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Macrophomina phaseolina: microbased biorefinery for gold nanoparticle production

Macrophomina phaseolina: microbased biorefinery for gold nanoparticle production Biofabrication of nanoparticles via the principles of green nanotechnology is a key issue addressed in nanobiotechnology research. There is a growing need for development of a synthesis method for producing biocompatible stable nanoparticles in order to avoid adverse effects in medical applications. We report the use of simple and rapid biosynthesis method for the preparation of gold nanoparticles using Macrophomina phaseolina (Tassi) Goid, a soil-borne pathogen. The effect of pH and temperature on the synthesis of gold nanoparticles by M. phaseolina was also assessed. Different techniques like UV-Visible Spectroscopy, Transmission Electron Microscopy (TEM), Dynamic light scattering (DLS) measurements, Fourier transform infrared (FTIR), and EDX were used to characterize the gold nanoparticles. The movement of these gold nanoparticles inside Escherichia coli (ATCC11103) along with effect on growth and viability was evaluated. The biogenic gold nanoparticle was synthesized at 37 °C temperature and neutral pH. UV-Visible Spectroscopy, TEM, EDX, and DLS measurements confirm the formation of 14 to 16 nm biogenic gold nanoparticles. FTIR substantiates the presence of protein capping on Macrophomina phaseolina-mediated gold nanoparticles. The non-toxicity of gold nanoparticles was confirmed by the growth and viability assay while the TEM images validated the entry of gold nanoparticles without disrupting the structural integrity of E. coli.Biogenic method for the synthesis of nanoparticles using fungi is novel, efficient, without toxic chemicals. These biogenic gold nanopar- ticles themselves are nontoxic to the microbial cells and offer a better substitute for drug delivery system. . . . . Keywords Nanotechnology Gold nanoparticles Bio-reduction Macrophomina phaseolina Transmission electron microscopy FTIR Introduction Iravani 2014). Gold nanoparticles have gathered the attention of nanotechnologists worldwide due to its stability, biocom- Nanomaterials are considered to be a distinguished constituent patibility, and resistance to oxidation (Alkilany and Murphy of the rapidly advancing field of nanotechnology (Salata 2010). Gold nanoparticles have numerous applications in bi- 2004; Narkeviciute et al. 2016). In recent times, immense ological fields including drug delivery and genetic engineer- importance has been laid on augmenting the biological meth- ing, cancer diagnostics, protein detection, and gene delivery odologies for the synthesis of nanomaterials as well as their (Arvizo et al. 2010;Yanget al. 2015;Daraee etal. 2016). characterization and abundant applications (Duran and Seabra A variety of physical and chemical methods have been 2009). The nanoparticles of profound interest are gold, silver, established to produce metal nanoparticles (Zhang et al. zinc, and titanium (Reddy et al. 2007; Reddy et al. 2011; 2007; Reddy et al. 2008; Hassan et al. 2014; Reddy et al. 2014). The chemical means include usage of chemical reduc- tants like citrate (Kimling et al. 2006), tryptophane (Akbarzadeh et al. 2009), PEG 4000 (Roy and Lahiri 2006), * Rajni Singh and amino acid derivatives (Sugunan and Dutta 2006; rsingh3@amity.edu; rajni_vishal@yahoo.com Ravindra 2009). But these expensive and hazardous chemicals led to general toxicity and can play havoc on the Amity Institute of Microbial Biotechnology, Amity University Uttar environment. Thus, there is a resurgence of interest in devel- Pradesh, Sector-125, Noida, U.P. 201313, India oping environmentally friendly processes. Biological systems ICAR-National Bureau of Agriculturally Important Microorganisms such as bacteria, yeasts, fungi, actinomycetes, and plants have (NBAIM), Mau Nath Bhanjan, Mau, Uttar Pradesh 275101, India 436 Ann Microbiol (2019) 69:435–445 been reportedly utilized for synthesis. The use of microbes as M. phaseolina is high as compared to other fungal species. nanofactories seems to be a very effective green and econom- This method is economical as less amount of enzyme is re- ical method for biogenic synthesis of gold nanoparticles (He quired for the generation of gold nanoparticles. Also this is of et al. 2007; Dhillon et al. 2012). Microbes have great potential ecological concern as the fungus is one of the most destructive to produce nanoparticles with features similar to chemically plant pathogen with a vast geographical distribution and is synthesized counterparts. There are several reports concerning especially problematic in tropical and subtropical countries the microbial synthesis of gold nanoparticles: intracellular with arid to semiarid climates in Africa, Asia, Europe, and synthesis by the actinomycete Rhodococcus sp. (Ahmad North and South America. The reductase enzymes produced et al. 2003c), fungus Hormoconis resinae (Varshney et al. by the fungus can be channelized towards the synthesis of 2009), and bacterium Acidithiobacillus thiooxidans (Lengke nanoparticles which could help in diverting its pathogenic et al. 2005); extracellular synthesis by actinomycete ability towards the biosynthesis of nanoparticles which could Thermomonospora sp. (Ahmad et al. 2003b) and fungus in turn help the growth of the plants. Fusarium oxysporum (Ahmad et al. 2003a) while intra- and Hence, the main objective of the present work was to pro- extracellular syntheses by the cyanobacterium Plectonema duce biogenic gold nanoparticles using M. phaseolina,deter- boryanum (Lengke et al. 2006a). The size and shape of gold mining the environmental parameters affecting its synthesis nanoparticles are affected by growth parameters such as pH, and their characterization. The toxicological profile of gold growth stage of the cells, and temperature (Gericke and nanoparticles was assessed by determining its effect on the Pinches 2006). Biogenic nanoparticles can be synthesized ei- growth and viability of the Escherichia coli (ATCC11103). ther internally or externally by the microbes but extracellular Their movement inside the bacterial cell was also studied for production is more practical in terms of ease of isolation. probable use in drug delivery. Rhizopus oryzae, Cylindrocladium floridanum, Verticillium sp., Hansenula anomala, Saccharomyces cerevisae, Candida guilliermondii, Yarrowia lipolytica, Aspergillus niger, Material and methods Pleurotus ostreatus, Penicillium brevicompactum, Neurospora crassa, Helminthosporum solani and Alternaria Materials alternate have been successfully utilized for biogenic gold nanoparticle synthesis (Siddiqi and Husen 2016). Potato dextrose agar and potato dextrose broth were pur- Metallic nanoparticle biosynthesis using fungus is more ad- chased from Hi-Media Laboratories Pvt. Ltd. Mumbai, vantageous as compared to bacterial synthesis of nanoparticles. India. Gold (III) chloride trihydrate (HAuCl ·3H O) was pur- 4 2 For large-scale production of nanoparticles, fungi and yeasts are chased from Sigma-Aldrich, USA. MiliQ water was used for preferred over other organisms as they would secrete more solution preparation. enzymes that would augment the biosynthesis of nanoparticle. Moreover the ease of scaling up and recovery, the effectiveness, Biosynthesis of gold nanoparticles and the presence of mycelia offering an increased surface area, are important factors (Kitching et al. 2015). The fungus used in the present study was selected from the Macrophomina phaseolina (Tassi) Goid is a vicious fungal laboratory stock cultures at Indian Institute of Agricultural pathogen infecting more than 500 plant types worldwide Research (IARI), New Delhi. Pure culture of the fungal strain (Mihail and Taylor 1995). It is an anamorphic Basidiomycetes was maintained on Potato dextrose agar slants. Macrophomina belonging to Macrophomina and Rhizoctonia, respectively. It was grown in 100 ml of potato dextrose broth at 28 °C under causes stem blight, charcoal rot, damping off, and stem rot in shaking condition (120 rpm) for 