation, the pellets were harvested and washed with double-deionized water, corresponding to live biomass. For the preparation of dead biomass, an appropriate amount of live biomass was autoclaved. Dried biomass was obtained by drying the fungal mat at 50 C until it became crispy. The dried mat was ground to obtain uniformly sized particles.
Biosorption isotherm and kinetic models Two equilibrium models [16] were used to fit Cu (II) biosorption isotherm experimental data, as follows. The linearized Langmuir isotherm model can be described by Eq. (3): Ce =qe D 1=.qm ¢b/ C Ce =qm
(3)
where qm is the monolayer sorption capacity of the sorbent (mg g¡1), and b is the Langmuir sorption constant (L mg¡1). The linearized Freundlich isotherm model can be described by Eq. (4): Inqe D InKF C 1=n ¢InCe
(4)
where KF is a constant relating the biosorption capacity and the term written as “1/n” is related to the adsorption intensity of the adsorbent. The kinetics of Cu (II) biosorption was analyzed using pseudo–first-order, and pseudo–second-order models. The linear pseudo–first-order model [17] can be represented by Eq. (5):
Evaluation of the effects of physicochemical factors on the adsorption efficiency of copper NPs onto the biosorbent
log.qe ¡ qt / D logqe ¡ K1 =2:303 ¢t
The effect of pH (2–6), temperature (20–60 C), contact time (5–360 min), initial copper concentration (50–500 mg L¡1), agitation rate (50–250 rpm), and biosorbent dose (0.15–1.0 g) on the removal of copper was analyzed. These
where qe (mg g¡1) and qt (mg g¡1) are the amount of adsorbed metal on the sorbent at the equilibrium time and at any time t, respectively, and K1 (1 min¡1) is the rate constant of the pseudo–first-order adsorption process. The
(5)
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linear pseudo–second-order model by Eq. (6):
[18]
can be represented
t=qt D 1=K2 ¢qe C t=qe
(6)
where K2 (g mg¡1 min¡1) is the equilibrium rate constant of the pseudo–second-order model.
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Biosynthesis of copper NPs by T. koningiopsis Only dead biomass of T. koningiopsis, which showed a higher capacity to adsorb copper metal ions than live and dried biomass, was used. Biosynthesis of copper NPs by dead T. koningiopsis biomass was investigated at a concentration of 100 mg L¡1 copper (II) using the equilibrium model data. The size, shape and location of the copper NPs on the biosorbent were determined by analyzing ultrathin sections by TEM (JEOL-1010, Peabody, MA, USA). Small fragments of the biological material obtained before and after the formation of copper NPs were mounted on pin stubs, coated with gold under vacuum, and examined by SEM (JEOL 6460 LV) equipped with EDS. The XPS analysis was carried out at a pressure of less than 5 £ 10¡7 Pa using a commercial spectrometer (UNISPECS UHV System, Berlin, Germany). The Mg-Ka line was used (hn D 1253.6 eV) and the analyzer pass energy was set to 10 eV. The inelastic background of the C 1s, O 1s, N 1s and the Cu 2p3/2 electron core-level spectra was subtracted using Shirley’s method. The composition (at.%) of the near surface region was determined with an accuracy of §10% from the ratio of the relative peak areas corrected by Scofield’s sensitivity factors of the corresponding elements. The binding energy scale of the spectra was corrected using the C 1s hydrocarbon component of the fixed value of 285.0 eV. The spectra were fitted without placing constraints using multiple Voigt profiles. The width at half maximum (FWHM) varied between 1.2 and 2.1 eV and the accuracy of the peak positions was § 0.1 eV.
