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Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization
JES-00943; No of Pages 8 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jes
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Yuanyuan Qu1,⁎, Wenli Shen1 , Xiaofang Pei1 , Fang Ma2,⁎, Shengnan You1 , Shuzhen Li1 , Jingwei Wang1 , Jiti Zhou1
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Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization
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1. State Key Laboratory of Fine Chemicals, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China 2. State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
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Article history:
Developing an eco-friendly approach for metallic nanoparticle synthesis is important in 17
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Received 3 May 2016
current nanotechnology research. In this study, green synthesis of gold nanoparticles 18
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Revised 22 August 2016
(AuNPs) was carried out by a newly isolated strain Trichoderma sp. WL-Go. UV–vis spectra of 19
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Accepted 7 September 2016
AuNPs showed a surface plasmon resonance peak at 550 nm, and transmission electron 20
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Available online xxxx
microscopy images revealed that the AuNPs were of varied shape with well dispersibility. 21
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Keywords:
pH 7–11. Moreover, the bio-AuNPs could efficiently catalyze the decolorization of various 23
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Gold nanoparticles
azo dyes. This research provided a new microbial resource candidate for green synthesis of 24
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Biosynthesis
AuNPs and demonstrated the potential application of bio-AuNPs for azo dye decolorization. 25
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Trichoderma
© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 26
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Azo dyes
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Decolorization
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The optimal conditions for AuNP synthesis were HAuCl4 1.0 mmol/L, biomass 0.5 g and 22
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Introduction
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Due to the advanced characteristics over the bulk materials, nanomaterials possess a great potential for innovative technology and are widely applied to every aspect of human life (Kuang et al., 2013). Among the various nanomaterials, gold nanoparticles (AuNPs) have attracted enormous attention because of their high oxidation resistance, biocompatibility and stability (Bhumkar et al., 2007; Gong and Mullins, 2009; Shukla et al., 2005). Various chemical and physical methods are available for AuNPs synthesis, however, the stability of nanoparticles and the use of toxic chemicals in AuNPs production processes raise intensive concerns (Das et al., 2010; Narayanan and Sakthivel, 2010). Therefore, there is an urgent need to develop a green process for AuNPs synthesis. Biological
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Published by Elsevier B.V. 27
methods of AuNPs synthesis have offered a reliable and ecofriendly alternative to physical and chemical methods (Zhang et al., 2011). It has been reported that many microbial resources such as bacteria, actinomycetes, fungi and yeasts have the potential to synthesize AuNPs (Zhang et al., 2011; Kitching et al., 2015; Shedbalkar et al., 2014). Among them, fungi are considered as better microbial resources for high production of nanoparticles due to their remarkable advantages for AuNP synthesis (Girard et al., 2013; Du et al., 2011). For instance, they secrete a large amount of proteins and secondary metabolites extracellularly, the extracellularly synthesized nanoparticles can be simply separated without the downstream processing (Mishra et al., 2014). However, less than 30 fungal species have been investigated for AuNP synthesis (Kitching et al., 2015), thus
⁎ Corresponding authors. E-mails: [email protected] (Yuanyuan Qu), [email protected] (Fang Ma).
http://dx.doi.org/10.1016/j.jes.2016.09.007 1001-0742/© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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Azo dyes used in this study were obtained from Dye Synthesize Laboratory, Dalian University of Technology. Hydrogen tetrachloroaurate (III) hydrate (HAuCl4·3H2O) was purchased from J&K Scientific Ltd. (China). All other chemicals were of analytical grade or above. The medium used in selection and cultivation was modified martin broth (MMB, pH 7), which consisted of (NH4)2SO4 1 g/L, KH2PO4 1 g/L, MgSO4·7H2O 0.5 g/L, and glucose 1 g/L. Solid medium contained 2.0% (w/v) agar in MMB. Tetracycline and terramycin (both 50 mg/L) were added to the medium as selective pressure for strain isolation. The media were autoclaved at 115°C, for 15 min before use.
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1.2. Isolation and identification of strain WL-Go
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The activated sludge samples from antibiotic-contaminated bioreactors were serially diluted and spread onto MMB agar plates. After incubation at 30°C for 4 days, colonies were selected and transferred into 25 mL MMB medium and cultivated at 30°C for 3 days with shaking at 150 r/min. After
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1.3. Biomass of strain WL-Go preparation and synthesis of 136 AuNPs 137
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Strain WL-Go was cultivated in 100 mL MMB medium at 30°C for 2 days with shaking at 150 r/min. After incubation, the biomass of strain WL-Go was prepared by filtration and washed three times using sterile distilled water. Appropriate concentration of wet biomass was resuspended in distilled water and incubated with 1.0 mmol/L HAuCl4 for 40 hr at 30°C. Control experiment without biomass was also performed. The effects of HAuCl4 concentration, biomass concentration and pH on AuNP synthesis were carried out as follows: (1) various concentrations of HAuCl4 (0.1, 0.5, 1.0, 2.0, 5.0 mmol/L) were added to the solutions containing 0.1 g biomass with final volume of 4 mL, (2) biomass (0.1, 0.2, 0.3, 0.4, 0.5 g) suspended in 4 mL sterile distilled water were incubated with 2.0 mmol/L HAuCl4 solution, (3) the reaction solution was adjusted to different pH (3, 5, 7, 9, 11) using HCl and NaOH. The above experiments were performed at 30°C for 40 hr.
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1.4. Characterization of AuNPs
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The formation of AuNPs was firstly examined by visual observation of color change of suspensions, and then monitored by UV–Vis spectrophotometer (Metash UV-9000, China) recording the spectra between 400 and 800 nm at the resolution of 1 nm. The morphology of AuNPs was characterized through a Tecnai G2 Spirit TEM (FEI, The Netherlands) with an accelerating voltage of 120 kV. For TEM analysis, 15 μL of samples were dropped on a carbon coated copper grid and dried at room temperature before analyses.