48 h. After incubation, the various plant species (Alexopoulos et al. 2007;Khan 2007). fungal mycelia was filtered out and washed thrice with sterile This fungus produces a mixture of hydrolytic enzymes capable distilled water to remove the traces of media components. The of degrading the plant cell wall to penetrate in host tissue. biomass was homogenized prior to the gold exposure experi- Genomic study of M. phaseolina has reported huge number ment. Five grams of wet biomass and cell-free filtrate were both of paralogs for the oxido-reductase class of enzymes (Islam challenged with chloroauric acidsolution(1mM) (0.0339gin et al. 2012). These oxido-reductase enzymes assist the fungus 100 ml) in the ratio 1:1 (v/v) and incubated at 28 °C and to colonize harsh environments and infect new plant hosts. The 180rpm alongwiththeir controls (cell-free filtrate, biomass, reductase enzymes are reported to catalyze the biogenic nano- and substrate solution alone) for 16 h. The optimal ratio was particle formation (Ramezani et al. 2010); however, there was determined after conducting experiments at different ratios and no report on the production of gold nanoparticles using was found to be 1:1 (data not included). The nanoparticles were M. phaseolina. recovered from the solution by centrifugation (10,000 rpm for The advantage of producing gold nanoparticles using 10 min). They were then washed with distilled water to obtain M. phaseolina is that the oxidoreductase activity of purified gold nanoparticles. The nanoparticles were preserved Ann Microbiol (2019) 69:435–445 437 Dynamic light-scattering measurements of the sample were 0.9 determined by Malvern Zetasizer Nano-S90 (Malvern 0.8 Instruments Ltd., Malvern, UK) to measure the hydrodynamic 0.7 4hrs size of gold nanoparticles in solution. 0.6 8hrs Fourier transform infrared (FTIR) spectra of the sample 0.5 12hrs were carried out with spectrophotometer (Jasco 410 Series). 0.4 −1 0.3 The measurements were done in the range of 4000–400 cm , 16hrs 0.2 using the KBr pellet technique, to identify the possible func- 0.1 tional groups present on the protein coating. The shape, size, and morphology of the gold nanopar- 400 500 600 700 800 ticles were analyzed using HRTEM (Philips, CM-10 mod- Wavelength (nm) el, AIIMS, New Delhi, India) with an operating voltage of Fig. 1 UV-Vis spectra from time-course experiment showing character- 100 kv. The gold nanoparticle films were prepared on istic peak at 540 nm indicated the synthesis of gold nanoparticles by the carbon-coated copper TEM grids for analysis. Pressure fungus M. phaseolina −5 maintained was 10 Pa and point to point resolution was 2 nm. Accelerating voltage at which it was operated in powdered form using freeze dryer (Scanvac). The yield of the was 100 kv HRTEM (Philips, CM-10 model, AIIMS, gold nanoparticles was also calculated [nanoparticle (dried New Delhi, India). powder) per milliliter reaction mixture]. All the studies have Energy dispersive X-ray analysis was done using Carl Zeiss been conducted in triplicates. EVO-40 at 20 kv to confirm the presence of gold nanoparticles and its chemical compositioninelectronmicrographs. Effect of pH and temperature on the formation of gold nanoparticles Effect of gold nanoparticles on growth and viability of E. coli Temperature optima Optimum temperature for the formation of gold nanoparticles was assessed by varying the incubation Two set of experiments were carried out to study the effect of temperature of the cell-free filtrate from 28 to 55 °C. gold nanoparticles on growth of E. coli. In the first set of experiment, 1% of E. coli culture (2.4 × 10 CFU/ml) and pH optima Optimum pH for the gold nanoparticle synthesis 100 μl of prepared gold nanoparticles were added into 25 ml was assessed by varying the pH of the cell-free filtrate with of the nutrient broth (Hi-Media Laboratories Pvt. Limited, buffers ranging from pH 5–9withanaccuracyof± 0.2 (so- Mumbai, India) and incubated at 37 °C in a rotor shaker at dium acetate buffer for (pH 5), phosphate buffer for pH 6–8 120 rpm. The samples were withdrawn at regular intervals (0, and sodium bicarbonate buffer for pH 9. 4, 8, 12, 16, 20, and 24 h) and the absorbance was measured at 620 nm using a UV-Vis spectrophotometer (Shimadzu model Characterization of gold nanoparticles 1700 UV-Vis). The experiments were carried out in triplicates andstandarderror wascalculated. After 16 h of incubation, the samples showing change in color In the second experiment, the autoclaved flask was inocu- were further characterized for the presence of nanoparticles. lated with 1% of the prepared inoculum and incubated at 3+ The reduction of Au ions was monitored by UV-Vis spectra 37 °C. Gold nanoparticles was added to the flask after 24 h of the solution using a UV-Vis spectrophotometer (Shimadzu and the flask was then kept for incubation for another 24 h. model 1700 UV-Vis). Sample aliquots of the solution at regular Samples were withdrawn aseptically from the second flask at intervals of 4 h were monitored in a range of 400 to 800 nm. 24 (+ 0 h, 4 h, 8 h, 12 h, and 24 h) and the absorbance was Fig. 2 A proposed model showing the bio-reduction of Cl Cl 3+ Au ions by NADPH dependent reductase for the biosynthesis of NDR NDR Cl gold nanoparticles by the fungus Cl + 0 M. phaseolina.NDR=NADPH Au dependent reductase Cl Cl Cl HCl Absorbance (a.u ) 438 Ann Microbiol (2019) 69:435–445 Fig. 3 DLS (Dynamic light scattering) Graph indicating monodispersity of gold nanoparticles synthesized by M. phaseolina measured at 620 nm using a UV-Vis spectrophotometer. The E. coli incubated with gold nanoparticles was taken in steril- cell viability was checked at regular time intervals using col- ized microfuge tubes and centrifuged at 10,000 rpm for ony forming units (CFU). The experiments were performed in 10 min. The culture without gold nanoparticles was taken as triplicates and standard error was calculated. control. The bacterial pellets collected after centrifugation were suspended in fixative and kept at 4 °C for 2 h. It was then centrifuged at 10,000 rpm for 10 min. The supernatant Sample preparation for transmission electron was discarded and the pellets were washed in 0.1 M PO microscopy buffer. The sample film was prepared on carbon-coated cop- per TEM grids. Negative staining was performed by using 6– Uptake of the gold nanoparticles into E. coli (ATCC11103) 10 μl of 2% aqueous phosphotungstic acid on the sample was determined by HRTEM. Twenty-four-hour-old culture of immediately before sample was dried (pH adjusted to 7.3 using 1 N NaOH). The grid was placed directly into grid (a) box and allowed to air-dry several hours and then analyzed by Transmission Electron Microscopy (TEM). Pressure main- 0.9 −5 0.8 tained was 10 Pa and accelerating voltage at which it was 0.7 pH 5 operated was 100 kv HRTEM (Philips, CM-10 model, 0.6 pH 6 AIIMS, New Delhi, India). 0.5 pH 7 0.4 pH 8 0.3 pH 9 0.2 Results and discussion 0.1 In the present study, the mycosynthesis of gold nanoparticles 400 500 600 700 800 Wavelength (nm) was detected by the visual color change in the reaction flasks at initial stage and was further confirmed using UV-Vis spec- (b) troscopy. The cell-free filtrate incubated with chloroauric acid changed its color from light yellow to deep pink. On the other 0.9 hand, the three control solutions retained their original color 0.8 (light yellow), suggesting the absence of nanoparticle synthe- 0.7 0.6 28°C sis. It is well-known that gold nanoparticles reveal striking 0.5 37°C color change from light yellow to pink/purple (Sawle et al. 0.4 45°C 2008; Bhambure et al. 2009). The color change was observed 0.3 55°C due to the phenomenon of surface plasmon resonance (SPR). 