Introduction

Gold nanoparticles (AuNPs) have immense applications in the fields of microbiology, physics, chemistry, medicine, and material science due to their distinctive electronic, optical, and catalytic properties (Arvizo et al. 2012; Guo and Wang 2007; He et al. 2014; Scari et al. 2012). Although wet-chemical procedure has been widely utilized for the synthesis of AuNPs due to its simplicity and high growth rate, the usage of toxic and hazardous organic reagents in the wet-chemical synthesis process has raised great concerns on the living organisms and the environment (Seo et al. 2015; Singh et al. 2016a, b). Therefore, it has been of increasing interest to develop an efficient and environmentally benign process to synthesize AuNPs. In recent years, green synthesis of AuNPs by microbes has gained much attention (Ahmad et al. 2013; Dhillon et al. 2012; Hulkoti and Taranath 2014; Park et al. 2016), which was considered as a safe, simple, nontoxic, biocompatible, and environment-friendly alternative to wet-chemical techniques because the microbes can serve as “green factory” for AuNP synthesis without addition of any reducing and capping agents (Kharissova et al. 2013; Moghaddam et al. 2015; Singh et al. 2016a, 2016b).

Many microbes, such as bacteria, actinomycetes, fungi, and yeasts, have been reported to synthesize AuNPs by reducing Au3+ (Kitching et al. 2015; Sugunan et al. 2007). For example, bacteria Geobacillus sp., Anabaena sp., and Lyngbya majuscula were reported to produce hexagonal and spherical AuNPs intracellularly by exposing cells to Au3+ (Brayner et al. 2007; Chakraborty et al. 2009; Correa-Llantén et al. 2013). Monodisperse AuNPs were extracellularly biosynthesized through reduction of aqueous chloroaurate ions by actinomycetes Thermomonospora sp. (Ahmad et al. 2003). Different fungal species, e.g., Aspergillus clavatus, Epicoccum nigrum, and Trichoderma sp. have been reported to synthesize AuNPs intracellularly or extracellularly (Qu et al. 2016; Sheikhloo et al. 2011; Verma et al. 2011). Yeasts like Pichia jadinii and Yarrowia lipolytica were also shown to have a good potential for AuNPs synthesis (Gericke and Pinches 2006; Pimprikar et al. 2009). In general, different microbes exhibit diverse ability to synthesize AuNPs, which is caused by varied oxidation-reduction enzymes and metabolites presented. As we know, fungi are able to secrete more proteins, produce more biomass, and show higher metal tolerance and bioaccumulation ability (Moghaddam et al. 2015), which may lead to possess superior ability to reduce soluble gold ions and synthesize insoluble nanocrystals (Kitching et al. 2015). However, the present researches were mostly focused on the characteristics of AuNPs synthesized by a single strain; there were few studies to comparing the characteristics of AuNPs biosynthesized by different microbes. Therefore, it is necessary to make some comparisons among different species of microbes to identify promising candidates for further study.

In this study, AuNPs were biosynthesized using different microbial cell-free extracts, which were obtained from three locally isolated strains of bacterium Labrys sp. WJW, yeast Trichosporon montevideense WIN, and filamentous fungus Aspergillus sp. WL-Au. The characterizations of AuNPs were performed by UV-Vis spectroscopy and transmission electron microscopy (TEM) analyses. The interactions of biomolecules present in the extracts with surfaces of synthesized AuNPs were confirmed by Fourier transform infrared spectroscopy (FTIR). The catalytic activities of AuNPs were determined by reduction of 4-NP in the presence of excess NaBH4. In this study, the characteristics of AuNPs synthesized by different species of microbes were comparatively studied in detail. It will provide some valuable reference for exploring efficient microbial resources to biosynthesize AuNPs.

