(656b) Potential Analysis for Biosynthesis of Copper Nanoparticles Using Fungus Hypocrea Lixii Cell Extract

 1.   Introduction

            Nowadays, there is a growing interest in the production of metal nanoparticles due to their numerous applications. Hence, a vast number of production methods have been developed and in the spotlight are biotechnological processes. They have been studied as an alternative to traditional processes in order to eliminate the use of components and to develop a cheap but clean and sustainable technology.

             The use of viable fungi biomass for the synthesis of nanoparticles is very common and the majority of studies explore the formation of gold and silver nanoparticles. A growing number of them describe the use of cell filtrate for the production of silver nanoparticles, indicating that the synthesis mechanism may be linked to molecules produced during cell growth.

            The vast majority of fungi used in studies with cell extracts are pathogens to plants and/or humans, which adds difficulties related to handling and discharge. Thus it is necessary to search for non-pathogenic alternatives that can be used in similar processes.

2.   Materials and Methods

            The fungus Hypocrea lixii used in the present work was kindly provided by the ICB – USP institute, through Dr. Márcia R. Salvadori. Fungus cells were grown in Erlenmeyer flasks of 500mL with 100mL of autoclaved Difco^TM Potato Dextrose medium. The flasks were incubated at 25°C and 150rpm for 120 hours. The cells were harvested, filtered using filter paper of 80g/m²and washed with double deionized water until no signs of culture medium was observed.

            After that procedure viable cells (V) were obtained. To obtain non-viable (NV) cells, an appropriate amount of viable cells was autoclaved at 121°C for 30 minutes.

            In order to obtain cells extracts, viable and non-viable cells were suspended in 100mL of double deionized water in 500 mL Erlenmeyer flasks and kept in shakers at the growth conditions for 24 hours. The cells were separated from the extracts by filtration using paper filters. Cell concentration was determined before suspension in water.

            The procedure used for nanoparticle synthesis consisted in adding an appropriate amount of CuCl₂(J. T. Baker®) solution to a sample contained in a falcon tube of 50mL. Samples were stored in fridges at 4°C.

             The identification and characterization of nanoparticle samples was performed using TEM imaging (Zeiss EM 900), spectrophotometry analysis (Shimadzu© UV-2600) with 0.5 nm resolution and working range from 190 to 800 nm and X-ray diffraction (Rigaku minicolection 300). In order to verify the interactions between samples and copper (II) ions mass spectrometry (Bruker© Ultraflextreme MALDI-TOF) and FTIR (Shimadzu© IRPrestige-21) with working range from 750 to 4000 cm^-1were used.

3.   Results and discussion

            In order to determine the potential use of fungus Hypocrea lixii cell extract for the production of copper nanoparticles, three methodologies were investigated. Due to evidence presented in recent studies, the following hypotheses were formulated: nanoparticle formation is a result of cell wall structure; nanoparticle formation is a result of molecules secreted by the fungus during cell growth; a combination of the preview. 

            The first approach consisted in investigating the interaction between cell extracts and copper (II) ions. The UV-Vis spectra of extracts with and without copper (II) ions were recorded. Viable cell extract and non-viable cell extract showed peaks in 255nm and 390nm (V) – 400nm (NV). After adding copper (II) ions, the peaks in 255nm were intensified and the peaks in the 400nm region were dislocated.

             The mass spectrometry analysis indicates that the non-viable cell extract has the highest protein concentration. Data obtained show significant interaction between cell extracts and copper (II) ions. Extracts were analyzed using X-ray diffraction to verify the presence of metallic copper and copper oxides as indicative of nanoparticle formation.

            The second approach was to use samples of viable and non-viable cells prior to suspension in water. The goal was to assess the hypothesis that is both the cell wall structure and molecules secreted by the fungus are the agents in nanoparticle synthesis. Through TEM imaging it was possible to observe the formation of nanoparticles with average diameter of 20nm in non-viable cells samples after only 1 hour of contact with CuCl₂ solution. This corroborates previous findings on the capacity of dead H. lixiibiomass of synthesizing metallic copper nanoparticles [1].  The particles were located outside and inside the cell wall.

            The third approach consisted in using viable and non-viable cells after extract removal to assess whether the cell wall structure alone is responsible for nanoparticle synthesis.

4.    Acknowledgments

                We thank FAPESP and CNPq for supporting this project.

5.   Bibliography

[1]      M. R. Salvadori, L. F. Lepre, R. a. Ando, C. a. Oller do Nascimento, and B. Corrêa, “Biosynthesis and Uptake of Copper Nanoparticles by Dead Biomass of Hypocrea lixii Isolated from the Metal Mine in the Brazilian Amazon Region,” PLoS One, vol. 8, no. 11, p. e80519, Nov. 2013.