0.2 3+ The bio-reduction of Au ions in aqueous solution was 0.1 observed by UV-Vis spectroscopic studies. The character- istic absorbance peak at 540 nm confirms the formation of 400 500 600 700 800 Wavelength ( nm ) gold nanoparticles. Similar absorbance peak have also been reported during the synthesis of gold nanoparticles Fig. 4 UV-Vis spectra showing the effect of different pH (a)and temperatures (b) on the synthesis of gold nanoparticles using fungal species such as Coriolus versicolor (Sanghi Absorbance ( a.u ) Absorbance (a.u) Ann Microbiol (2019) 69:435–445 439 Fig. 5 FTIR indicating the various biological groups present on the gold nanoparticles synthesized from fungus M. phaseolina and Verma 2010)and Cylindrocladium floridanum mechanism of the cell. In order to protect themselves from (Narayanan and Sakthivel 2013). The absorbance value the exposure of metal salts, it secretes various proteins and of the band was further observed with increase in time enzymes which detoxify the metal ions through reduction (Fig. 1). The prolonged incubation resulted in continual leading to the generation of nanoparticles (Siddiqi and reduction of the metal ions into nanoparticles as justified Husen 2016). The extracellular synthesis begins with by the further increase in the absorbance value of the band. biosorption of the metal ions onto the cell wall enzymes by The yield of the gold nanoparticles synthesized was found electrostatic interaction with positively charged groups of the to be 10 mg/ml of the reaction mixture used. cell wall of the fungus and then reduction in the presence of Although the exact mechanism of gold nanoparticle syn- enzymes secreted out by the cell. A number of enzymes are thesis is not known, but it is proposed that the biofabrication known to be involved in the biosynthesis of nanoparticle in- of gold nanoparticle by fungus involves the bio-reduction of cluding nitrate reductase, sulfate reductase, NADH-dependent metal salts to elemental metal. It is further stabilized by organ- reductase, and sulfite reductase (Ramezani et al. 2010; ic molecules secreted by the fungus. The synthesis of metal Kitching et al. 2015). The diagrammatic representation is giv- nanoparticles by fungus can be attributed to self-defense en in Fig. 2. Fig. 6 HRTEM image showing spherical shaped gold nanoparticles. (→) indicates protein coating on gold nanoparticles 440 Ann Microbiol (2019) 69:435–445 Table 1 Synthesis of gold nanoparticle using different biological routes Gold nanoparticle producer Biological route Shape Size (nm) References Bacteria Brevibacterium casei Spherical 10–50 Kalishwaralal et al. (2010) Escherichia coli Triangles, hexagons 20–30 Brown et al. (2000) Plectonemaboryanum Cubic < 10–25 Lengke et al. (2006a) Plectonema boryanum UTEX 485 Octahedral 10 nm–6 μmLengkeetal.(2006b) Rhodococcus sp. Spherical 5–15 Ahmad et al. (2003c) Rhodopseudomonas capsulate Spherical 10–20 He et al. (2007) Sargassum wightii Planar 8–12 Singaravelu et al. (2007) Shewanella algae 10–20 Konishi et al. (2007) Shewanella oneidensis Spherical 12 ± 5 Suresh et al. (2011) Ureibacillus thermosphaericus Spherical 35–75 Juibari et al. (2015) Fungi Macrophomina phaseolina Spherical 14–16 Present study Alternaria alternate Spherical, triangular, hexagonal 12 ± 5 Sarkar et al. (2012) Aspergillus niger Spherical, elliptical 12.8 ± 5.6 Bhambure et al. (2009) Aspergillus niger Polydispersed 10–20 Xie et al. (2007) Aspergillus oryzae var. viridis Various shapes (cell-free filtrate), 10–60 Binupriya et al. (2010) mostly spherical (biomass) Aspergillus sydowii Spherical 8.7–15.6 Vala (2015) Aureobasidium pullulans Spherical 29 ± 6 Zhang et al. (2011) Candida albicans Monodispersed spherical 5 Ahmad et al. (2013) Candida albicans Spherical 20–40 Chauhan et al. (2011) Candida albicans Non-spherical 60–80 SathishKumar et al. (2011) Colletotrichum sp. Spherical 8–40 Shankar et al. (2003) Coriolus versicolor Spherical and ellipsoidal 20–100, 100–300 Sanghi and Verma (2010) Cylindrocladium floridanum Spherical 19.05 Narayanan and Sakthivel (2013) Cylindrocladium floridanum Spherical 5–35 Narayanan and Sakthivel (2011a, b) Fusarium oxysporum Spherical, triangular 2–50 Mandal et al. (2006) Fusarium semitectum Spherical 10–80 Sawle et al. (2008) Hormoconis resinae Spherical 3–20 Mishra et al. (2010) Helminthosporum solani Spheres, rods, triangles, pentagons, 2–70 Kumar et al. (2008) pyramids, stars Neurospora crassa Spherical 32 Castro et al. (2011) Penicillium brevicompactum Spherical 10–50 Mishra et al. (2011) Penicillium rugulosum Spherical 20–40 Mishra et al. (2012) Penicillium sp. 1–208 Spherical 30–50 Du et al. (2011) Phanerochaete chrysosporium Spherical 10–100 Sanghi et al. (2011) Pichia jadinii Spherical < 100 Gericke and Pinches (2006) Rhizopus oryzae Spherical 16–25 Das et al. (2012) Saccharomyces cerevisiae Spherical > 100 Lin et al. (2005) Sclerotium rolfsii Spherical 25.2 ± 6.8 Narayanan and Sakthivel (2011a, b) Trichoderma koningii Small spheres to polygons 30–40 Maliszewska et al. (2009) Trichoderma koningii Spheres 10–14 Maliszewska (2013) Verticillium sp. Spherical 20 ± 8 Mukherjee et al. (2001) Verticillium volvacea Triangular, spherical, hexagonal 20–150 Philip (2009) Yarrowia lipolytica NCIM 3589 Hexagonal, triangular 15 Agnihotri et al. (2009) Dynamic light scattering (DLS) showed the fairly well- biomolecules involved in the reduction and capping of the defined dimensions and good monodispersity of synthesized newly formed nanoparticles. gold nanoparticles with an average size of 69 nm with the It was witnessed that at low pH (5 and 6), slight change in polydispersity index (PDI) of 0.2 (Fig. 3). the color observed after 4 h and the absorbance was 0.28 and The effects of different pH and temperature on the for- 0.31, respectively, whereas the color intensity enhanced at mation of nanoparticles were observed using UV-visible high pH (7 and 8) as indicated by the increase in absorbance spectroscopy (Fig. 4a, b). Good SPR absorption bands were (0.59 and 0.79 respectively) after 4 h. Hence, pH 8 support observed at pH 7 and 8, the maximum being at pH 8 spec- enhanced synthesis of gold nanoparticles. Previous studies ifying the favorable support of neutral pH in the synthesis of have also reported that pH plays a significant role on the gold nanoparticles. The most important effect of the reaction synthesis and size variation of gold nanoparticles (Xie et al. pH is its ability to modify the charge on the various 2007; Das et al. 2012; Jain and Mehata 2017). Ann Microbiol (2019) 69:435–445 441 Fig. 7 EDX spectrum of gold nanoparticles confirming the presence of elemental gold −1 −1 −1 −1 −1 The reaction temperature also has significant effect on the 3848 cm ,3341cm ,2062cm ,1636cm ,1495 cm , synthesis of gold nanoparticles. It was found that the optimum and some minor bands. The absorption bands located at −1 synthesis of gold nanoparticles took place at 37 °C. Gold 3848 cm are assigned as the absorption peak of the N-H −1 nanoparticles were also synthesized at 28 °C. It might be be- group (Gupta et al. 2015). The band at 3341 cm can be cause of generation of adequate activation energy at 37 °C to ascribed to O–H stretch of alcohols and phenols (Mishra propel the catalytic activity of reductase enzyme for synthesis et al. 2011; Basavegowda et al. 2013). The band at −1 of gold nanoparticles. At 45 and 55 °C, a small peak was 2062 cm corresponds to aromatic C-H stretching and char- −1 observed in the absorption spectra which indicated a lesser acteristic band at 1636 cm is for stretching peaks of amide I synthesis of gold nanoparticles; it may be due to the inactiva- group of polypeptides/proteins (Basavegowda et al. 2013). −1 tion of the reductase enzymes of fungi at higher temperature. The band at 1495 cm may be assigned to N-H bending of Scientific evidences have also highlighted the role of temper- amide II band of polypeptides/proteins (Garidel and Schott ature in determining the nanoparticle shape and size (Gericke and Pinches 2006; Xie et al. 2007). 