Materials and methods

Chemicals, media, and strains

Hydrogen tetrachloroaurate (III) hydrate (HAuCl4·3H2O) was purchased from J&K Scientific Ltd. (China). 4-NP and NaBH4 were obtained from Sinopharm Chemical Regent Beijing Co., Ltd. (China). All other reagents were of analytical grade.

Filamentous fungus Aspergillus WL-Au and yeast Trichosporon montevideense WIN were isolated from antibiotic-contaminated reactor in our laboratory, and bacteria Labrys WJW was isolated from graphene oxide degradation reactor in our laboratory. Strain WL-Au was aerobically cultured at 30 °C for 3 days in the modified martin broth (MMB, pH 7), containing 1 g/L (NH4)2SO4, 1 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, and 2 g/L glucose, which was autoclaved at 115 °C for 15 min before use. Strains WJW and WIN were aerobically cultured at 30 °C for 2 days in lysogeny broth medium (LB, pH 7), containing 10 g/L NaCl, 5 g/L yeast extract and 10 g/L peptone, which was autoclaved at 121 °C for 20 min before use.

Biosynthesis of AuNPs by different microbial cell-free extracts

The microbial cell-free extracts used for AuNPs synthesis were prepared as follows: cells of strains WJW and WIN were firstly harvested after being cultivated for 2 days by centrifugation (10,000×g for 10 min at 4 °C), and cells of strain WL-Au was harvested by sieving through a qualitative filter paper. Then, the cells were washed with sterile deionized water to remove medium components before use. Subsequently, cells were re-suspended in phosphate sodium buffer (50 mM, pH 7) and lysed by ultrasonication (Ultrasonic Processor CPX 750, USA) for 40 min. After centrifugation of the broken cracking liquid samples at 10,000×g for 20 min (4 °C), the supernatants were then treated by filtering through 0.45-μm syringe Millipore filters and the filtrate samples were collected as microbial cell-free extracts. The protein concentrations of microbial cell-free extracts were regulated to 150 mg/L by the Bradford assays before use. The HAuCl4 solutions were added to the microbial cell-free extracts with final concentrations of 1 mM for AuNPs synthesis, and then the reaction mixtures were incubated in dark at 30 °C.

Characterization of AuNPs synthesized by different microbial cell-free extracts

The synthesis of AuNPs were determined by UV-Vis spectrophotometer (Metash UV-9000, China) recording the spectra between 400 and 800 nm at the resolution of 1 nm. The concentrations of synthesized AuNPs were measured by inductively coupled plasma optical emission spectrometer (ICP-OES, Perkin-Elmer Optima 2000 DV, USA). The shapes and sizes of biogenic AuNPs were characterized by TEM (FEI Tecnai G220 S-Twin, USA) with an accelerating voltage of 120 kV. FTIR spectra were recorded by a Shimadzu IRPrestige-21 spectrophotometer (Japan) with the wavelength ranging from 500 to 4000 cm−1 to investigate the conjugation of proteins on the nanoparticles surfaces.

Catalytic activities of different biogenic AuNPs for 4-NP reduction

The catalytic activities of different biogenic AuNPs were measured by reducing 4-NP in the presence of excess NaBH4. In a typical assay, 0.1 mL of 2 mM stock solution of 4-NP and 0.4 mL of 30 mM freshly prepared NaBH4 solution were mixed in a 3-mL standard quartz cuvette. The biogenic AuNPs with final concentrations of 1 mg/L quantified by ICP-OES were added to initiate the reactions. The final reaction volumes were adjusted to 2 mL with sterile water. To monitor the reduction of 4-NP, samples were taken at intervals and recorded by UV-Vis spectra in the range of 300–500 nm. The catalytic reduction rates of 4-NP by different biogenic AuNPs were calculated through measuring the decrease of UV-Vis absorbance at 401 nm. All the reactions were performed in triplicate at ambient temperature.

