Bioprospection on different and less commonly studied environments allows us to analyse the microbial diversity and encounter microbes specialised in certain bioproducts, like metal nanoparticles. When compared with physical–chemical methods, the microbial biosynthesis of nanoparticles by microorganisms is faster, cheaper, more effective, and without the involvement of hazardous chemicals (Durán et al. 2016; Rahimi et al. 2016). In this study, an initial batch of 20 fungal strain, isolated from sugar cane plantation soil, was screened for its biogenic capacity of producing AgNP by reducing silver nitrate, and four fungal strains (Rhizopus arrhizus IPT1011, Rhizopus arrhizus IPT1013, Trichoderma gamsii IPT853, and Aspergillus niger IPT856) were detected to be capable of biosynthesizing AgNP. According to our measurements, the four selected strains were capable of extracellular biosynthesis of AgNP of uniform size and round-shaped, with diameters in the range of 30–100 nm. Extracellular secretion of enzymes by fungi allows to easily recover those enzymes, which in our study were then used for nanoparticles synthesis, turning this into an effortless biological method.
The exact mechanism of AgNP synthesis by fungi is not yet clearly known but previous studies have indicated that NADH and NADH-dependent nitrate reductases are important factors in the biosynthesis of metal nanoparticles (Ahmad et al. 2003; Hamedi et al. 2013). In the present study, the activity of nitrate reductase was measure for all 20 strains, capable and non-capable of biosynthesizing AgNP. The lack of activity in the cell filtrates for the strains that did not show reduction of silver nitrate supports the hypothesis of enzyme based biosynthesis.
Furthermore, the differences in intensity, shown in Fig. 4, are linked to the structuring process of the silver crystal. The more regular and larger the crystallite formed, the greater the intensity and the smaller the width of the Brag peaks. This structure of the crystals is dependent on the chemical environment, pressure, temperature and time; as these last three factors were constant for all the samples, we can then say that the chemical environment of the fungi was responsible for the differentiation of the structures obtained, which is linked to the Nitrate reductase activity profiles. This activity (Fig. 5), is very low in the initial moments for the strain IPT853 (Trichoderma gamsii), while for IPT1011 (Rhizopus arrhizus) it is more pronounced. We know that the initial moments of nucleation of the nanoparticles dictate the structure and stability of the crystal, and these differences are what caused the appearance of the oxides in other samples. However, all profiles detected by XRD reflect Ag, AgO, AgO2 and carbon particles, with no other contaminant species crystallized.
The AgNP produced by the Rhizopus arrhizus, Trichoderma gamsii and Aspergillus niger strains in this study were found to be active against E. coli, S. aureus, and P. aeruginosa. Multiple bactericidal mechanisms can act in synergy to confer a broad spectrum of activity against different types of bacteria. It is known that the antimicrobial activity of AgNP is due to the formation of insoluble compounds by inactivation of sulfhydryl groups in the cell wall and disruption of membrane bound enzymes and lipids resulting in cell lysis (Dorau et al. 2004). And it has also been reported that the process may involve the binding of AgNP to external proteins to create pores, interfering with DNA replication or forming reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anions, and hydroxyl radicals (Duncan 2011; Durán et al. 2016; Jung et al. 2008).
The AgNP of smaller dimensions in this study, produced by R. arrhizus, were shown to be the most efficient against the bacteria tested. In fact, several studies have shown that AgNP activity is strongly dependent on the NP size (Wu et al. 2014; Tamayo et al. 2014; Rahimi et al. 2016).
The correlation between the bactericidal effect and AgNP concentrations is bacterial class dependent (Chernousova and Epple 2013). Just like in previous studies (Zhang et al. 2014), E. coli was more affected by AgNP than P. aeruginosa and the inhibitory effect on the growth of S. aureus was less marked than in E. coli as previously found by Wu et al. (2014). This strengthens several previous investigations (Pal et al. 2007; Fayaz et al. 2010; Devi and Joshi 2014), that found that Gram-positive and -negative bacteria have different susceptibility to AgNP, probably due to differences in their membranes and cell walls (Feng et al. 2000).
The fact that bacterial resistance to elemental silver is extremely rare (Silver 2003) emphasizes the increased interest in using AgNP as potent antimicrobial agent in biomedical applications. The increase in publications on this topic, like our own research, will benefit future research and development of cost-effective metal nanoparticles production, with desirable therapeutic effects.
The presence of nitrate reductase in the supernatant supports the hypothesis of its strong influence on the mycogenic synthesis process. All the four biosynthesised AgNP were characterised and showed potential antimicrobial activity noted through growth inhibition of several bacterial species. Furthermore, we observed a direct relation between the concentration of AgNP and antimicrobial capacity.
Further analyses are needed to fully understand the mycogenic synthesis process and the mechanisms involved in AgNP production by these strains. Nevertheless, our study proves the importance of exploring more environments and analyse their microbial community to discover novel and/or better bio-products. This will have high impact on society and health, as the world is facing a massive increase of microbial resistance to most of the known and commercially available drugs and antibiotics.