0.5 Figure 5 showed FTIR measurements of gold nanoparticles to determine the presence of biological groups on the gold 0.45 nanoparticles. The FTIR spectrum showed bands at 0.4 0.35 1.2 0.3 0.25 E coli control 0.2 Au NP treated E coli 0.8 0.15 0.6 Au NP treated E coli 0.1 E coli control 0.4 0.05 0.2 048 12 16 20 24 Time (h) 048 12 16 20 24 28 Fig. 9 Growth of pre-grown cultures of Escherichia coli in presence of Time (h) gold nanoparticles. Escherichia coli was incubated for 24 h reaching 0.85 Fig 8 Growth of Escherichia coli in the presence of 0.4% of gold OD units, then 0.4% of gold nanoparticles was added, cultures were nanoparticles incubated for 24 h more Absorbance (a. u) Absorbance ( a. u) 442 Ann Microbiol (2019) 69:435–445 2006). The spectral data supports the role of protein in en- non-toxic nature of the gold nanoparticles on the growth and abling stability of the newly synthesized gold nanoparticles viability of E. coli cells. These results strongly support the by capping them. This protein capping would enable the an- previously published data (Gupta et al. 2015; Roy et al. 2016). choring of drug or other chemicals inside the human system. The synthesized gold nanoparticles were assessed for inter- Moreover, the non-toxic layer of protein will promote the nalization efficacy using E. coli cells. The TEM studies of the increased uptake and retaining of the drug inside the cells E. coli cells treated with gold nanoparticles displayed the pres- (Kitching et al. 2015). ence of gold nanoparticles within the cytoplasm. Figure 10 The representative HRTEM images (Fig. 6)showed spher- confirms the movement of gold nanoparticles inside the cell ical shaped gold nanoparticles ranging between 14 and 16 nm membrane barriers and accumulation inside the cell without with distinct coating on the surface. Various other fungal spe- affecting the structural integrity and shape of the cells (Lin cies have also reported to be producing spherical gold nano- et al. 2010). This indicates the biocompatible nature of the particles in similar range (Sawle et al. 2008; Mishra et al. synthesized gold nanoparticles unlike the chemically synthe- 2011;Maliszewska 2013). Other scientists have also reported sized ones which are reported to be toxic to the cells (Li et al. different shaped gold nanoparticles by physical, biological, 2014;Feng etal. 2015). and chemical means (Table 1) (Kimling et al. 2006; A prevalent observation is that positively charged nanopar- Akbarzadeh et al. 2009;Hassanetal. 2014; Reddy et al. ticles are taken up more easily by cells than neutral or nega- 2014; Menon et al. 2017). tively charged nanoparticles. The reason being cited as the Figure 7 shows the EDX spectrum of the gold nanoparti- electro-adhesion interaction between positively charged nano- cles. The presence of strong peak around 2.3 and 9.6 keV is particles and negatively charged lipopolysaccharides on the typical of adsorption of gold nanoparticles due to surface plas- bacterial outer membrane leads to the attachment of nanopar- mon resonance (Sarkar et al. 2012). The EDX spectrometry ticles onto bacterial surface (Jacobson et al. 2015). But this confirmed the presence of elemental gold as the main constit- explanation does not take into account a critical component: uent. The peaks of carbon and oxygen were also noted which the nanoparticle protein corona. Nevertheless, there are re- may be due to the protein capping on the nanoparticles. ports of cellular uptake of negatively charged nanoparticles The gold nanoparticles were assessed for biocompatibility which indicates that the electrostatic interactions play only a studies by incubation with E. coli cells. The analysis of the partial role in the cell-nanoparticle interaction (Schweiger growth curve revealed the variation in optical density due to et al. 2012;Forestetal. 2015). Our experimental evidence the effect of gold nanoparticles (Fig. 8). In the initial 2 h, both also supports the fact that negatively charged nanoparticles gold nanoparticle–treated and non-treated cultures showed the are also taken up by the bacterial cell. lag phase. After 2 h, there was a steady rise in the growth of Once the nanoparticles are introduced into the biological gold nanoparticle–treated culture and it was maintained systems, it become surrounded by a wide variety of biomole- throughout its exponential phase. Figure 9 represents the cules which can get adsorbed onto its surface and cover the growth of E. coli cells when gold nanoparticles were added nanoparticles. This formation of protein corona can alter the after 24 h of incubation of bacteria, i.e., at the late stationary original properties of nanoparticles in terms of hydrodynamic phase of the growth. The addition of gold nanoparticles mar- diameter and zeta potential of the nanoparticle. The new nano- ginally enhanced the growth of E. coli cells. These indicate the particle interface generated as a result can reorient its Fig. 10 HRTEM image of movement of enzymatically synthesized gold nanoparticles inside Escherichia coli without disrupting the shape and structural integrity of the cell. (→) indicates gold nanoparticles Ann Microbiol (2019) 69:435–445 443 Alkilany AM, Murphy CJ (2010) Toxicity and cellular uptake of gold interaction with the cell surface and can in turn affect the cell nanoparticles: what we have learned so far? J Nanopart Res 12: response and dissemination of the nanoparticle inside the cell 2313–2333. https://doi.org/10.1007/s11051-010-9911-8 (Forest et al. 2015). The present work introduces the concept Arvizo R, Bhattacharya R, Mukherjee P (2010) Gold nanoparticles: op- of using Macrophomina phaseolina, which is otherwise a vi- portunities and challenges in nanomedicine. Expert Opin Drug Deliv 7:753–763. https://doi.org/10.1517/17425241003777010 cious fungal pathogen, for biosynthesis of production of stable Basavegowda N, SobczakKupiec A, Malina D, Yathirajan HS, and uniform shaped gold nanoparticles in large amount. We Keerthi VR, Chandrashekar N, Dinkar S, Liny P (2013) Plant have successfully assessed the effect of environmental condi- mediated synthesis of gold nanoparticles using fruit extracts of tions such as pH and temperature on the biogenic synthesis of Ananas comosus (L.) (pineapple) and evaluation of biological activities. Adv Mater Lett 4:332–337. https://doi.org/10.5185/ gold nanoparticles. The gold nanoparticles synthesized are amlett.2012.9423 non-toxic in nature as demonstrated by their biocompatibility Bhambure R, Bule M, Shaligram N, Kamat M, Singhal R (2009) studies (growth and viability). The unhindered entry of the Extracellular biosynthesis of gold nanoparticles using Aspergillus nanoparticles inside the bacterial cell highlights the possible niger—its characterization and stability. Chem Eng Technol 32: avenue of exploring them as drug carriers to improve drug 1036–1041 Binupriya AR, Sathishkumar M, Vijayaraghavan K, Yun SI (2010) delivery system. Bioreduction of trivalent aurum to nanocrystalline gold particles by active and inactive cells and cell free extract of Aspergillus oryzae Compliance with ethical standards var. viridis. J Hazard Mater 177:539–545 Brown S, Sarikaya M, Johnson EA (2000) A genetic analysis of crystal Conflict of interest The authors declare that they have no conflict of growth. J Mol Biol 299:725–735 interest. Castro LE, Vilchis NAR, Avalos BM (2011) Biosynthesis of silver, gold and bimetallic nanoparticles using the filamentous fungus Neurospora crassa. Colloids Surf B Biointerfaces 83:42–48 Research involving human participants and/or animals (if applicable) This article does not contain any studies with human par- Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC, Azam ticipants or animals performed by any of the authors. A, Owais M (2011) Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer. Int J Nanomedicine 6:2305–2319 Informed consent For this type of study informed consent is not Daraee H, Eatemadi A, Abbasi E, Aval SF, Kouhi M, Akbarzadeh A required. (2016) Application of gold nanoparticles in biomedical and drug delivery. Artif Cells Nanomed Biotechnol 44:410–422. https://doi. Publisher’sNote Springer Nature remains neutral with regard to jurisdic- org/10.3109/21691401.2014.955107 tional claims in published maps and institutional affiliations. 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Macrophomina phaseolina: microbased biorefinery for gold nanoparticle production

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Springer Journals