Results and discussion The effect of different copper concentrations (50–2000 mg L¡1) was tested in agar medium and the results showed that T. koningiopsis exhibited high tolerance to copper (1057 mg L¡1). It has previously been demonstrated that fungi are potential biosorbents of heavy metals,[19,20] and this fact was confirmed in the present study. Fungi offer a wide range of chemical groups that can attract and sequester metals present in the biomass. The cell wall of fungi consists of structural polysaccharides, proteins and lipids that contain metal-binding functional groups.[20]
Effects of physicochemical factors on biosorption The removal of copper by T. koningiopsis biomass is influenced by physicochemical factors such as biomass concentration, pH, temperature, contact time, rate of agitation, and metal ion concentration. The amount of biosorbent is an important parameter to determine the sorbent-sorbate equilibrium of the system.[21,22] Figure 1a shows that dead biomass was more efficient in removing copper compared to live and dried biomass. This finding indicates that dead biomass possesses a higher affinity for copper than live and dried biomass. The use of live biomass for the binding of metal ions depends on nutrient availability, environmental conditions and cell age.[23] In addition, live biomass may be subject to the toxic effect of heavy metals when present at high concentrations. Therefore, dead biomass is preferred to overcome these disadvantages.[24] The efficiency of ionization of functional groups present on the surface of the biomass cell wall depends on the pH of the solution.[25] The influence of pH on copper uptake is shown in Figure 1b. Maximum removal occurred at pH 5 for the three types of biomass. With respect to temperature, maximum removal of copper was observed at a temperature of 40 C for the three types of biomass (Fig. 1c). According to the theory of adsorption, adsorption decreases with increasing temperature, as molecules adsorbed earlier on a surface tend to desorb from the surface at higher temperatures.[26] The graph obtained for the three types of biomass showed sigmoid kinetics (Fig. 1d), characterizing an enzyme-catalyzed reaction. The results showed that this was a rapid process, with more than 90% of the NPs formed by dead biomass within 60 min of the reaction. Regarding agitation speed, optimum copper removal was observed at 150 rpm for the three types of biomass (Fig. 1e). At higher agitation speeds, vortex phenomena occur and the suspension is no longer homogenous, a fact impairing metal removal.[27] The initial metal concentration, provides an important driving force to overcome all mass transfer resistance of the metal between the aqueous and solid phases.[28] The percentage of copper adsorption decreased with increasing metal concentration (50–500 mg L¡1) for the three types of biomass (Fig. 1f). The same has been observed for the removal of zinc by fungi at concentrations ranging from 100–400 mg L¡1.[29]
Biosorption isotherm and kinetics models The experimental Cu (II) isotherm was tested using the Langmuir and Freundlich models. The Langmuir isotherms for Cu (II) biosorption obtained for the three types of T. koningiopsis biomass are shown in Figures 2a, 2b and 2c. Table 1 shows the isotherm constants, maximum loading capacity estimated by the Langmuir and Freundlich models, and regression coefficients. The Langmuir
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5 10 15 20 25 30 40 60 120 180 240 300 360
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a
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Live Dried Dead 50 100 150 200 300 400 500 Amount of copper (mg/L)
Fig. 1. Effects of physicochemical factors on live, dried and dead biomass of T. koningiopsis. (a) Amount of biosorbent; (b) pH; (c) temperature; (d) contact time; (e) agitation rate; and (f) initial copper concentration.
adsorption model was the best to fit the experimental biomass data. The dead biomass of T. koningiopsis (21.1 mg g¡1) had a similar or higher adsorption capacity than those described for other known biosorbents, such as, Pleurotus pulmonaris, Schizophyllum commune, Penicillium spp, Rhizopus arrhizus, Trichoderma viride, Pichia stiptis, Pycnoporus sanguineus with adsorption rates of 6.2, 1.52, 15.08, 19.0, 19.6, 15.85 and 2.76 mg g¡1 respectively.[14,30–34] Comparison with biosorbents of bacterial origin showed that the
Cu (II) adsorption rate of T. koningiopsis is similar to that of Bacillus subtilis IAM 1026 (20.8 mg g¡1).[35] In contrast the filamentous fungus T. koningiopsis exhibited a higher rate of metal ion adsorption than the algae Cladophora spp (14.28 mg g¡1).[36] The kinetic results suggested that Cu (II) adsorption by T. koningiopsis was best described using the pseudo–second-order kinetic model (Figs. 2d, 2e and 2f). This adsorption kinetics is typical of the adsorption of divalent metals onto biosorbents.[37] The constants and regression coefficients are shown in Table 2.
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Fig. 2. Langmuir plots for live (a), dried (b) and dead (c) biomass of T. koningiopsis. Pseudo–second-order models for live (d), dried (e) and dead (f) biomass.