Results and discussion

UV-Vis spectra of AuNPs synthesized by different microbial cell-free extracts

After incubation of the microbial cell-free extracts with HAuCl4 for 7 days, the formation of AuNPs was preliminary visually observed by colors, which changed from pale yellow to ruby red and purple as shown in Fig. 1 insets. Meanwhile, the UV-Vis spectra of different reaction solutions were recorded in the range of 400–800 nm, which could act as a reliable method to confirm the presence of AuNPs based on their optical transition and electronic band structure (Arumugam and Berchmans 2011). As shown in Fig. 1, the UV-Vis spectra of different reaction solutions respectively showed characteristic absorption peaks at 538 nm (WJW), 539 nm (WIN), and 543 nm (WL-Au) corresponding to the surface plasmon resonance of AuNPs. It is indicated that the AuNPs were biosynthesized by the cell-free extracts from these microbes (Das et al. 2012), named as WJW-AuNPs, WIN-AuNPs, and WL-Au-AuNPs, respectively. In addition, the stability of these biogenic AuNPs was also investigated. It was found that WJW-AuNPs, WIN-AuNPs, and WL-Au-AuNPs were stable even after being stored for 3 months at 4 °C. The UV-Vis spectra showed no evident changes in the wavelength and intensity of SPR bands, which indicated that the as-synthesized AuNPs were stable and well dispersed in solution without obvious aggregation. This suggested that the interactions between as-synthesized AuNPs and biomolecules in the microbial cell-free extracts take important roles in preventing aggregation and stabilization of the formed AuNPs (Das et al. 2012).

Fig. 1
figure 1

UV-Vis spectra of AuNPs synthesized by incubation 1 mM HAuCl4 with 150 mg/L microbial cell-free extracts from strains WJW, WIN, and WL-Au. Insets showed the colors of different biogenic AuNPs

Full size image

Shapes and particle sizes of AuNPs synthesized by different microbial cell-free extracts

The morphologies of AuNPs formed by different microbial cell-free extracts were investigated through TEM analysis. As shown in Fig. 2, the major shapes of these biogenic AuNPs were common sphere or pseudo-sphere. However, the WL-Au-AuNPs were relatively uniform and nearly monodisperse. The selected area electron diffraction (SAED) patterns (Fig. 2 insets) of all the three biogenic AuNPs indicated that the biosynthesized AuNPs were highly crystalline in nature (Emmanuel et al. 2014). In addition, the size distributions of different AuNPs were calculated by analytical procedure. As shown in Fig. 3, the size distributions of WJW-AuNPs, WIN-AuNPs, and WL-Au-AuNPs were 4.3–52.1, 8.4–38.5, and 3.2–20.6 nm, respectively, which demonstrated the relatively uniform size of WL-Au-AuNPs compared with others. Meanwhile, the average sizes were calculated as 18.8 nm (WJW-AuNPs), 22.2 nm (WIN-AuNPs), and 9.5 nm (WL-Au-AuNPs), respectively. It was generally assumed that there were two main processes in the AuNP biosynthesis by microbes: (i) Au3+ reduction to Au0 and (ii) Au0 stabilized by biomolecules and formed AuNPs. In the process of Au3+ reduction, Au3+ were reduced by proteins, reducing sugars, and other metabolites, and therefore synthesized Au0 nucleus extracellularly or intracellularly. The Au0 nucleus would continuously grow and finally become aggregations if without addition of capping and stabilizing agents. In the process of AuNP biosynthesis by microbes, biomacromolecules were speculated to act as capping and stabilizing agents, and they can stabilize the growing Au0 nucleus and form AuNPs. The morphology of synthesized AuNPs was closely associated with the types of biomacromolecules (Kitching et al. 2015; Dhillon et al. 2015; Das et al. 2010). These results suggested that the filamentous fungus had a superior potential for synthesis of uniform and monodisperse AuNPs, which should provide insights into the green synthesis of metal nanoparticles.