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Copyright © 2019 by Università degli studi di Milano
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
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1869-2044
DOI
10.1007/s13213-018-1434-z
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Abstract

Biofabrication of nanoparticles via the principles of green nanotechnology is a key issue addressed in nanobiotechnology research. There is a growing need for development of a synthesis method for producing biocompatible stable nanoparticles in order to avoid adverse effects in medical applications. We report the use of simple and rapid biosynthesis method for the preparation of gold nanoparticles using Macrophomina phaseolina (Tassi) Goid, a soil-borne pathogen. The effect of pH and temperature on the synthesis of gold nanoparticles by M. phaseolina was also assessed. Different techniques like UV-Visible Spectroscopy, Transmission Electron Microscopy (TEM), Dynamic light scattering (DLS) measurements, Fourier transform infrared (FTIR), and EDX were used to characterize the gold nanoparticles. The movement of these gold nanoparticles inside Escherichia coli (ATCC11103) along with effect on growth and viability was evaluated. The biogenic gold nanoparticle was synthesized at 37 °C temperature and neutral pH. UV-Visible Spectroscopy, TEM, EDX, and DLS measurements confirm the formation of 14 to 16 nm biogenic gold nanoparticles. FTIR substantiates the presence of protein capping on Macrophomina phaseolina-mediated gold nanoparticles. The non-toxicity of gold nanoparticles was confirmed by the growth and viability assay while the TEM images validated the entry of gold nanoparticles without disrupting the structural integrity of E. coli.Biogenic method for the synthesis of nanoparticles using fungi is novel, efficient, without toxic chemicals. These biogenic gold nanopar- ticles themselves are nontoxic to the microbial cells and offer a better substitute for drug delivery system. . . . . Keywords Nanotechnology Gold nanoparticles Bio-reduction Macrophomina phaseolina Transmission electron microscopy FTIR Introduction Iravani 2014). Gold nanoparticles have gathered the attention of nanotechnologists worldwide due to its stability, biocom- Nanomaterials are considered to be a distinguished constituent patibility, and resistance to oxidation (Alkilany and Murphy of the rapidly advancing field of nanotechnology (Salata 2010). Gold nanoparticles have numerous applications in bi- 2004; Narkeviciute et al. 2016). In recent times, immense ological fields including drug delivery and genetic engineer- importance has been laid on augmenting the biological meth- ing, cancer diagnostics, protein detection, and gene delivery odologies for the synthesis of nanomaterials as well as their (Arvizo et al. 2010;Yanget al. 2015;Daraee etal. 2016). characterization and abundant applications (Duran and Seabra A variety of physical and chemical methods have been 2009). The nanoparticles of profound interest are gold, silver, established to produce metal nanoparticles (Zhang et al. zinc, and titanium (Reddy et al. 2007; Reddy et al. 2011; 2007; Reddy et al. 2008; Hassan et al. 2014; Reddy et al. 2014). The chemical means include usage of chemical reduc- tants like citrate (Kimling et al. 2006), tryptophane (Akbarzadeh et al. 2009), PEG 4000 (Roy and Lahiri 2006), * Rajni Singh and amino acid derivatives (Sugunan and Dutta 2006; rsingh3@amity.edu; rajni_vishal@yahoo.com Ravindra 2009). But these expensive and hazardous chemicals led to general toxicity and can play havoc on the Amity Institute of Microbial Biotechnology, Amity University Uttar environment. Thus, there is a resurgence of interest in devel- Pradesh, Sector-125, Noida, U.P. 201313, India oping environmentally friendly processes. Biological systems ICAR-National Bureau of Agriculturally Important Microorganisms such as bacteria, yeasts, fungi, actinomycetes, and plants have (NBAIM), Mau Nath Bhanjan, Mau, Uttar Pradesh 275101, India 436 Ann Microbiol (2019) 69:435–445 been reportedly utilized for synthesis. The use of microbes as M. phaseolina is high as compared to other fungal species. nanofactories seems to be a very effective green and econom- This method is economical as less amount of enzyme is re- ical method for biogenic synthesis of gold nanoparticles (He quired for the generation of gold nanoparticles. Also this is of et al. 2007; Dhillon et al. 2012). Microbes have great potential ecological concern as the fungus is one of the most destructive to produce nanoparticles with features similar to chemically plant pathogen with a vast geographical distribution and is synthesized counterparts. There are several reports concerning especially problematic in tropical and subtropical countries the microbial synthesis of gold nanoparticles: intracellular with arid to semiarid climates in Africa, Asia, Europe, and synthesis by the actinomycete Rhodococcus sp. (Ahmad North and South America. The reductase enzymes produced et al. 2003c), fungus Hormoconis resinae (Varshney et al. by the fungus can be channelized towards the synthesis of 2009), and bacterium Acidithiobacillus thiooxidans (Lengke nanoparticles which could help in diverting its pathogenic et al. 2005); extracellular synthesis by actinomycete ability towards the biosynthesis of nanoparticles which could Thermomonospora sp. (Ahmad et al. 2003b) and fungus in turn help the growth of the plants. Fusarium oxysporum (Ahmad et al. 2003a) while intra- and Hence, the main objective of the present work was to pro- extracellular syntheses by the cyanobacterium Plectonema duce biogenic gold nanoparticles using M. phaseolina,deter- boryanum (Lengke et al. 2006a). The size and shape of gold mining the environmental parameters affecting its synthesis nanoparticles are affected by growth parameters such as pH, and their characterization. The toxicological profile of gold growth stage of the cells, and temperature (Gericke and nanoparticles was assessed by determining its effect on the Pinches 2006). Biogenic nanoparticles can be synthesized ei- growth and viability of the Escherichia coli (ATCC11103). ther internally or externally by the microbes but extracellular Their movement inside the bacterial cell was also studied for production is more practical in terms of ease of isolation. probable use in drug delivery. Rhizopus oryzae, Cylindrocladium floridanum, Verticillium sp., Hansenula anomala, Saccharomyces cerevisae, Candida guilliermondii, Yarrowia lipolytica, Aspergillus niger, Material and methods Pleurotus ostreatus, Penicillium brevicompactum, Neurospora crassa, Helminthosporum solani and Alternaria Materials alternate have been successfully utilized for biogenic gold nanoparticle synthesis (Siddiqi and Husen 2016). Potato dextrose agar and potato dextrose broth were pur- Metallic nanoparticle biosynthesis using fungus is more ad- chased from Hi-Media Laboratories Pvt. Ltd. Mumbai, vantageous as compared to bacterial synthesis of nanoparticles. India. Gold (III) chloride trihydrate (HAuCl ·3H O) was pur- 4 2 For large-scale production of nanoparticles, fungi and yeasts are chased from Sigma-Aldrich, USA. MiliQ water was used for preferred over other organisms as they would secrete more solution preparation. enzymes that would augment the biosynthesis of nanoparticle. Moreover the ease of scaling up and recovery, the effectiveness, Biosynthesis of gold nanoparticles and the presence of mycelia offering an increased surface area, are important factors (Kitching et al. 2015). The fungus used in the present study was selected from the Macrophomina phaseolina (Tassi) Goid is a vicious fungal laboratory stock cultures at Indian Institute of Agricultural pathogen infecting more than 500 plant types worldwide Research (IARI), New Delhi. Pure culture of the fungal strain (Mihail and Taylor 1995). It is an anamorphic Basidiomycetes was maintained on Potato dextrose agar slants. Macrophomina belonging to Macrophomina and Rhizoctonia, respectively. It was grown in 100 ml of potato dextrose broth at 28 °C under causes