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1.5. Decolorization of azo dyes by bio-AuNPs
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The bio-AuNPs used for decolorization were prepared by ultrasonication of the reaction mixture containing strain WL-Go and the synthesized AuNPs. The potential catalytic activity of bio-AuNPs for decolorization of azo dyes was demonstrated using acid, reactive and cationic azo dyes. The dyes were added to the bio-AuNPs solution with final concentration of 50 mg/L, and then incubated at 30°C with shaking at 150 r/min. Control experiment was carried out under identical conditions using ultrasonicated WL-Go cells instead of the bio-AuNPs when decolorization of 50 mg/L Acid Brilliant Scarlet GR. The effect of initial concentration (25, 50, 100, 200 mg/L) of Acid Brilliant Scarlet GR on decolorization was also investigated.
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screening and enriching for about two months, one colony designed as WL-Go was selected for further characterization and identified by 26S rRNA gene sequence analysis. Genomic DNA of strain WL-Go was used to amplify the 26S rRNA gene by polymerase chain reaction (PCR), and then the PCR product was sequenced by TaKaRa Biotechnology Co. Ltd. (Dalian, China). The BLAST program was used to compare the sequence with those in GenBank, and the related sequences were aligned using Clustal X (1.8). The aligned sequences were used to construct phylogenetic tree using Neighbor-joining method by MEGA (Version 5.1) with 1000 bootstrap replicates (Qu et al., 2010).
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it is necessary to develop more fungal resources for AuNP synthesis. Besides, the control of size, shape and dispersity is a major challenge in the process of AuNP biosynthesis. Studies have reported that some factors, such as substrate concentrations, biomass concentrations and pH, could affect the size, shape and dispersity of AuNPs (Gericke and Pinches, 2006; Pimprikar et al., 2009; Mishra et al., 2011). As is known, particle size directly affects the properties of nanomaterial and thus the size control on AuNP biosynthesis is significantly important. Azo dyes are the largest chemical class of dyes with amounts of structural and color variety (Tony et al., 2009). Disposal of dyes-containing effluents has caused serious damage to the environment. Since the color contents in dyes adsorb and reflect sunlight entering the contaminated wastewater, they significantly affect the photosynthesis and the growth of aquatic organisms (Champagne and Ramsay, 2010). Some efforts about the green methods for azo dye pollutant treatment have been published. A variety of microbes, such as Shewanella decolorationis S12, Bacillus and Pseudomonas species, could be efficiently used for the treatment of azo dye pollutants (Chen et al., 2011; Fang et al., 2015; Xu et al., 2007). Nanoremediation as an alternative could well improve the efficiency of the degradation process (Kumari and Philip, 2015). However, the application of biogenic AuNPs on dye degradation has been rarely reported. In the present study, a newly isolated Trichoderma strain was exploited to green synthesis of AuNPs. The characteristics of AuNPs were determined by UV–Vis spectrophotometer and transmission electron microscopy (TEM). Different parameters that may affect the biosynthesis of AuNPs were investigated to obtain the optimal synthesis conditions. In addition, the catalytic characteristics of the as-prepared bio-AuNPs were evaluated by decolorization of various azo dyes.
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Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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1.6. Analytical methods
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The decolorization process was monitored by scanning the characteristic absorption wavelength (λmax) of azo dyes with UV–Vis spectrophotometer after centrifugation at 10,000g for 5 min at different time intervals. The decolorization rate was calculated using the following equation:
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Decolorization ð%Þ ¼ ðA0 −A1 Þ=A0 100
2. Results and discussion
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2.1. Isolation and identification of strain WL-Go
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A fungal strain designated as WL-Go was successfully isolated from the activated sludge sample (Dalian, China). Strain WL-Go was tubular hyphae and obtuse spores as observed by scanning electron microscope, and its colonies showed green concentric round striate on agar plate (Fig. 1a). Based on the analysis of 26S rRNA gene sequence, strain WL-Go
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The AuNPs were synthesized by incubating strain WL-Go with HAuCl4. The color of the reaction solution turned violet after incubating strain WL-Go with HAuCl4 for 4 hr, and the UV–vis spectrum of the reaction solution showed an absorption peak at 550 nm (Fig. 2a), which indicated the formation of AuNPs (Kalishwaralal et al., 2009). TEM images showed that the synthesized AuNPs were well-dispersed with diverse shapes such as spheres (the major shape), triangles and hexagons. In addition, the spherical particles tended to be smaller than the particles with polygonal and triangular shape (Fig. 2b). Trichoderma is a well-known genus that can secret various kinds and large amounts of extracellular enzymes and metabolites, thus it can serve as an excellent candidate for industrial scale production of metal nanoparticles (Vahabi
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2.2. Biosynthesis of AuNPs by strain WL-Go
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where A0 and A1 represented the initial and final absorbance of the dye, respectively. The intermediate products of Acid Brilliant Scarlet GR reduction by bio-AuNPs were performed with HPLC-MS as described previously (Tan et al., 2013).
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exhibited 100% homology to Trichoderma (Genebank accession number KM242362), thus strain WL-Go was identified as Trichoderma. The phylogenic tree demonstrating the relationship between strain WL-Go and other strains is shown in Fig. 1b. The partial 26S rRNA gene sequence (500 bp) of strain WL-Go was deposited in GenBank database under accession number KP676894. Strain WL-Go has been deposited in the China General Microbiological Culture Collection Center (CGMCC; Beijing) under accession number CGMCC 10456.