Fig. 2
figure 2

TEM and SAED images of the biogenic AuNPs synthesized by microbial cell-free extracts from strains WJW, WIN, and WL-Au

Full size image
Fig. 3
figure 3

Size distribution histograms of the biogenic AuNPs synthesized by microbial cell-free extracts from strains WJW, WIN, and WL-Au

Full size image

FTIR analyses of different biogenic AuNPs

The capping effects of biomolecules in different microbial cell-free extracts for AuNP synthesis were investigated by FTIR analysis (Fig. 4). The bands at 3442 cm−1 (WJW-AuNPs), 3329 cm−1 (WIN-AuNPs), and 3300 cm−1 (WL-Au-AuNPs) were due to –N–H stretching vibrations arising from the peptide linkages (Mukherjee et al. 2008). The bands at 2798 cm−1, 2924 cm−1 (WL-Au-AuNPs), and 2920 cm−1 (WIN-AuNPs) could have resulted from stretching vibrations of –C–H groups of aliphatic acids (Kora et al. 2012). The bands at 1635~1653, 1500~1540, and 1026~1041 cm−1 were attributed to the stretching vibrations of –C=C, –N=O, and –S=O groups, respectively (Ashour et al. 2015). The additional band at 1751 cm−1 in the WL-Au-AuNPs spectrum indicated the presence of –C=O group (Gajbhiye et al. 2009). This suggested that the microbial-synthesized AuNPs had a strong binding ability with many functional biomolecules, such as proteins, esters, and carboxylic acids, which could serve as capping and stabilizing agents, thereby offering stability to particles by preventing agglomeration and growth of nanocrystals (Au0) (Ashour et al. 2015). Researchers have made many efforts to investigate the chemicals involved in the AuNP stabilization. FTIR analysis is one of the most commonly used methods, which could detect the functional groups bonded to the synthesized AuNP surfaces. For example, Faramarzi and Forootanfar found that –OH/–NH functional groups and carbonyl group were used in bioconjugation and immobilization of the AuNPs synthesized by laccase from Paraconiothyrium variabile (Faramarzi and Forootanfar 2011). In addition, Binupriya et al. detected the amide I and II of proteins on the synthesized AuNP surfaces using FTIR analysis. They pointed out that proteins could bind to AuNPs through either free amine groups or crysteine residues in the proteins, and therefore, stabilization of the AuNPs by surface-bound proteins is a possibility (Binupriya et al. 2010). In this study, many functional groups including –N–H, –C–H, –C=C, –N=O, –S=O, and –C=O groups were detected in the synthesized AuNP surfaces, indicating these groups possibly participated in the AuNP stabilization. In addition, it was also found that the surfaces of WL-Au-AuNPs attached more biological groups. Filamentous fungi had advantages over bacteria and yeasts for AuNP biosynthesis due to the large amounts of secreted proteins with diverse functions, which could lead to the formation of homogeneous nanoparticles with enhanced stability (Kitching et al. 2015).

Fig. 4
figure 4

FTIR spectra of the biogenic AuNPs synthesized by microbial cell-free extracts from strains WJW, WIN, and WL-Au