stem blight, charcoal rot, damping off, and stem rot in shaking condition (120 rpm) for 48 h. After incubation, the various plant species (Alexopoulos et al. 2007;Khan 2007). fungal mycelia was filtered out and washed thrice with sterile This fungus produces a mixture of hydrolytic enzymes capable distilled water to remove the traces of media components. The of degrading the plant cell wall to penetrate in host tissue. biomass was homogenized prior to the gold exposure experi- Genomic study of M. phaseolina has reported huge number ment. Five grams of wet biomass and cell-free filtrate were both of paralogs for the oxido-reductase class of enzymes (Islam challenged with chloroauric acidsolution(1mM) (0.0339gin et al. 2012). These oxido-reductase enzymes assist the fungus 100 ml) in the ratio 1:1 (v/v) and incubated at 28 °C and to colonize harsh environments and infect new plant hosts. The 180rpm alongwiththeir controls (cell-free filtrate, biomass, reductase enzymes are reported to catalyze the biogenic nano- and substrate solution alone) for 16 h. The optimal ratio was particle formation (Ramezani et al. 2010); however, there was determined after conducting experiments at different ratios and no report on the production of gold nanoparticles using was found to be 1:1 (data not included). The nanoparticles were M. phaseolina. recovered from the solution by centrifugation (10,000 rpm for The advantage of producing gold nanoparticles using 10 min). They were then washed with distilled water to obtain M. phaseolina is that the oxidoreductase activity of purified gold nanoparticles. The nanoparticles were preserved Ann Microbiol (2019) 69:435–445 437 Dynamic light-scattering measurements of the sample were 0.9 determined by Malvern Zetasizer Nano-S90 (Malvern 0.8 Instruments Ltd., Malvern, UK) to measure the hydrodynamic 0.7 4hrs size of gold nanoparticles in solution. 0.6 8hrs Fourier transform infrared (FTIR) spectra of the sample 0.5 12hrs were carried out with spectrophotometer (Jasco 410 Series). 0.4 −1 0.3 The measurements were done in the range of 4000–400 cm , 16hrs 0.2 using the KBr pellet technique, to identify the possible func- 0.1 tional groups present on the protein coating. The shape, size, and morphology of the gold nanopar- 400 500 600 700 800 ticles were analyzed using HRTEM (Philips, CM-10 mod- Wavelength (nm) el, AIIMS, New Delhi, India) with an operating voltage of Fig. 1 UV-Vis spectra from time-course experiment showing character- 100 kv. The gold nanoparticle films were prepared on istic peak at 540 nm indicated the synthesis of gold nanoparticles by the carbon-coated copper TEM grids for analysis. Pressure fungus M. phaseolina −5 maintained was 10 Pa and point to point resolution was 2 nm. Accelerating voltage at which it was operated in powdered form using freeze dryer (Scanvac). The yield of the was 100 kv HRTEM (Philips, CM-10 model, AIIMS, gold nanoparticles was also calculated [nanoparticle (dried New Delhi, India). powder) per milliliter reaction mixture]. All the studies have Energy dispersive X-ray analysis was done using Carl Zeiss been conducted in triplicates. EVO-40 at 20 kv to confirm the presence of gold nanoparticles and its chemical compositioninelectronmicrographs. Effect of pH and temperature on the formation of gold nanoparticles Effect of gold nanoparticles on growth and viability of E. coli Temperature optima Optimum temperature for the formation of gold nanoparticles was assessed by varying the incubation Two set of experiments were carried out to study the effect of temperature of the cell-free filtrate from 28 to 55 °C. gold nanoparticles on growth of E. coli. In the first set of experiment, 1% of E. coli culture (2.4 × 10 CFU/ml) and pH optima Optimum pH for the gold nanoparticle synthesis 100 μl of prepared gold nanoparticles were added into 25 ml was assessed by varying the pH of the cell-free filtrate with of the nutrient broth (Hi-Media Laboratories Pvt. Limited, buffers ranging from pH 5–9withanaccuracyof± 0.2 (so- Mumbai, India) and incubated at 37 °C in a rotor shaker at dium acetate buffer for (pH 5), phosphate buffer for pH 6–8 120 rpm. The samples were withdrawn at regular intervals (0, and sodium bicarbonate buffer for pH 9. 4, 8, 12, 16, 20, and 24 h) and the absorbance was measured at 620 nm using a UV-Vis spectrophotometer (Shimadzu model Characterization of gold nanoparticles 1700 UV-Vis). The experiments were carried out in triplicates andstandarderror wascalculated. After 16 h of incubation, the samples showing change in color In the second experiment, the autoclaved flask was inocu- were further characterized for the presence of nanoparticles. lated with 1% of the prepared inoculum and incubated at 3+ The reduction of Au ions was monitored by UV-Vis spectra 37 °C. Gold nanoparticles was added to the flask after 24 h of the solution using a UV-Vis spectrophotometer (Shimadzu and the flask was then kept for incubation for another 24 h. model 1700 UV-Vis). Sample aliquots of the solution at regular Samples were withdrawn aseptically from the second flask at intervals of 4 h were monitored in a range of 400 to 800 nm. 24 (+ 0 h, 4 h, 8 h, 12 h, and 24 h) and the absorbance was Fig. 2 A proposed model showing the bio-reduction of Cl Cl 3+ Au ions by NADPH dependent reductase for the biosynthesis of NDR NDR Cl gold nanoparticles by the fungus Cl + 0 M. phaseolina.NDR=NADPH Au dependent reductase Cl Cl Cl HCl Absorbance (a.u ) 438 Ann Microbiol (2019) 69:435–445 Fig. 3 DLS (Dynamic light scattering) Graph indicating monodispersity of gold nanoparticles synthesized by M. phaseolina measured at 620 nm using a UV-Vis spectrophotometer. The E. coli incubated with gold nanoparticles was taken in steril- cell viability was checked at regular time intervals using col- ized microfuge tubes and centrifuged at 10,000 rpm for ony forming units (CFU). The experiments were performed in 10 min. The culture without gold nanoparticles was taken as triplicates and standard error was calculated. control. The bacterial pellets collected after centrifugation were suspended in fixative and kept at 4 °C for 2 h. It was then centrifuged at 10,000 rpm for 10 min. The supernatant Sample preparation for transmission electron was discarded and the pellets were washed in 0.1 M PO microscopy buffer. The sample film was prepared on carbon-coated cop- per TEM grids. Negative staining was performed by using 6– Uptake of the gold nanoparticles into E. coli (ATCC11103) 10 μl of 2% aqueous phosphotungstic acid on the sample was determined by HRTEM. Twenty-four-hour-old culture of immediately before sample was dried (pH adjusted to 7.3 using 1 N NaOH). The grid was placed directly into grid (a) box and allowed to air-dry several hours and then analyzed by Transmission Electron Microscopy (TEM). Pressure main- 0.9 −5 0.8 tained was 10 Pa and accelerating voltage at which it was 0.7 pH 5 operated was 100 kv HRTEM (Philips, CM-10 model, 0.6 pH 6 AIIMS, New Delhi, India). 0.5 pH 7 0.4 pH 8 0.3 pH 9 0.2 Results and discussion 0.1 In the present study, the mycosynthesis of gold nanoparticles 400 500 600 700 800 Wavelength (nm) was detected by the visual color change in the reaction flasks at initial stage and was further confirmed using UV-Vis spec- (b) troscopy. The cell-free filtrate incubated with chloroauric acid changed its color from light yellow to deep pink. On the other 0.9 hand, the three control solutions retained their original color 0.8 (light yellow), suggesting the absence of nanoparticle synthe- 0.7 0.6 28°C sis. It is well-known that gold nanoparticles reveal striking 0.5 37°C color change from light yellow to pink/purple (Sawle et al. 0.4 45°C 2008; Bhambure et al. 2009). The color change was observed 0.3 55°C due to the phenomenon of surface plasmon resonance (SPR). 