Fig. 1 – (a) The images of hypha (i), spores (ii) and agar plate (iii) of strain WL-Go. (b) Phylogenetic tree of Trichoderma sp. WL-Go and related species. The GenBank accession number corresponded to each microorganism was exhibited in parentheses. Bootstrap values were showed at each branch. Scale bar indicated 0.002 Jukes-cantor distances. Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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et al., 2011). AuNP synthesis by Trichoderma was conducted previously. For instance, gold nanospheres with uniform diameter were synthesized by Trichoderma asperellum (Mukherjee et al., 2012). Spherical AuNPs with the particle sizes ranging from 26 to 34 nm were synthesized by Trichoderma harzianum (Tripathi et al., 2014). Moreover, rapid biosynthesis of AuNPs was achieved using cell free extract of Trichoderma viride (Mishra et al., 2014). These studies indicated that Trichoderma strains had excellent ability on AuNP biosynthesis. The strain Trichoderma sp. WL-Go could serve as a new potential microbial resource for biosynthesis of AuNPs.
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Fig. 2 – (a) UV–vis spectra of the dispersed AuNPs synthesized by strain WL-Go after 40 hr incubation, HAuCl4 solution, and control strain WL-Go. (b) The TEM image of AuNPs synthesized by strain WL-Go.
Fig. 3 – UV–Vis spectra of the dispersed AuNPs synthesized under different conditions: (a) HAuCl4 concentrations (0.1, 0.5, 1.0, 2.0, 5.0 mM), (b) strain WL-Go biomass (0.1, 0.2, 0.3, 0.4, 0.5 g) and (c) pH (3, 5, 7, 9, 11) of reaction solutions. AuNPs: gold nanoparticles.
2.3. Effect of HAuCl4 concentrations on AuNPs synthesis by strain WL-Go The effect of HAuCl4 concentrations on AuNPs synthesis is shown in Fig. 3a. Suspensions treated with 0.5, 1.0 and 2.0 mmol/L HAuCl4 for 40 hr showed violet or pink color, and
the absorption peaks at 550 nm were also recorded, which confirmed the formation of AuNPs (Song et al., 2009). Furthermore, a strong surface plasmon resonance (SPR) band at 550 nm was obtained when the HAuCl4 concentration was
Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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t1:1 t1:3 t1:2
2.4. Effect of biomass concentrations on AuNP synthesis by strain WL-Go The effect of biomass concentrations on AuNP synthesis is shown in Fig. 3b. All the reaction solutions were changed to purple color, which indicated that the AuNPs were synthesized at different biomass concentrations. The SPR absorbance of reaction solution exhibited an apparent broadening at maximum wavelength (550 nm) when using 0.1 g biomass. The generation of broad SPR band was probably due to the formation of large sized AuNPs, e.g., sphere, triangle and hexagon, resulting in the transverse interaction of radiance
2.5. Effect of pH on AuNPs synthesis by strain WL-Go
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The effect of pH on AuNPs synthesis is shown in Fig. 3c. It was observed that the rate of AuNPs synthesis enhanced by increasing the pH of solutions. The color changed within 2 hr under extremely alkaline condition (pH 12), whereas it took more than 4 hr under acidic condition (pH 3). In addition, the SPR band was broad at pH 3, while it became relative sharp and strong at pH 7–11. The better performances of AuNP synthesis at pH 7–11 were probably attributed to the higher stability of the capping proteins secreted by the fungus under neutral or alkaline conditions, while protonation of the carboxylic groups in proteins might occur under acidic condition (Bastús et al., 2011). This phenomenon was also observed by Mishra et al., who confirmed that the cell free extract of Trichoderma viride could synthesize AuNPs at pH 7 and 9, but not at pH 5 (Mishra et al., 2011).
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(Kreibig and Genzel, 1985; Sujitha and Kannan, 2013). However, it was noteworthy that the absorbance band became narrower and blue shift when the biomass concentration increased to 0.5 g. The reason should be that higher concentration of biomass would provide more biomolecules to reduce gold ions, thus smaller size particles could be synthesized, leading to the blue shift in absorption peak (Haiss et al., 2007).
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1.0 mmol/L. Therefore, the optimal HAuCl4 concentration for AuNP synthesis was 1.0 mmol/L. Gold particles in large sizes were synthesized attaching on the tube wall when the HAuCl4 concentration increased to 5.0 mmol/L. The formation of large particles was probably due to the lack of biomolecules required for capping and efficient stabilization of the synthesized nanoparticles (Kumari and Philip, 2015). A similar observation was also reported on AuNP synthesis using Verticillium luteoalbum. It revealed that when HAuCl4 concentration was below 500 mg/L, the particle size was around 20 nm, but then the particle size distributed from 50 nm to several hundred nanometers when HAuCl4 concentration was above 500 mg/L. In addition, massive particle aggregates could be found on the cells (Gericke and Pinches, 2006).
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Table 1 – Decolorization of azo dyes (50 mg/L) by bio-AuNPs.
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Azo dyes
λmax (nm)
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Acid Brilliant Scarlet GR
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94.7
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Acid Red B
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84.8
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Acid Orange G
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Acid Black 1
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Reactive Red X-3B
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Reactive Black
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Reactive Red
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Cation Red
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AuNPs: gold nanoparticles.
