Synthesis of Nanomaterials by Microorganisms, Their Characterization and Applications

Abstract

Nanotechnology has endorsed enormous development in material science to formulate innovative products by manipulating matter at nano-scale (1-100 nm). Due to certain limitations associated with conventional physico-chemical synthesis protocols, novel techniques are still being pursued for fabrication of nanoparticles. In them biological synthesis of nanoparticles using different microorganisms has been considered comparatively novel, eco-friendly safe and cost-effective. However, this technique is still immature in terms of fabricating nanoparticles with high quality [size and shape (monodispersity)] and quantity in limited time scale in order to cater real benefits. So, the current research work has investigated the ability of different fungi in synthesizing nanoparticles (NPs) of different metals under varying conditions. Besides, it evaluated the nature and scope of nanoparticles in different applications. The first phase of the study assessed NPs synthesis ability of different fungi for different metals under varying operational conditions. Primarily, NPs synthesis ability of a metal tolerant fungus species Fusarium oxysporum was screened out by reacting its biomass (10 g/50 ml) (Age = 6 days) with salts of different metals on shaker (150 rpm) incubator (28 °C). The results indicated synthesis of only silver (Ag), gold (Au) and platinum (Pt) NPs as evident from change in coloration patterns correspond to transformation in surface plasmon resonance of the colloidal suspensions at their respective wavelengths of 420, 530 and 230 nm under UV-Vis spectroscopy. Based upon these preliminary observations, metal tolerant fungal species including Fusarium oxysporum, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus and Aspergillus terreus were separately investigated for AgNPs synthesis in two separate strategies using; fungal biomass and their culture filtrates. The UV-Vis spectroscopic analysis exhibited AgNPs peak around 400-420 nm in all cases. Overall, NPs synthesis increased with time from 2-96 hr. Its rate was comparatively higher with A. fumigatus during 2-4 hr. While, in case of A. niger and A. flavus, it was more noticeable at 24 hr. The culture filtrate from A. flavus proved to be most efficient in term of producing AgNPs with less polydispersity (5-30 nm). XRD crystallography and Debye-Scherrer formulae demonstrated that AgNPs produced by fungi were crystalline in nature and in acceptable nanometer range in both the strategies. TEM further confirmed the size range of AgNPs varying from 3-80 nm and mostly they were spherical in shape. Comparatively, synthesis of NPs by using fungal cultural filtrate was more effective as it avoided any reaction artifact related to directly exposing the biomass with metal salts, besides; NPs were easily purified in this procedure. Furthermore, production of culture filtrate of A. niger under varying pH revealed pH 5.8 as most suitable for fabrication of high quality AgNPs with less polydispersity (7-27 nm) and at other optimum reaction conditions i.e., temperature 30 oC, precursor salt conc. 0.1M, in 96 hr incubation. Moreover, agitated (150 rpm) reaction condition proved to be more effective for the fabrication of NPs than static condition. In similar reaction conditions, the sizes of Au and Pt NPs varied from 10- 35 and 10-20 respectively. Zetasizer nano ZS (Malvern) further revealed maximum colloidal stability and mobility in Au, followed by Pt and least in case of Ag NPs. In terms of applications, biologically (B) synthesized AgNPs were evaluated as an antibacterial, antifungal agent. Individual and combined antibacterial activities of the five traditional antibiotics and B AgNPs were checked against eight different multi drug resistant bacterial pathogens utilizing Kirby-Bauer disc-diffusion technique. The decreasing order of antibacterial activity (zone of inhibition in mm) of antibiotics, AgNPs and their conjugates against bacterial isolates (group, average) found to be; ciprofloxacin + AgNPs (23) > imipenem + AgNPs (21) > gentamycin + AgNPs (18.5) > vancomycin + AgNPs (15.5) > AgNPs (14.75) > imipenem (13.66) > trimethoprim + AgNPs (13.5) > ciprofloxacin (12.5) > gentamycin (11) > vancomycin (4) > trimethoprim (0). Generally, synergistic outcome of nanoparticles and antibiotics ensued a 0.2-7 (average = 2.8) fold upsurge in antibacterial action. Similarly, AgNPs showed antifungal activities and were slightly higher with B AgNPs (13-15) than C AgNPs (8-12) against four different fungi. AgNPs showed varying anti-oxidant, cytotoxic and phytotoxic activities. Antioxidant activities in terms of free radical (DPPH) scavenging (%) rates per 30 min were; 19.8 and 17.9 at 1000 ppm and were significantly reduced to 5.79 and 5.3 at 100 ppm with biologically (B) synthesized and commercial (C) AgNPs respectively. Brine shrimp assay revealed AgNPs cytotoxicity which increased with increase in conc. of NPs (10- 1000 ppm) and time (0-72 hr). Comparatively, the mortality rate of nauplii (larvae) (n =10) was slightly higher with C AgNPs (90 %) than B AgNPs (80 %) after 72 hr. In phytotoxicity assays, AgNPs at varying conc. i.e., 10, 100 & 1000 ppm showed 35-55 % inhibition in radish seed germination. It was relatively higher at 100 ppm AgNPs after 5 days and the results were non-significantly differed in B AgNPs and C AgNPs. Nevertheless, increase in conc. of AgNPs helped stimulating roots and shoots lengths. In tissue engineering perspective and for stimulation of stem cells growth, NIH3T3 fibroblast cells were incorporated in methacrylated gelatin hydrogels containing AuNPs. Water retension, mechanical, degradation and microscopic analysis of this Au-GelMA hydrogels were measured and proved to be bio-compatible. Au-GelMA nanocomposite material hydrogels provided better environment and significantly triggered cellular viability and growth compared to simple GelMA hydrogels without AuNPs (control). Both B AgNPs and C AgNPs played role in the transformation of recalcitrant aromatic compounds i.e. azo dyes (100 ppm concentration) Acid red 151 and Orange II when treated separately and in combination with fungus (A. niger) under shaking conditions (150 rpm) at 30 oC. UV-visible spectroscopy and FTIR determined 75-95 % reduction or transformation of dyes in 96 to 120 hr reaction time. It was maximum with A. niger + B AgNPs, followed by B AgNPs & C AgNPs and least in case of A. niger. Finally, the anti-biofouling ability of the AgNPs was determined in ultra- filtration polysulfone (Psf) membranes by incubating them with sludge for 45 days. Scanning electron microscopy exhibited considerably less biofilm development on AgNPs incorporated Psf membrane than normal Psf membrane used as control. Moreover, these results were supported by less bacterial growth (36: 288 CFU) and corresponding variations in FTIR spectra of the biofilm covered Psf membranes.