0.2 3+ The bio-reduction of Au ions in aqueous solution was 0.1 observed by UV-Vis spectroscopic studies. The character- istic absorbance peak at 540 nm confirms the formation of 400 500 600 700 800 Wavelength ( nm ) gold nanoparticles. Similar absorbance peak have also been reported during the synthesis of gold nanoparticles Fig. 4 UV-Vis spectra showing the effect of different pH (a)and temperatures (b) on the synthesis of gold nanoparticles using fungal species such as Coriolus versicolor (Sanghi Absorbance ( a.u ) Absorbance (a.u) Ann Microbiol (2019) 69:435–445 439 Fig. 5 FTIR indicating the various biological groups present on the gold nanoparticles synthesized from fungus M. phaseolina and Verma 2010)and Cylindrocladium floridanum mechanism of the cell. In order to protect themselves from (Narayanan and Sakthivel 2013). The absorbance value the exposure of metal salts, it secretes various proteins and of the band was further observed with increase in time enzymes which detoxify the metal ions through reduction (Fig. 1). The prolonged incubation resulted in continual leading to the generation of nanoparticles (Siddiqi and reduction of the metal ions into nanoparticles as justified Husen 2016). The extracellular synthesis begins with by the further increase in the absorbance value of the band. biosorption of the metal ions onto the cell wall enzymes by The yield of the gold nanoparticles synthesized was found electrostatic interaction with positively charged groups of the to be 10 mg/ml of the reaction mixture used. cell wall of the fungus and then reduction in the presence of Although the exact mechanism of gold nanoparticle syn- enzymes secreted out by the cell. A number of enzymes are thesis is not known, but it is proposed that the biofabrication known to be involved in the biosynthesis of nanoparticle in- of gold nanoparticle by fungus involves the bio-reduction of cluding nitrate reductase, sulfate reductase, NADH-dependent metal salts to elemental metal. It is further stabilized by organ- reductase, and sulfite reductase (Ramezani et al. 2010; ic molecules secreted by the fungus. The synthesis of metal Kitching et al. 2015). The diagrammatic representation is giv- nanoparticles by fungus can be attributed to self-defense en in Fig. 2. Fig. 6 HRTEM image showing spherical shaped gold nanoparticles. (→) indicates protein coating on gold nanoparticles 440 Ann Microbiol (2019) 69:435–445 Table 1 Synthesis of gold nanoparticle using different biological routes Gold nanoparticle producer Biological route Shape Size (nm) References Bacteria Brevibacterium casei Spherical 10–50 Kalishwaralal et al. (2010) Escherichia coli Triangles, hexagons 20–30 Brown et al. (2000) Plectonemaboryanum Cubic < 10–25 Lengke et al. (2006a) Plectonema boryanum UTEX 485 Octahedral 10 nm–6 μmLengkeetal.(2006b) Rhodococcus sp. Spherical 5–15 Ahmad et al. (2003c) Rhodopseudomonas capsulate Spherical 10–20 He et al. (2007) Sargassum wightii Planar 8–12 Singaravelu et al. (2007) Shewanella algae 10–20 Konishi et al. (2007) Shewanella oneidensis Spherical 12 ± 5 Suresh et al. (2011) Ureibacillus thermosphaericus Spherical 35–75 Juibari et al. (2015) Fungi Macrophomina phaseolina Spherical 14–16 Present study Alternaria alternate Spherical, triangular, hexagonal 12 ± 5 Sarkar et al. (2012) Aspergillus niger Spherical, elliptical 12.8 ± 5.6 Bhambure et al. (2009) Aspergillus niger Polydispersed 10–20 Xie et al. (2007) Aspergillus oryzae var. viridis Various shapes (cell-free filtrate), 10–60 Binupriya et al. (2010) mostly spherical (biomass) Aspergillus sydowii Spherical 8.7–15.6 Vala (2015) Aureobasidium pullulans Spherical 29 ± 6 Zhang et al. (2011) Candida albicans Monodispersed spherical 5 Ahmad et al. (2013) Candida albicans Spherical 20–40 Chauhan et al. (2011) Candida albicans Non-spherical 60–80 SathishKumar et al. (2011) Colletotrichum sp. Spherical 8–40 Shankar et al. (2003) Coriolus versicolor Spherical and ellipsoidal 20–100, 100–300 Sanghi and Verma (2010) Cylindrocladium floridanum Spherical 19.05 Narayanan and Sakthivel (2013) Cylindrocladium floridanum Spherical 5–35 Narayanan and Sakthivel (2011a, b) Fusarium oxysporum Spherical, triangular 2–50 Mandal et al. (2006) Fusarium semitectum Spherical 10–80 Sawle et al. (2008) Hormoconis resinae Spherical 3–20 Mishra et al. (2010) Helminthosporum solani Spheres, rods, triangles, pentagons, 2–70 Kumar et al. (2008) pyramids, stars Neurospora crassa Spherical 32 Castro et al. (2011) Penicillium brevicompactum Spherical 10–50 Mishra et al. (2011) Penicillium rugulosum Spherical 20–40 Mishra et al. (2012) Penicillium sp. 1–208 Spherical 30–50 Du et al. (2011) Phanerochaete chrysosporium Spherical 10–100 Sanghi et al. (2011) Pichia jadinii Spherical < 100 Gericke and Pinches (2006) Rhizopus oryzae Spherical 16–25 Das et al. (2012) Saccharomyces cerevisiae Spherical > 100 Lin et al. (2005) Sclerotium rolfsii Spherical 25.2 ± 6.8 Narayanan and Sakthivel (2011a, b) Trichoderma koningii Small spheres to polygons 30–40 Maliszewska et al. (2009) Trichoderma koningii Spheres 10–14 Maliszewska (2013) Verticillium sp. Spherical 20 ± 8 Mukherjee et al. (2001) Verticillium volvacea Triangular, spherical, hexagonal 20–150 Philip (2009) Yarrowia lipolytica NCIM 3589 Hexagonal, triangular 15 Agnihotri et al. (2009) Dynamic light scattering (DLS) showed the fairly well- biomolecules involved in the reduction and capping of the defined dimensions and good monodispersity of synthesized newly formed nanoparticles. gold nanoparticles with an average size of 69 nm with the It was witnessed that at low pH (5 and 6), slight change in polydispersity index (PDI) of 0.2 (Fig. 3). the color observed after 4 h and the absorbance was 0.28 and The effects of different pH and temperature on the for- 0.31, respectively, whereas the color intensity enhanced at mation of nanoparticles were observed using UV-visible high pH (7 and 8) as indicated by the increase in absorbance spectroscopy (Fig. 4a, b). Good SPR absorption bands were (0.59 and 0.79 respectively) after 4 h. Hence, pH 8 support observed at pH 7 and 8, the maximum being at pH 8 spec- enhanced synthesis of gold nanoparticles. Previous studies ifying the favorable support of neutral pH in the synthesis of have also reported that pH plays a significant role on the gold nanoparticles. The most important effect of the reaction synthesis and size variation of gold nanoparticles (Xie et al. pH is its ability to modify the charge on the various 2007; Das et al. 2012; Jain and Mehata 2017). Ann Microbiol (2019) 69:435–445 441 Fig. 7 EDX spectrum of gold nanoparticles confirming the presence of elemental gold −1 −1 −1 −1 −1 The reaction temperature also has significant effect on the 3848 cm ,3341cm ,2062cm ,1636cm ,1495 cm , synthesis of gold nanoparticles. It was found that the optimum and some minor bands. The absorption bands located at −1 synthesis of gold nanoparticles took place at 37 °C. Gold 3848 cm are assigned as the absorption peak of the N-H −1 nanoparticles were also synthesized at 28 °C. It might be be- group (Gupta et al. 2015). The band at 3341 cm can be cause of generation of adequate activation energy at 37 °C to ascribed to O–H stretch of alcohols and phenols (Mishra propel the catalytic activity of reductase enzyme for synthesis et al. 2011; Basavegowda et al. 2013). The band at −1 of gold nanoparticles. At 45 and 55 °C, a small peak was 2062 cm corresponds to aromatic C-H stretching and char- −1 observed in the absorption spectra which indicated a lesser acteristic band at 1636 cm is for stretching peaks of amide I synthesis of gold nanoparticles; it may be due to the inactiva- group of polypeptides/proteins (Basavegowda et al. 2013). −1 tion of the reductase enzymes of fungi at higher temperature. The band at 1495 cm may be assigned to N-H bending of Scientific evidences have also highlighted the role of temper- amide II band of polypeptides/proteins (Garidel and Schott ature in determining the nanoparticle shape and size (Gericke and Pinches 2006; Xie et al. 2007). 