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Molecular structure
Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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The application of bio-AuNPs in decolorization of azo dyes was investigated. The bio-AuNPs could decolor various azo dyes as shown in Table 1, including four acid azo dyes (82.9%– 94.7% in 120 min), three reactive azo dyes (46.1%–73.3% in 100–180 min) and one cation azo dye (41.7% in 180 min). The highest decolorization efficiency was 94.7% for Acid Brilliant Scarlet GR, thus it was chosen as the model dye for further investigation. Decolorization processes of Acid Brilliant Scarlet GR with different concentrations by the bio-AuNPs are shown in Fig. 4. It was observed that the decolorization efficiency of Acid Brilliant Scarlet GR was more than 90% in 40 min when the dye concentration was 25 mg/L. It was still of high efficient
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3. Conclusions In this study, green synthesis of AuNPs by a newly isolated strain Trichoderma sp. WL-Go was investigated. The UV–vis and TEM analysis were employed to investigate the characteristics of the biosynthesized AuNPs. The effects of different
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Fig. 4 – Effects of initial concentration (25, 50, 100, 200 mg/L) of Acid Brilliant Scarlet GR on decolorization.
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(more than 90% in 100 min) when the concentration of Acid Brilliant Scarlet GR increased to 50 mg/L, however, only 4.8% Acid Brilliant Scarlet GR were decolored by ultrasonicated WL-Go cells (Fig. S1). It was suggested that the AuNPs greatly improved the decolorization rate with high catalytic activities. The byproducts of Acid Brilliant Scarlet GR decolorization were analyzed by HPLC-MS, and two possible decolorization intermediates were detected (Fig. 5). 3, 7-Dihydroxyoctahydronaphthalene-2,6-dione (I) was corresponding to the mass peak of 215.1658 ([M–H+]), and naphthol (II) was confirmed with m/z ion peak of 143.1078 ([M–H+]), both of which were detected as the intermediates of Acid Brilliant Scarlet GR degradation in previous studies (Tan et al., 2013; Gomi et al., 2011). Generally, reductive cleavage of azo groups (\N_N\) was the first step of azo dye degradation (dos Santos et al., 2007). Therefore, the Acid Brilliant Scarlet GR was probably firstly transformed to the corresponding amines, such as 1-aminonaphthylene-2hydroxy-3, 6-disulfonic acid (III), which, however, was not detected in this study. Then, the intermediate III was probably oxidized to the compound I under aerobic conditions (Tan et al., 2013), and intermediate II could be generated from product I through removing a hydroxyl group and two aldehyde groups. The results suggested the potential application of bio-AuNPs for effective decolorization of azo dyes.
Fig. 5 – The mass spectra of intermediates formed from Acid Brilliant Scarlet GR removal by bio-AuNPs, I: 3, 7-dihydroxyoctahydro-naphthalene-2, 6-dione; II: naphthol. AuNPs: gold nanoparticles. Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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This work was supported by the National Natural Science Foundation of China (Nos. 21176040 and 51508068), the Program for New Century Excellent Talents in University (No. NCET-13-0077), the Fundamental Research Funds for the Central Universities (No. DUT14YQ107), and the Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. ESK201529).
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Haiss, W., Thanh, N.T.K., Aveyard, J., Fernig, D.G., 2007. Determination of size and concentration of gold nanoparticles from UV–vis spectra. Anal. Chem. 79 (11), 4215–4221. Kalishwaralal, K., Deepak, V., Pandian, S.R.K., Gurunathan, S., 2009. Biological synthesis of gold nanocubes from Bacillus licheniformis. Bioresour. Technol. 100 (21), 5356–5358. Kitching, M., Ramani, M., Marsili, E., 2015. Fungal biosynthesis of gold nanoparticles: mechanism and scale up. Microb. Biotechnol. 8 (6), 904–917. Kreibig, U., Genzel, L., 1985. Optical absorption of small metallic particles. Surf. Sci. 156, 678–700. Kuang, Y., Zhou, Y., Chen, Z.L., Megharaj, M., Naidu, R., 2013. Impact of Fe and Ni/Fe nanoparticles on biodegradation of phenol by the strain Bacillus fusiformis (BFN) at various pH values. Bioresour. Technol. 136, 588–594. Kumari, M.M., Philip, D., 2015. Degradation of environment pollutant dyes using phytosynthesized metal nanocatalysts. Spectrochim. Acta A 135, 632–638. Mishra, A., Kumari, M., Pandey, S., Chaudhry, V., Gupta, K.C., Nautiyal, C.S., 2014. Biocatalytic and antimicrobial activities of gold nanoparticles synthesized by Trichoderma sp. Bioresour. Technol. 166, 235–242. Mishra, A., Tripathy, S.K., Yun, S.I., 2011. Bio-synthesis of gold and silver nanoparticles from Candida guilliermondii and their antimicrobial effect against pathogenic bacteria. J. Nanosci. Nanotechnol. 11 (1), 243–248. Mukherjee, P., Roy, M., Mandal, B.P., Choudhury, S., Tewari, R., Tyagi, A.K., et al., 2012. Synthesis of uniform gold nanoparticles using non-pathogenic bio-control agent: evolution of morphology from nano-spheres to triangular nanoprisms. J. Colloid Interface Sci. 367 (1), 148–152. Narayanan, K.B., Sakthivel, N., 2010. Biological synthesis of metal nanoparticles by microbes. Adv. Colloid Interf. Sci. 156 (1–2), 1–13. Pimprikar, P.S., Joshi, S.S., Kumar, A.R., Zinjarde, S.S., Kulkarni, S.K., 2009. Influence of biomass and gold salt concentration on nanoparticle synthesis by the tropical marine yeast Yarrowia lipolytica NCIM 3589. Colloids Surf. B: Biointerfaces 74 (1), 309–316. Qu, Y.Y., Shi, S.N., Ma, F., Yan, B., 2010. Decolorization of reactive dark blue KR by the synergism of fungus and bacterium using response surface methodology. Bioresour. Technol. 101 (21), 8016–8023. Shedbalkar, U., Singh, R., Wadhwani, S., Gaidhani, S., Chopade, B.A., 2014. Microbial synthesis of gold nanoparticles: current status and future prospects. Adv. Colloid Interf. Sci. 209, 40–48. Shukla, R., Bansal, V., Chaudhary, M., Basu, A., Bhonde, R.R., Sastry, M., 2005. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview. Langmuir 21 (23), 10644–10654. Song, J.Y., Jang, H.K., Kim, B.S., 2009. Biological synthesis of gold nanoparticles using Magnolia kobus and Diopyros kaki leaf extracts. Process Biochem. 44 (10), 1133–1138. Sujitha, M.V., Kannan, S., 2013. Green synthesis of gold nanoparticles using citrus fruits (Citrus limon, Citrus reticulata and Citrus sinensis) aqueous extract and its characterization. Spectrochim. Acta A 102, 15–23. Tan, L., Ning, S.X., Zhang, X.W., Shi, S.N., 2013. Aerobic decolorization and degradation of azo dyes by growing cells of a newly isolated yeast Candida tropicalis TL-F1. Bioresour. Technol. 138, 307–313. Tony, B.D., Goyal, D., Khanna, S., 2009. Decolorization of textile azo dyes by aerobic bacterial consortium. Int. Biodeterior. Biodegrad. 63 (4), 462–469. Tripathi, R.M., Gupta, R.K., Singh, P., Bhadwal, A.S., Shrivastav, A., Kumar, N., et al., 2014. Ultra-sensitive detection of mercury (II) ions in water sample using gold nanoparticles synthesized by Trichoderma harzianum and their mechanistic approach. Sensors Actuators B Chem. 204, 637–646.
