Biofabrication and Applications of Metal Nanoparticles from Indigenous Fungi

Abstract

Hypothesis: The innovative field of nanoscience, has remarkable ability to influence various fields of humans’ lives. The fusion of microbiology, biotechnology and nanoscience has given birth to the field of bionanoscience which has enabled scientists to explore the potential of microbial processes and systems to fabricate novel nanostructures of biological importance. Use of biological procedures, eliminate the use of expensive chemicals, intensive energy use and are eco-friendly in nature as compared to the chemical and physical approaches. Nanotechnology today offers new approaches by altering the physical and chemical properties of various types of materials to create effective antimicrobial products. Applications of metal nanoparticles especially silver nanoparticles are wide in range from bioremediation, biosensing to biomedicine. Fungi belong to versatile group of microorganisms which can withstand adverse life circumstances. Among different biological routes, fungus-mediated green approach of synthesis of nanomaterials includes advantages of fast growth, high amounts of extracellular biomolecules, easy processing, economic viability and easy biomass handling. Experiments: This study involves the use of indigenous fungi for synthesis of mycogenic silver nanoparticles (Ag NPmyc) and to explore the mechanisms of this synthesis (reducing and capping agents). For this purpose, in the first part, fungi were isolated from different soils, their silver tolerance and extracellular silver reduction potential was determined. In second part, thermophilic fungus Thermomyces lanuginosus STm was used to synthesize Ag NPmyc. The size and shape of Ag NPmyc were characterized using ultraviolet-visible spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction techniques. The most significant process variables affecting mycosynthesis of Ag NPmyc were screened using statistical experimental design of Placket Berman model. The mechanism of mycosynthesis and capping on mycogenic nanoparticles was explored using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Liquid chromatography–mass spectrometry (LC-MS/MS). In third part, Ag NPmyc were synthesized using Aspergillus flavus SZ2 extracellularly and role of nitrate reductase (NR) in synthesis of mycogenic silver nanoparticles was determined. Nitrate reductase was purifed and characterized. In fourth part, Ag NPmyc were synthesize using Aspergillus oryzae SZ1, the organismic level acute cytotoxicity effect of Ag NPmyc was determined against brine shrimps. The potent synergistic effect of Ag NPmyc along with antifungal fluconazole on planktonic growth and biofilm formation on six fluconazole resistant clinical isolates of Candida species (C. albicans, C. galabrata, C. parapsilosis, C. krusie, C. tropicalis, C. albicans ATCC 24433) was investigated. The combined effect of fluconazole and Ag NPmyc on exopolymeric substance (EPS) and secretion of secreted aspartyl proteinases (SAP) of planktonic and biofilm matrix composition of six Candida sp. was investigated. In fifth part, Ag NPmyc were synthesized using silver tolerant oleaginous fungus Mucor circinelloides SZ3, biocompatibility of Ag NPmyc was studied through determination of antioxidant potential and the effect of different capping agents on stability and antibacterial potential of Ag NPmyc was observed. Findings: In the first part, four indigenous fungi were selected based upon their ability to tolerate high concentration of silver ions. These fungi included Aspergillus oryzae SZ1, Aspergillus flavus SZ2, Mucor circinelloides SZ3, and thermophilic fungus Thermomyces lanuginosus STm. The minimum inhibitory concentration in case of silver tolerance was found to be 2000 mg/l in case of A. oryzae SZ1, A. flavus SZ2 and T. lanuginosus whereas in case of M. circinelloides SZ3 wasn’t able to grow beyond 1000mg/l. The characteristic spectra of surface plasmon resonance (SPR) for silver reduction was observed as 405 nm for M. circinelloides, 430 nm for A. oryzae, 450 nm for A. flavus and 410 nm in case of T. lanuginosus. The optimum growth conditions: M. circinelloides pH 5, for A. oryzae pH 7, for A. flavus pH 7 and for