Full size image

Catalytic activities of different biogenic AuNPs

To evaluate the catalytic activities of different biogenic AuNPs, 4-NP reduction assays were performed by AuNPs with excess NaBH4, which was studied as a model reaction. Though the reduction of 4-NP to 4-AP by NaBH4 was thermodynamically favorable, the presence of the kinetic barrier due to large potential difference between donor and acceptor molecules reduced the feasibility. AuNPs could help transfer the electrons from BH4− ions to the nitro group of 4-NP, reducing it to 4-AP (Gangula et al. 2011). For all experiments, the initial concentrations of 4-NP, NaBH4, and AuNPs were kept at 0.1, 10, and 1 mg/L, respectively. Since the absorption peak of 4-NP shifted from 317 to 401 nm in the presence of NaBH4, the change in the absorption spectra at 401 nm was monitored to track the reaction process (Gangula et al. 2011). Figure 5a showed the UV-Vis spectra of 4-NP reduction by WL-Au-AuNPs, which was chosen as a representative to analyze the catalytic reduction of 4-NP by the biogenic AuNPs. The SPR absorption band at 401 nm decreased rapidly after addition of the biogenic AuNPs, corresponding to the disappearance of the p-nitrophenolate ion. Meanwhile, a new absorption peak at 310 nm appeared, indicating the formation of 4-AP (Panigrahi et al. 2007). The reduction reactions were also repeated by WJW-AuNPs and WIN-AuNPs. Figure 5b showed the catalytic reduction rates of 4-NP by different biogenic AuNPs. The reactions could be completed within 16, 16, and 10 min, respectively when using WJW-AuNPs, WIN-AuNPs, and WL-Au-AuNPs as the catalysts. However, in the control experiment without addition of AuNP catalyst, the absorbance at 401 nm decreased slightly even after 2 days.

Fig. 5
figure 5

a UV-Vis spectra of 4-NP reduction catalyzed by WL-Au-AuNPs. b Plots of 4-NP adsorption peak intensity versus reaction time catalyzed by different biogenic AuNPs. c Plots of ln(C/C0) versus reaction time for different biogenic AuNPs

Full size image

Since the concentrations of NaBH4 were much higher than those of 4-NP, the catalytic reductions should follow pseudo-first-order kinetics (Das et al. 2012). The apparent rate constants (k app ) of different biogenic AuNPs were calculated as shown in Fig. 5c. The k app values of WJW-AuNPs, WIN-AuNPs, and WL-Au-AuNPs were 0.27, 0.23, and 0.37 min−1, respectively, which were comparable to those of reported biogenetic AuNPs. For example, the biogenic AuNPs synthesized by Cylindrocladium floridanum and stem extract of Breynia rhamnoides could function as efficient catalysts for 4-NP reduction with reaction rate constants of 0.03 min−1 (Narayanan and Sakthivel 2011) and 0.55 min−1 (Gangula et al. 2011), respectively. The AuNPs synthesized by Prunus domestica fruit extract had reaction rate constants of 0.11 to 0.31 min−1 for 4-NP reduction when diverse volume of AuNPs were used (Dauthal and Mukhopadhyay 2012). The WL-Au-AuNPs showed the highest catalytic rate of 0.37 min−1 with a smallest average size of 9.5 nm, which may be due to the higher ratio of surface area to volume. The result was also determined by Das et al. (2012), who found that the surface area to volume ratio of AuNPs could affect their catalytic activities. This research demonstrated that the filamentous fungi showed superior ability to produce well-dispersed and uniform AuNPs with high catalytic activity (Dhillon et al. 2012; Kitching et al. 2015). It might provide a well-founded pathway for exploring efficient microbes to green synthesize AuNPs with high catalytic performances.

Conclusions

In this study, three different microbes, containing one bacterium, one yeast, and one filamentous fungus were employed to green synthesize AuNPs. The formation of AuNPs were determined by color change and UV-Vis spectra of reaction solutions. TEM and SAED analyses showed that crystalline AuNPs of similar sphere or pseudo-sphere were formed by different microbial cell-free extracts but the sizes of different AuNPs were diverse (WL-Au-AuNPs 9.5 nm, WJW-AuNPs 18.8 nm, and WIN-AuNPs 22.2 nm). FTIR analyses demonstrated that the biomolecules were involved in stabilizing the synthesized AuNPs. The as-synthesized AuNPs exhibited high catalytic activities for 4-NP reduction with catalytic rates of 0.37 min−1 (WL-Au-AuNPs), 0.27 min−1 (WJW-AuNPs), and 0.23 min−1 (WIN-AuNPs). In conclusion, filamentous fungus WL-Au-mediated AuNPs were more uniform and better dispersed and exhibited superior catalytic activity than other biogenic AuNPs.