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parameters such as HAuCl4 concentrations, biomass concentrations and pH on AuNPs synthesis were investigated, which would provide some useful data for oriented biosynthesis of AuNPs. In addition, the applications of bio-AuNPs were studied by decolorization of various azo dyes, which suggested that the biogenetic AuNPs should have a good potential application for the azo dye decolorization. This study should provide a further insight for the fungal mediated synthesis of AuNPs. Supplementary data to this article can be found online at doi:10.1016/j.jes.2016.09.007.
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Vahabi, K., Mansoori, G.A., Karimi, S., 2011. Biosynthesis of silver nanoparticles by fungus Trichoderma reesei (a route for large-scale production of AgNPs). Insciences J. 1 (1), 65–79. Xu, M.Y., Guo, J., Kong, X.Y., Chen, X.J., Sun, G.P., 2007. Fe(III)-enhanced azo reduction by Shewanella decolorationis S12. Appl. Microbiol. Biotechnol. 74 (6), 1342–1349.
Zhang, X.L., Yan, S., Tyagi, R.D., Surampalli, R.Y., 2011. Synthesis of nanoparticles by microorganisms and their application in enhancing microbiological reaction rates. Chemosphere 82 (4), 489–494.
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Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jes
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Yuanyuan Qu1,⁎, Wenli Shen1 , Xiaofang Pei1 , Fang Ma2,⁎, Shengnan You1 , Shuzhen Li1 , Jingwei Wang1 , Jiti Zhou1
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Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization
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1. State Key Laboratory of Fine Chemicals, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China 2. State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
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Article history:
Developing an eco-friendly approach for metallic nanoparticle synthesis is important in 17
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Received 3 May 2016
current nanotechnology research. In this study, green synthesis of gold nanoparticles 18
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Revised 22 August 2016
(AuNPs) was carried out by a newly isolated strain Trichoderma sp. WL-Go. UV–vis spectra of 19
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Accepted 7 September 2016
AuNPs showed a surface plasmon resonance peak at 550 nm, and transmission electron 20
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Available online xxxx
microscopy images revealed that the AuNPs were of varied shape with well dispersibility. 21
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Keywords:
pH 7–11. Moreover, the bio-AuNPs could efficiently catalyze the decolorization of various 23
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Gold nanoparticles
azo dyes. This research provided a new microbial resource candidate for green synthesis of 24
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Biosynthesis
AuNPs and demonstrated the potential application of bio-AuNPs for azo dye decolorization. 25
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Trichoderma
© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 26
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Azo dyes
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Decolorization
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The optimal conditions for AuNP synthesis were HAuCl4 1.0 mmol/L, biomass 0.5 g and 22
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Introduction
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Due to the advanced characteristics over the bulk materials, nanomaterials possess a great potential for innovative technology and are widely applied to every aspect of human life (Kuang et al., 2013). Among the various nanomaterials, gold nanoparticles (AuNPs) have attracted enormous attention because of their high oxidation resistance, biocompatibility and stability (Bhumkar et al., 2007; Gong and Mullins, 2009; Shukla et al., 2005). Various chemical and physical methods are available for AuNPs synthesis, however, the stability of nanoparticles and the use of toxic chemicals in AuNPs production processes raise intensive concerns (Das et al., 2010; Narayanan and Sakthivel, 2010). Therefore, there is an urgent need to develop a green process for AuNPs synthesis. Biological
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Published by Elsevier B.V. 27
methods of AuNPs synthesis have offered a reliable and ecofriendly alternative to physical and chemical methods (Zhang et al., 2011). It has been reported that many microbial resources such as bacteria, actinomycetes, fungi and yeasts have the potential to synthesize AuNPs (Zhang et al., 2011; Kitching et al., 2015; Shedbalkar et al., 2014). Among them, fungi are considered as better microbial resources for high production of nanoparticles due to their remarkable advantages for AuNP synthesis (Girard et al., 2013; Du et al., 2011). For instance, they secrete a large amount of proteins and secondary metabolites extracellularly, the extracellularly synthesized nanoparticles can be simply separated without the downstream processing (Mishra et al., 2014). However, less than 30 fungal species have been investigated for AuNP synthesis (Kitching et al., 2015), thus
⁎ Corresponding authors. E-mails: [email protected] (Yuanyuan Qu), [email protected] (Fang Ma).