T. lanuginosus pH 5. In case of temperature, the optimum values were found to be 20 ºC for M. circinelloides, 30 ºC for A. oryzae and A. flavus and 50 ºC for T. DRSML QAU xvi lanuginosus. After 18S sequencing, the fungi were given accession numbers from NCBI: A. oryzae SZ1 (MH664050), A. flavus SZ2 (MH664051) and M. circinelloides SZ3 (MH664052). In second part, glucoamylase protein (67 kDa) secreted extracellularly by T. lanuginosus STm was discovered to be the capping protein surrounding Ag NPmyc. Ag NPmyc against brine shrimps, were found to be less toxic as compared to ionic form of silver. In third part, the size and morphology Ag NPmyc synthesized using Aspergillus flavus SZ2 as determined by TEM, was found to be spherical shaped with particle size range of 1 to 70 nm. Inhibition in NR activity along with silver reduction by sodium azide suggested the involvement of NR in Ag NPmyc production. Positive correlation (R = 0.855) between specific activity of NR (U/mg) and Ag NPmyc optical density at 450 nm, indicating the involvement of NR in the mycosynthesis of Ag NPmyc by reducing silver nitrate. Specific activity of NR was improved from 15.54 to 17.77 U/mg after Sephadex-100 gel filtration. The Km and Vmax of NR were calculated as 13.18 mg/ml and 0.07 U/ml (μmol/ml/min). The size of the NR gene was found to be 2604 kb and two protein bands of 70 and 45 kDa showed its dimer nature. In fourth part, Ag NPmyc synthesized using Aspergillus oryzae SZ1 were found to be non cytotoxic to brine shrimps. The results showed that combinational use of Ag NPmyc with FLC exhibited strong in vitro antifungal synergy (FICI values ranged from 0.2812 to 0.375) against the majority of the Candida strains tested with gradual decrease in CFU till 12 hours. In case of percentage inhibition in biofilm metabolic activity (XTT) of six Candida species, >60% reduction of metabolic activity was observed in different Candida sp. at 16 μg/ml FLC in combination with Ag NPmyc. The EPS inhibition (%) in planktonic cells ranged from 2 to 73, 3–82 and 1–19 by 15 ppm Ag NPmyc/FLC, 25 ppm Ag NPmyc/FLC and FLC alone, respectively. Whereas in case of biofilm forms, EPS inhibition (%) ranged from 1 to 84, 1–93 and 1–32 by 15 ppm Ag NPmyc/FLC, 25 ppm Ag NPmyc/FLC and FLC alone, respectively. The results obtained, showed that combinations of 15 ppm and 25 ppm Ag NPmyc with FLC effectively inhibited the SAP in Candida spp. with an inhibition range of 1–82% and 1–79%, respectively in case of planktonic cells. Whereas 1–78% and 2–92% in case of biofilm cells as compared to less inhibition (<20%) seen in case of FLC treated Candida spp. planktonic and biofilm cells. In fifth part, Ag NPmyc synthesized using oleaginous fungus Mucor circinelloides SZ3 were found to be spherical and triangular shaped with particle size range of 10 to 50 nm. The FTIR spectra revealed the peaks for amide I, amide II, lipids, chitin/chitosan, and polyphosphate. The antioxidant activity of silver nanoparticles at different concentrations time intervals was studied and found that 100 μl sample of 5 ppm concentration showed a very good free radical (DPPH) scavenging activity with time. Tween 80 and CTAB gave highest stability as determined by high intensity peaks of surface plasmon resonance and highest antibacterial activity. Conclusions: The study concludes that biological methods involving fungi give an economical and simple step approach towards synthesizing silver nanoparticles. It is interesting to note that in case of Ag NPmyc synthesized by extracellular titre of thermophilic fungus, the adsorbed thermostable protein glucoamylase can possibly help in resuming activity of Ag NPmyc at high industrial temperatures. Current study revealed a new antifungal potential of mycogenic silver nanoparticles (Ag NPmyc) via synergistic combination for anti virulence against multi drug resistant pathogens. Synergistic combined drug therapy along with multi-target strategy, potentially reduce the usage dose of drug thus lowering its toxicity and resistance against it thus increasing the drug-efficacy. Key words: Ag NPmyc, Thermomyces lanuginosus, Aspergillus oryzae, oleaginous fungus Mucor circinelloides, glucoamylase, nitrate reductase, aspartyl proteinase enzyme (SAP)