Table 1. Adsorption constants from simulations with Langmuir and Freundlich models. Langmuir model b Type of qm biomass (mg g¡1) (L mg¡1) Live Dried Dead
6.0 10.0 21.1
0.021 0.021 0.043
Table 2. Kinetic parameters for adsorption of copper. Pseudo–first-order
Freundlich model R2 0.989 0.984 0.984
KF (mg g¡1) 16 n 0.41 0.44 1.10
R2
0.41 0.815 0.47 0.817 0.57 0.898
Type of biomass Live Dried Dead
Pseudo–second-order
K1 (1 min¡1)
R2
K2 (g mg¡1 min¡1)
R2
4.83 £ 10¡3 4.37 £ 10¡3 2.53 £ 10¡3
0.395 0.469 0.139
18.90 £ 10¡3 15.26 £ 10¡3 19.35 £ 10¡3
0.981 0.986 0.987
Fungal bioremediation of wastewater
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Biosynthesis and characterization of copper NPs using dead biomass of T. koningiopsis Knowledge of the mechanisms underlying the formation of NPs by biological systems is important in order to develop more credible and reproducible processes for their biosynthesis. For this purpose a fraction of dead T. koningiopsis biomass was examined by TEM, which revealed the presence of NPs in the cell wall, but not in the cytoplasm or cytoplasmic membrane. No NPs were detected in the control. The ultrastructural changes observed included shrinking of the cytoplasm in the control and in biomass impregnated with copper, probably as a result of the autoclaving process (Figs. 3a and 3b). From the point of view of applications, this extracellular location offers a major advantage compared to an intracellular synthesis process. Because the NPs formed inside the biomass require an additional processing step for their release from the biomass by ultrasound treatment or by reaction with suitable detergents,[38] the extracellular location permits the fast and easy removal of large amounts of NPs and the reutilization of biomass in the production process. In this study,
1291 the copper NPs had an average diameter of 87.5 nm and a predominantly spherical shape, although, few aggregates of NPs with an average diameter of 328.27 nm (Fig. 3c) were also observed. The presence of copper NPs was confirmed by spot profile SEM-EDS measurements. Figures 4a and 4b show the SEM photomicrographs before and after the biosorption of Cu (II) by the fungal biomass. Surface modifications could be seen that were mainly due to an increase in irregularity after the binding of copper NPs onto the surface of dead biomass. The spot profile EDS spectra recorded in the region of the mycelium examined indicated signals from copper (Figs. 5a and 5b) to the fungus. Furthermore, the C, N and O peaks correspond to the presence of proteins as capping material on the surface of the copper NPs and other biopolymers present in the sample. These peaks are likely due to proteins secreted by the fungus. The XPS spectra Figure 6 shows C 1s, N 1s, O 1s and Cu 2p core level after biosynthesis of copper NPs by dead biomass of T. koningiopsis. As can be seen in the high resolution carbon spectra (C 1s), the components of higher binding energy were deconvoluted within four elements. The main component
Fig. 3. TEM micrographs of T. koningiopsis sections. (a) Control (without copper). (b) Section of the fungus showing the extracellular localization of copper nanoparticles (arrow). (c) Aggregated of nanoparticles (arrow).
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Fig. 4. Dead biomass of T. koningiopsis analyzed by SEM-EDS. (a) Control (without copper), and (b) biomass exposed to copper.
at 284.8 eV can be attributed to the hydrocarbon chains of the cellular phase, the peak at 286.5 eV to the a- carbon, the peak at 288.0 eV to the carbonyl groups and, finally, the peak at 289.0 eV to the carboxylic groups of the peptides/proteins bound to copper NPs.[39] The deconvoluted spectra of oxygen (O 1s), showed peaks at 531.4 eV, 532.7 eV and 533.2 eV and corresponded to the peaks found in the C 1s spectra. The nitrogen spectra (N 1s) had two components, the main component at 400.0 eV and a lower one at 402.0 eV. In the O 1s and N 1s spectra, major binding energies were
observed at 532.7 eV and 400.0 eV, respectively, confirming the presence of proteins involving copper NPs. These findings suggest the possibility that these agents act as capping agents.[39] The Cu 2p core level showed a sharp peak at 932.8 eV, corresponding to the Cu 2p3/2 level characteristic of Cu(0).[40–42] The presence of a CuO (Cu (II)) phase can be ruled out because of the lack of a signal at 933.7 eV. The presence of Cu2O can also be ruled out by the fact that no Cu 2p satellite peak appears with Cu2O.[43,44] These observations indicate the presence and binding of
Fig. 5. EDS spectra of dead biomass of T. koningiopsis. (a) Before exposure to copper solution, and (b) after exposure to the metal confirming the presence of copper.