http://dx.doi.org/10.1016/j.jes.2016.09.007 1001-0742/© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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Azo dyes used in this study were obtained from Dye Synthesize Laboratory, Dalian University of Technology. Hydrogen tetrachloroaurate (III) hydrate (HAuCl4·3H2O) was purchased from J&K Scientific Ltd. (China). All other chemicals were of analytical grade or above. The medium used in selection and cultivation was modified martin broth (MMB, pH 7), which consisted of (NH4)2SO4 1 g/L, KH2PO4 1 g/L, MgSO4·7H2O 0.5 g/L, and glucose 1 g/L. Solid medium contained 2.0% (w/v) agar in MMB. Tetracycline and terramycin (both 50 mg/L) were added to the medium as selective pressure for strain isolation. The media were autoclaved at 115°C, for 15 min before use.
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1.2. Isolation and identification of strain WL-Go
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The activated sludge samples from antibiotic-contaminated bioreactors were serially diluted and spread onto MMB agar plates. After incubation at 30°C for 4 days, colonies were selected and transferred into 25 mL MMB medium and cultivated at 30°C for 3 days with shaking at 150 r/min. After
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1.3. Biomass of strain WL-Go preparation and synthesis of 136 AuNPs 137
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Strain WL-Go was cultivated in 100 mL MMB medium at 30°C for 2 days with shaking at 150 r/min. After incubation, the biomass of strain WL-Go was prepared by filtration and washed three times using sterile distilled water. Appropriate concentration of wet biomass was resuspended in distilled water and incubated with 1.0 mmol/L HAuCl4 for 40 hr at 30°C. Control experiment without biomass was also performed. The effects of HAuCl4 concentration, biomass concentration and pH on AuNP synthesis were carried out as follows: (1) various concentrations of HAuCl4 (0.1, 0.5, 1.0, 2.0, 5.0 mmol/L) were added to the solutions containing 0.1 g biomass with final volume of 4 mL, (2) biomass (0.1, 0.2, 0.3, 0.4, 0.5 g) suspended in 4 mL sterile distilled water were incubated with 2.0 mmol/L HAuCl4 solution, (3) the reaction solution was adjusted to different pH (3, 5, 7, 9, 11) using HCl and NaOH. The above experiments were performed at 30°C for 40 hr.
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1.4. Characterization of AuNPs
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The formation of AuNPs was firstly examined by visual observation of color change of suspensions, and then monitored by UV–Vis spectrophotometer (Metash UV-9000, China) recording the spectra between 400 and 800 nm at the resolution of 1 nm. The morphology of AuNPs was characterized through a Tecnai G2 Spirit TEM (FEI, The Netherlands) with an accelerating voltage of 120 kV. For TEM analysis, 15 μL of samples were dropped on a carbon coated copper grid and dried at room temperature before analyses.
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1.5. Decolorization of azo dyes by bio-AuNPs
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The bio-AuNPs used for decolorization were prepared by ultrasonication of the reaction mixture containing strain WL-Go and the synthesized AuNPs. The potential catalytic activity of bio-AuNPs for decolorization of azo dyes was demonstrated using acid, reactive and cationic azo dyes. The dyes were added to the bio-AuNPs solution with final concentration of 50 mg/L, and then incubated at 30°C with shaking at 150 r/min. Control experiment was carried out under identical conditions using ultrasonicated WL-Go cells instead of the bio-AuNPs when decolorization of 50 mg/L Acid Brilliant Scarlet GR. The effect of initial concentration (25, 50, 100, 200 mg/L) of Acid Brilliant Scarlet GR on decolorization was also investigated.
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screening and enriching for about two months, one colony designed as WL-Go was selected for further characterization and identified by 26S rRNA gene sequence analysis. Genomic DNA of strain WL-Go was used to amplify the 26S rRNA gene by polymerase chain reaction (PCR), and then the PCR product was sequenced by TaKaRa Biotechnology Co. Ltd. (Dalian, China). The BLAST program was used to compare the sequence with those in GenBank, and the related sequences were aligned using Clustal X (1.8). The aligned sequences were used to construct phylogenetic tree using Neighbor-joining method by MEGA (Version 5.1) with 1000 bootstrap replicates (Qu et al., 2010).
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it is necessary to develop more fungal resources for AuNP synthesis. Besides, the control of size, shape and dispersity is a major challenge in the process of AuNP biosynthesis. Studies have reported that some factors, such as substrate concentrations, biomass concentrations and pH, could affect the size, shape and dispersity of AuNPs (Gericke and Pinches, 2006; Pimprikar et al., 2009; Mishra et al., 2011). As is known, particle size directly affects the properties of nanomaterial and thus the size control on AuNP biosynthesis is significantly important. Azo dyes are the largest chemical class of dyes with amounts of structural and color variety (Tony et al., 2009). Disposal of dyes-containing effluents has caused serious damage to the environment. Since the color contents in dyes adsorb and reflect sunlight entering the contaminated wastewater, they significantly affect the photosynthesis and the growth of aquatic organisms (Champagne and Ramsay, 2010). Some efforts about the green methods for azo dye pollutant treatment have been published. A variety of microbes, such as Shewanella decolorationis S12, Bacillus and Pseudomonas species, could be efficiently used for the treatment of azo dye pollutants (Chen et al., 2011; Fang et al., 2015; Xu et al., 2007). Nanoremediation as an alternative could well improve the efficiency of the degradation process (Kumari and Philip, 2015). However, the application of biogenic AuNPs on dye degradation has been rarely reported. In the present study, a newly isolated Trichoderma strain was exploited to green synthesis of AuNPs. The characteristics of AuNPs were determined by UV–Vis spectrophotometer and transmission electron microscopy (TEM). Different parameters that may affect the biosynthesis of AuNPs were investigated to obtain the optimal synthesis conditions. In addition, the catalytic characteristics of the as-prepared bio-AuNPs were evaluated by decolorization of various azo dyes.