0.5 Figure 5 showed FTIR measurements of gold nanoparticles to determine the presence of biological groups on the gold 0.45 nanoparticles. The FTIR spectrum showed bands at 0.4 0.35 1.2 0.3 0.25 E coli control 0.2 Au NP treated E coli 0.8 0.15 0.6 Au NP treated E coli 0.1 E coli control 0.4 0.05 0.2 048 12 16 20 24 Time (h) 048 12 16 20 24 28 Fig. 9 Growth of pre-grown cultures of Escherichia coli in presence of Time (h) gold nanoparticles. Escherichia coli was incubated for 24 h reaching 0.85 Fig 8 Growth of Escherichia coli in the presence of 0.4% of gold OD units, then 0.4% of gold nanoparticles was added, cultures were nanoparticles incubated for 24 h more Absorbance (a. u) Absorbance ( a. u) 442 Ann Microbiol (2019) 69:435–445 2006). The spectral data supports the role of protein in en- non-toxic nature of the gold nanoparticles on the growth and abling stability of the newly synthesized gold nanoparticles viability of E. coli cells. These results strongly support the by capping them. This protein capping would enable the an- previously published data (Gupta et al. 2015; Roy et al. 2016). choring of drug or other chemicals inside the human system. The synthesized gold nanoparticles were assessed for inter- Moreover, the non-toxic layer of protein will promote the nalization efficacy using E. coli cells. The TEM studies of the increased uptake and retaining of the drug inside the cells E. coli cells treated with gold nanoparticles displayed the pres- (Kitching et al. 2015). ence of gold nanoparticles within the cytoplasm. Figure 10 The representative HRTEM images (Fig. 6)showed spher- confirms the movement of gold nanoparticles inside the cell ical shaped gold nanoparticles ranging between 14 and 16 nm membrane barriers and accumulation inside the cell without with distinct coating on the surface. Various other fungal spe- affecting the structural integrity and shape of the cells (Lin cies have also reported to be producing spherical gold nano- et al. 2010). This indicates the biocompatible nature of the particles in similar range (Sawle et al. 2008; Mishra et al. synthesized gold nanoparticles unlike the chemically synthe- 2011;Maliszewska 2013). Other scientists have also reported sized ones which are reported to be toxic to the cells (Li et al. different shaped gold nanoparticles by physical, biological, 2014;Feng etal. 2015). and chemical means (Table 1) (Kimling et al. 2006; A prevalent observation is that positively charged nanopar- Akbarzadeh et al. 2009;Hassanetal. 2014; Reddy et al. ticles are taken up more easily by cells than neutral or nega- 2014; Menon et al. 2017). tively charged nanoparticles. The reason being cited as the Figure 7 shows the EDX spectrum of the gold nanoparti- electro-adhesion interaction between positively charged nano- cles. The presence of strong peak around 2.3 and 9.6 keV is particles and negatively charged lipopolysaccharides on the typical of adsorption of gold nanoparticles due to surface plas- bacterial outer membrane leads to the attachment of nanopar- mon resonance (Sarkar et al. 2012). The EDX spectrometry ticles onto bacterial surface (Jacobson et al. 2015). But this confirmed the presence of elemental gold as the main constit- explanation does not take into account a critical component: uent. The peaks of carbon and oxygen were also noted which the nanoparticle protein corona. Nevertheless, there are re- may be due to the protein capping on the nanoparticles. ports of cellular uptake of negatively charged nanoparticles The gold nanoparticles were assessed for biocompatibility which indicates that the electrostatic interactions play only a studies by incubation with E. coli cells. The analysis of the partial role in the cell-nanoparticle interaction (Schweiger growth curve revealed the variation in optical density due to et al. 2012;Forestetal. 2015). Our experimental evidence the effect of gold nanoparticles (Fig. 8). In the initial 2 h, both also supports the fact that negatively charged nanoparticles gold nanoparticle–treated and non-treated cultures showed the are also taken up by the bacterial cell. lag phase. After 2 h, there was a steady rise in the growth of Once the nanoparticles are introduced into the biological gold nanoparticle–treated culture and it was maintained systems, it become surrounded by a wide variety of biomole- throughout its exponential phase. Figure 9 represents the cules which can get adsorbed onto its surface and cover the growth of E. coli cells when gold nanoparticles were added nanoparticles. This formation of protein corona can alter the after 24 h of incubation of bacteria, i.e., at the late stationary original properties of nanoparticles in terms of hydrodynamic phase of the growth. The addition of gold nanoparticles mar- diameter and zeta potential of the nanoparticle. The new nano- ginally enhanced the growth of E. coli cells. These indicate the particle interface generated as a result can reorient its Fig. 10 HRTEM image of movement of enzymatically synthesized gold nanoparticles inside Escherichia coli without disrupting the shape and structural integrity of the cell. (→) indicates gold nanoparticles Ann Microbiol (2019) 69:435–445 443 Alkilany AM, Murphy CJ (2010) Toxicity and cellular uptake of gold interaction with the cell surface and can in turn affect the cell nanoparticles: what we have learned so far? J Nanopart Res 12: response and dissemination of the nanoparticle inside the cell 2313–2333. https://doi.org/10.1007/s11051-010-9911-8 (Forest et al. 2015). The present work introduces the concept Arvizo R, Bhattacharya R, Mukherjee P (2010) Gold nanoparticles: op- of using Macrophomina phaseolina, which is otherwise a vi- portunities and challenges in nanomedicine. Expert Opin Drug Deliv 7:753–763. https://doi.org/10.1517/17425241003777010 cious fungal pathogen, for biosynthesis of production of stable Basavegowda N, SobczakKupiec A, Malina D, Yathirajan HS, and uniform shaped gold nanoparticles in large amount. We Keerthi VR, Chandrashekar N, Dinkar S, Liny P (2013) Plant have successfully assessed the effect of environmental condi- mediated synthesis of gold nanoparticles using fruit extracts of tions such as pH and temperature on the biogenic synthesis of Ananas comosus (L.) (pineapple) and evaluation of biological activities. Adv Mater Lett 4:332–337. https://doi.org/10.5185/ gold nanoparticles. The gold nanoparticles synthesized are amlett.2012.9423 non-toxic in nature as demonstrated by their biocompatibility Bhambure R, Bule M, Shaligram N, Kamat M, Singhal R (2009) studies (growth and viability). The unhindered entry of the Extracellular biosynthesis of gold nanoparticles using Aspergillus nanoparticles inside the bacterial cell highlights the possible niger—its characterization and stability. Chem Eng Technol 32: avenue of exploring them as drug carriers to improve drug 1036–1041 Binupriya AR, Sathishkumar M, Vijayaraghavan K, Yun SI (2010) delivery system. Bioreduction of trivalent aurum to nanocrystalline gold particles by active and inactive cells and cell free extract of Aspergillus oryzae Compliance with ethical standards var. viridis. J Hazard Mater 177:539–545 Brown S, Sarikaya M, Johnson EA (2000) A genetic analysis of crystal Conflict of interest The authors declare that they have no conflict of growth. J Mol Biol 299:725–735 interest. Castro LE, Vilchis NAR, Avalos BM (2011) Biosynthesis of silver, gold and bimetallic nanoparticles using the filamentous fungus Neurospora crassa. Colloids Surf B Biointerfaces 83:42–48 Research involving human participants and/or animals (if applicable) This article does not contain any studies with human par- Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC, Azam ticipants or animals performed by any of the authors. A, Owais M (2011) Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer. Int J Nanomedicine 6:2305–2319 Informed consent For this type of study informed consent is not Daraee H, Eatemadi A, Abbasi E, Aval SF, Kouhi M, Akbarzadeh A required. (2016) Application of gold nanoparticles in biomedical and drug delivery. Artif Cells Nanomed Biotechnol 44:410–422. https://doi. Publisher’sNote Springer Nature remains neutral with regard to jurisdic- org/10.3109/21691401.2014.955107 tional claims in published maps and institutional affiliations. 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Published: Jan 9, 2019

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