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Fungal bioremediation of wastewater
Fig. 6. XPS spectra of C 1s, N 1s, O 1s and Cu 2p core-level binding energies after the biosynthesis of copper NPs by T. koningiopsis.
proteins to copper NPs. In dead biomass, proteins are probably released from the cell during the autoclaving process and bind to the cell surface. This finding indicates that proteins are bound to the surface of spherical copper NPs, thereby acting as stabilizing agents. However, the type of protein involved in the interactions with copper NPs remains to be determined. This information may permit the development of more efficient processes for the biosynthesis of copper NPs.
Conclusion In the present work we explored for the first time the extracellular biosynthesis and removal of copper NPs from aqueous solution using dead biomass of the fungus T. koningiopsis. Using T. koningiopsis as the reducing agent, a suitable strategy was developed for the rapid, inexpensive, environmental friendly and easily scalable biosynthesis and removal of copper NPs. The biosynthesis
1294 mediated by the fungus provides a promising approach for scaling up industrial and technological of the synthesis of copper NPs and appears to be a good candidate for industrial application in mycoremediation of copper NPs from contaminated water systems.
Salvadori et al.
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[14]
Acknowledgments The authors are very much thankful to the Laboratory of Photoelectron Spectroscopy (LEFE), Universidade Estadual Paulista “J ulio de Mesquita Filho” (UNESP), Araraquara, S~ ao Paulo, Brazil, by the performing the XPS analysis in this research work.
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Funding
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This work was supported by the Funda¸c ~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP grant 2010/ 52305-1).
[19] [20]
References
[22]
[1] Bhattacharya, D.; Gupta, R.K. Nanotechnology and potential of microorganisms. Crit. Rev. Biotechnol. 2005, 25(4), 199–204. [2] Varshney, R.; Bhadauria, S.; Gaur, M.S. A review: Biological synthesis of silver and copper nanoparticles. Nano. Biomed. Eng. 2012, 4(2), 99–106. [3] Lee, Y.; Choi, J.; Lee, K.J.; Stott, N.E.; Kim, D. Large-scale synthesis of copper nanoparticles by chemically controlled reduction for application of inkjet-printed electronics. Nanotechnology 2008, 19(41), 415604–415611. [4] Song, X.; Sun, S.; Zhang, W.; Yin, Z. A method for the synthesis of spherical copper nanoparticles in the organic phase. J. Coll. Interf. Sci. 2004, 273(2), 463–469. [5] Li, Y.; Liang, J.; Tao, Z.; Chen, J. CuO particles and plates: Synthesis and gas-sensor application. Mater. Res. Bull. 2007, 43(9), 2380–2385. [6] Guo, Z.; Liang, X.; Pereira, T.; Scaffaro, R.; Hahn, H.T. CuO nanoparticle filled viny-ester resin nanocomposites: Fabrication, characterization and property analysis. Compos. Sci. Tech. 2007, 67, 2036–2044. [7] Evans, P., Matsunaga, H.; Kiguchi, M. Large-scale application of nanotechnology for wood protection. Nat. Nanotechnol. 2008, 3, 577. [8] Saha, S.; Sarkar, J.; Chattopadhyay, D.; Patra, S.; Chakraborty, A.; Acharya, K. Production of silver nanoparticles by a phytopathogenic fungus Bipolaris nodulosa and its antimicrobial activity. Dig. J. Nanomater. Bios. 2010, 5(4), 887–895. [9] Han, R.; Zhang, J.; Zou, W.; Xiao, H.; Shi, J.; Liu, H. Biosorption of copper(II) and lead(II) from aqueous solution by chaff in a fixed-bed column. J. Hazard. Mater. 2006, 133(3), 262–268. [10] Volesky, B. Detoxification of metal bearing effluents: biosorption for the next century. Hydrometallurgy 2001, 59(1), 203–216. [11] Anand, P.; Isar, J.; Saran, S.; Saxena, R.K. Bioaccumulation of copper by Trichoderma viride. Bioresour. Technol. 2006, 97(1), 1018–1025. [12] Salvadori, M.R.; Lepre, L.F.; Ando, R.A.; do Nascimento, C.A. O.; Corr^ea, B. Biosynthesis and uptake of copper nanoparticles by