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Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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The decolorization process was monitored by scanning the characteristic absorption wavelength (λmax) of azo dyes with UV–Vis spectrophotometer after centrifugation at 10,000g for 5 min at different time intervals. The decolorization rate was calculated using the following equation:
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Decolorization ð%Þ ¼ ðA0 −A1 Þ=A0 100
2. Results and discussion
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2.1. Isolation and identification of strain WL-Go
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A fungal strain designated as WL-Go was successfully isolated from the activated sludge sample (Dalian, China). Strain WL-Go was tubular hyphae and obtuse spores as observed by scanning electron microscope, and its colonies showed green concentric round striate on agar plate (Fig. 1a). Based on the analysis of 26S rRNA gene sequence, strain WL-Go
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The AuNPs were synthesized by incubating strain WL-Go with HAuCl4. The color of the reaction solution turned violet after incubating strain WL-Go with HAuCl4 for 4 hr, and the UV–vis spectrum of the reaction solution showed an absorption peak at 550 nm (Fig. 2a), which indicated the formation of AuNPs (Kalishwaralal et al., 2009). TEM images showed that the synthesized AuNPs were well-dispersed with diverse shapes such as spheres (the major shape), triangles and hexagons. In addition, the spherical particles tended to be smaller than the particles with polygonal and triangular shape (Fig. 2b). Trichoderma is a well-known genus that can secret various kinds and large amounts of extracellular enzymes and metabolites, thus it can serve as an excellent candidate for industrial scale production of metal nanoparticles (Vahabi
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where A0 and A1 represented the initial and final absorbance of the dye, respectively. The intermediate products of Acid Brilliant Scarlet GR reduction by bio-AuNPs were performed with HPLC-MS as described previously (Tan et al., 2013).
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exhibited 100% homology to Trichoderma (Genebank accession number KM242362), thus strain WL-Go was identified as Trichoderma. The phylogenic tree demonstrating the relationship between strain WL-Go and other strains is shown in Fig. 1b. The partial 26S rRNA gene sequence (500 bp) of strain WL-Go was deposited in GenBank database under accession number KP676894. Strain WL-Go has been deposited in the China General Microbiological Culture Collection Center (CGMCC; Beijing) under accession number CGMCC 10456.
Fig. 1 – (a) The images of hypha (i), spores (ii) and agar plate (iii) of strain WL-Go. (b) Phylogenetic tree of Trichoderma sp. WL-Go and related species. The GenBank accession number corresponded to each microorganism was exhibited in parentheses. Bootstrap values were showed at each branch. Scale bar indicated 0.002 Jukes-cantor distances. Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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et al., 2011). AuNP synthesis by Trichoderma was conducted previously. For instance, gold nanospheres with uniform diameter were synthesized by Trichoderma asperellum (Mukherjee et al., 2012). Spherical AuNPs with the particle sizes ranging from 26 to 34 nm were synthesized by Trichoderma harzianum (Tripathi et al., 2014). Moreover, rapid biosynthesis of AuNPs was achieved using cell free extract of Trichoderma viride (Mishra et al., 2014). These studies indicated that Trichoderma strains had excellent ability on AuNP biosynthesis. The strain Trichoderma sp. WL-Go could serve as a new potential microbial resource for biosynthesis of AuNPs.
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Fig. 2 – (a) UV–vis spectra of the dispersed AuNPs synthesized by strain WL-Go after 40 hr incubation, HAuCl4 solution, and control strain WL-Go. (b) The TEM image of AuNPs synthesized by strain WL-Go.
Fig. 3 – UV–Vis spectra of the dispersed AuNPs synthesized under different conditions: (a) HAuCl4 concentrations (0.1, 0.5, 1.0, 2.0, 5.0 mM), (b) strain WL-Go biomass (0.1, 0.2, 0.3, 0.4, 0.5 g) and (c) pH (3, 5, 7, 9, 11) of reaction solutions. AuNPs: gold nanoparticles.
2.3. Effect of HAuCl4 concentrations on AuNPs synthesis by strain WL-Go The effect of HAuCl4 concentrations on AuNPs synthesis is shown in Fig. 3a. Suspensions treated with 0.5, 1.0 and 2.0 mmol/L HAuCl4 for 40 hr showed violet or pink color, and
the absorption peaks at 550 nm were also recorded, which confirmed the formation of AuNPs (Song et al., 2009). Furthermore, a strong surface plasmon resonance (SPR) band at 550 nm was obtained when the HAuCl4 concentration was
Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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2.4. Effect of biomass concentrations on AuNP synthesis by strain WL-Go The effect of biomass concentrations on AuNP synthesis is shown in Fig. 3b. All the reaction solutions were changed to purple color, which indicated that the AuNPs were synthesized at different biomass concentrations. The SPR absorbance of reaction solution exhibited an apparent broadening at maximum wavelength (550 nm) when using 0.1 g biomass. The generation of broad SPR band was probably due to the formation of large sized AuNPs, e.g., sphere, triangle and hexagon, resulting in the transverse interaction of radiance
2.5. Effect of pH on AuNPs synthesis by strain WL-Go
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The effect of pH on AuNPs synthesis is shown in Fig. 3c. It was observed that the rate of AuNPs synthesis enhanced by increasing the pH of solutions. The color changed within 2 hr under extremely alkaline condition (pH 12), whereas it took more than 4 hr under acidic condition (pH 3). In addition, the SPR band was broad at pH 3, while it became relative sharp and strong at pH 7–11. The better performances of AuNP synthesis at pH 7–11 were probably attributed to the higher stability of the capping proteins secreted by the fungus under neutral or alkaline conditions, while protonation of the carboxylic groups in proteins might occur under acidic condition (Bastús et al., 2011). This phenomenon was also observed by Mishra et al., who confirmed that the cell free extract of Trichoderma viride could synthesize AuNPs at pH 7 and 9, but not at pH 5 (Mishra et al., 2011).