[21]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
dead biomass of Hypocrea lixii isolated from the metal mine in the Brazilian Amazon region. Plos One 2013, 8(11), 1–8. Honary, S.; Barabadi, H.; Gharaei-Fathabad, E.; Naghib, F. Green synthesis of copper oxide nanoparticles using Penicillium aurantiogriseum, Penicillium citrinum and Penicillium waksmanii. Dig. J. Nanomater. Bios. 2012, 7(3), 999–1005. Kumar, B.N.; Seshadri, N.; Ramana, D.K.V.; Seshaiah, K.; Reddy, A.V.R. Equilibrium, thermodynamic and kinetic studies on Trichoderma viride biomass as biosorbent for the removal of Cu (II) from water. Separ. Sci. Technol. 2011, 46, 997–1004. Ahmad, I.; Ansari, M.I.; Aqil, F. Biosorption of Ni, Cr and Cd by metal tolerante Aspergillus niger and Penicillium sp using single and multi-metal solution. Ind. J. Exp. Biol. 2006, 44(1), 73–76. Volesky, B. Biosorption process simulation tools. Hydrometallurgy 2003, 71(1), 179–190. Lagergren, S. About the theory of so called adsorption of soluble substances. Kung. Sven. Veten. Hand. 1898, 24, 1–39. Ho, Y.S.; Mckay, G. Pseudo–second-order model for sorption process. Proc. Biochem. 1999, 34, 451–465. Volesky, B.; Holan, Z.R. Biosorption of heavy metals. Biotechnol. Prog. 1995, 11, 235–250. Veglio, F.; Beolchini, F. Removal of metals by biosorption: a review. Hydrometallurgy 1997, 44(3), 301–316. Hanif, M.A.; Nadeem, R.; Bhatti, H.N.; Ahmad, N.R.; Ansari, T. M. Nickel biosorption by Cassia fistula (Golden shower) biomass. J. Hazard. Mater. 2007, 139(2), 345–355. Sari, A.; Tuzen, M.; Uluuozlu, O.D.; Soylak, M. Biosorption of Pb (II) and Ni(II) from aqueous solution by lichen (Cladoni furcata) biomass. Biochem. Eng. J. 2007, 37(2), 151–158. Kapoor, A.; Viraraghavan, T. Fungal biosorption. An alternative treatment option for heavy metals bearing wastewater. A review. Bioresour. Technol. 1995, 53(3), 195–206. Butter, T.J.; Evison, L.M.; Hancoch, H.F.S.; Matis, K.A.; Philipson, A.; Sheikh, A.J.; Zouboulis, A.I. The removal of cadmium from dilute aqueous solution by biosorption and electrolysis at laboratory scale. Water Res. 1998, 32(2), 400–406. Ozer, A.; Ozer, D. Comparative study of the biosorption of Pb(II), Ni(II) and Cr(VI) ions onto S. cerevisiae: Determination of biosorption heats. J. Hazard. Mater. 2003, 100(3), 219–229. Iftikhar, A.R.; Bhatti, H.N.; Hanif, M.A.; Nadeem, R. Kinetic and thermodynamic aspects of Cu(II) and Cr(III) removal from aqueous solution using rose waste biomass. J. Hazard. Mater. 2009, 161(3), 941–947. Liu, Y.G.; Fan, T.; Zeng, G.M.; Li, X.; Tong, Q.; Ye, F.; Zhou, M.; Xu, W.H.; Huang, Y.E. Removal of cadmium and zinc ions from aqueous solution by living Aspergillus niger. Trans. Nonferrous. Met. Soc. China 2006, 16(3), 681–686. Xuejiang, W.; Siqing, X.; Ling, C.; Jianfu, Z.; Jeanmarc, C.; Renault, N.J. Biosorption of cadmium(II) and lead(II) ions from aqueous solution onto dried activated sluge. J. Environ. Sci. 2006, 18(5), 840–844. Faryal, R.; Lodhi, A.; Hameed, A. Isolation, characterization and biosorption of zinc by indigenous fungal strains Aspergillus fumigatus RH05 and Aspergillus flavus RH07. Pak. J. Bot. 2006, 38(4), 817–832. Veit, M.T.; Tavares, C.R.G.; Gomes-da-Costa, S.M.; Guedes, T. A. Adsorption isotherms of copper (II) for two species of dead fungi biomasses. Proc. Biochem. 2005, 40(10), 3303–3308. Du, A.; Cao, L..; Zhang, R.; Pan, R. Effects of a copper-resistant fungus on copper adsorption and chemical forms in soils. Water Air Soil Poll. 2009, 201(4), 99–107. Rome, L.; Gadd, D.M. Copper adsorption by Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum. Appl. Microbiol. Biotechnol. 1987, 26(1), 84–90.
Downloaded by [University of Waterloo] at 12:58 09 October 2014
Fungal bioremediation of wastewater [33] Yilmazer, P.; Saracoglu, N. Bioaccumulation and biosorption of copper (II) and chromium (III) from aqueous solutions by Pichia stiptis yeast. J. Chem. Technol. Biot. 2009, 84, 604–610. [34] Yahaya, Y.A.; Matdom, M.; Bhatia, S. Biosorption of copper (II) onto immobilized cells of Pycnoporus sanguineus from aqueous solution: Equilibrium and Kinetic studies. J. Hazard. Mater. 2008, 161(1), 189–195. [35] Nakajima, A.; Yasuda, M.; Yokoyama, H.; Ohya-Nishiguchi, H.; Kamada, H. Copper sorption by chemically treated Micrococcus luteus cells. World. J. Microb. Biot. 2001, 17(4), 343–347. € [36] Elmacy, A.; Yonar, T.; Ozengin, N. Biosorption characteristics of copper (II), chromium (III), nickel (II) and lead (II) from aqueous solutions by Chara sp and Cladophora sp. Water. Environ. Res. 2007, 79, 1000–1005. [37] Reddad, Z.; Gerent, C.; Andres, Y.; LeCloirec, P. Adsorption of several metal ions onto a low-cost biosorbents: kinetic and equilibrium studies. Environ. Sci. Technol. 2002, 36(9), 2067– 2073. [38] Bhainsa, K.C.; D’Souza, S.F. Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Coll. Surf. B 2006, 47(1), 160–164.
1295 [39] Bansal, V.; Ahamad, A.; Sastry, M. Fungus-mediated biotransformation of amorphous silica in rice husk to nanocrystalline silica. J. Am. Chem. Soc. 2006, 128(43), 14059–14066. [40] Naumkin, A.V.; Kraut-Vass, A.; Gaarenstroom, S.W.; Powell, C. J. NIST X-ray Photoelectron Spectroscopy Database: NIST Standard Reference Database 20, v. 4.1. National Institute of Standards and Technology: Gaithersburg, MD, 2012. Available at http:// www.srdata.nist.gov/XPS/ (accessed Dec 2013). [41] Briggs, D.; Seah, M.P. Practical Surface Analysis, Auger and X-ray Photoelectron Spectroscopy. John Wiley & Sons: Chichester, UK, 1990. [42] Jeong, S.; Woo, K.; Kim, D.; Lim, S.; Kim, J.S.; Shin, H.; Xia, Y.; Moon, J. Controlling the thickness of the surface oxide layer on Cu nanoparticles for the fabrication of conductive structures by ink-jet printing. Adv. Funct. Mater. 2008, 18(5), 679–686. [43] Zhang, J.; Wang, Y.; Cheng, P.; Yao, Y.L. Effect of pulsing parameters on laser ablative cleaning of copper oxides. J. Appl. Phys. 2006, 99(1), 1–11. [44] Ghodselahi, T.; Vesaghi, M.A.; Shafielkhani, A.; Bachizadeh, A.; Lameii, M. XPS study of the Cu@Cu2O core-shell nanoparticle. Appl. Surf. Sci. 2008, 255(5), 2730–2734.