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(Kreibig and Genzel, 1985; Sujitha and Kannan, 2013). However, it was noteworthy that the absorbance band became narrower and blue shift when the biomass concentration increased to 0.5 g. The reason should be that higher concentration of biomass would provide more biomolecules to reduce gold ions, thus smaller size particles could be synthesized, leading to the blue shift in absorption peak (Haiss et al., 2007).
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1.0 mmol/L. Therefore, the optimal HAuCl4 concentration for AuNP synthesis was 1.0 mmol/L. Gold particles in large sizes were synthesized attaching on the tube wall when the HAuCl4 concentration increased to 5.0 mmol/L. The formation of large particles was probably due to the lack of biomolecules required for capping and efficient stabilization of the synthesized nanoparticles (Kumari and Philip, 2015). A similar observation was also reported on AuNP synthesis using Verticillium luteoalbum. It revealed that when HAuCl4 concentration was below 500 mg/L, the particle size was around 20 nm, but then the particle size distributed from 50 nm to several hundred nanometers when HAuCl4 concentration was above 500 mg/L. In addition, massive particle aggregates could be found on the cells (Gericke and Pinches, 2006).
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Table 1 – Decolorization of azo dyes (50 mg/L) by bio-AuNPs.
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Acid Red B
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Acid Orange G
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Acid Black 1
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Reactive Red X-3B
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Reactive Black
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Reactive Red
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Cation Red
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AuNPs: gold nanoparticles.
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Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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The application of bio-AuNPs in decolorization of azo dyes was investigated. The bio-AuNPs could decolor various azo dyes as shown in Table 1, including four acid azo dyes (82.9%– 94.7% in 120 min), three reactive azo dyes (46.1%–73.3% in 100–180 min) and one cation azo dye (41.7% in 180 min). The highest decolorization efficiency was 94.7% for Acid Brilliant Scarlet GR, thus it was chosen as the model dye for further investigation. Decolorization processes of Acid Brilliant Scarlet GR with different concentrations by the bio-AuNPs are shown in Fig. 4. It was observed that the decolorization efficiency of Acid Brilliant Scarlet GR was more than 90% in 40 min when the dye concentration was 25 mg/L. It was still of high efficient
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3. Conclusions In this study, green synthesis of AuNPs by a newly isolated strain Trichoderma sp. WL-Go was investigated. The UV–vis and TEM analysis were employed to investigate the characteristics of the biosynthesized AuNPs. The effects of different
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Fig. 4 – Effects of initial concentration (25, 50, 100, 200 mg/L) of Acid Brilliant Scarlet GR on decolorization.
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(more than 90% in 100 min) when the concentration of Acid Brilliant Scarlet GR increased to 50 mg/L, however, only 4.8% Acid Brilliant Scarlet GR were decolored by ultrasonicated WL-Go cells (Fig. S1). It was suggested that the AuNPs greatly improved the decolorization rate with high catalytic activities. The byproducts of Acid Brilliant Scarlet GR decolorization were analyzed by HPLC-MS, and two possible decolorization intermediates were detected (Fig. 5). 3, 7-Dihydroxyoctahydronaphthalene-2,6-dione (I) was corresponding to the mass peak of 215.1658 ([M–H+]), and naphthol (II) was confirmed with m/z ion peak of 143.1078 ([M–H+]), both of which were detected as the intermediates of Acid Brilliant Scarlet GR degradation in previous studies (Tan et al., 2013; Gomi et al., 2011). Generally, reductive cleavage of azo groups (\N_N\) was the first step of azo dye degradation (dos Santos et al., 2007). Therefore, the Acid Brilliant Scarlet GR was probably firstly transformed to the corresponding amines, such as 1-aminonaphthylene-2hydroxy-3, 6-disulfonic acid (III), which, however, was not detected in this study. Then, the intermediate III was probably oxidized to the compound I under aerobic conditions (Tan et al., 2013), and intermediate II could be generated from product I through removing a hydroxyl group and two aldehyde groups. The results suggested the potential application of bio-AuNPs for effective decolorization of azo dyes.
Fig. 5 – The mass spectra of intermediates formed from Acid Brilliant Scarlet GR removal by bio-AuNPs, I: 3, 7-dihydroxyoctahydro-naphthalene-2, 6-dione; II: naphthol. AuNPs: gold nanoparticles. Please cite this article as: Qu, Y., et al., Biosynthesis of gold nanoparticles by Trichoderma sp. WL-Go for azo dyes decolorization, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.09.007
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This work was supported by the National Natural Science Foundation of China (Nos. 21176040 and 51508068), the Program for New Century Excellent Talents in University (No. NCET-13-0077), the Fundamental Research Funds for the Central Universities (No. DUT14YQ107), and the Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. ESK201529).
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parameters such as HAuCl4 concentrations, biomass concentrations and pH on AuNPs synthesis were investigated, which would provide some useful data for oriented biosynthesis of AuNPs. In addition, the applications of bio-AuNPs were studied by decolorization of various azo dyes, which suggested that the biogenetic AuNPs should have a good potential application for the azo dye decolorization. This study should provide a further insight for the fungal mediated synthesis of AuNPs. Supplementary data to this article can be found online at doi:10.1016/j.jes.2016.09.007.
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