Verma et al. Nanoscale Res Lett 2011, 6:16 http://www.nanoscalereslett.com/content/6/1/16 Page 6 of 7 Figure 7 AFM images of gold nanotriangles synthesized by the reduction of 1 mM HAuCl4 by endophytic A. clavatus after 48 h (a–b) and 72 h (c–d). Vertical stacking and clustering was observed with advancement of reaction period (a, c) within nanoparticles. Surface roughness of the nanoparticles was measured 25–30 nm (b, d). show surface roughness of about 30 nm (Figure 7b) and 25 nm (Figure 7d), interestingly stacking faults of nanotriangles was observed as they grow in size, which results in an over- all increase in the surface roughness. The Figure 7a, c also tested and verified the results already obtained in short incubated samples. The gold nanoparticles are not resolved into single crystals due to the vertical stacking of nanotrian- gles, and thus the measurement and estimation of size for single nanoparticle with AFM remains intricating. Although the exact mechanism of the synthesis of nanomaterials are not known, but it was observed that when fungal biomass was treated with 1 mM aqueous solution of HAuCl4, there are negligible amount of nanoparticles present in the solu- tion (Figure 2), the biomass instead changed into purple colour, this clearly indicates that the reduction in gold ions takes place intracellular (surface reduction). For getting some more insight into the exact mechanism of bioreduc- tion, a TEM analysis of fungal cells had been performed that are challenged with aqueous gold ions for 72 h. The thin sections of fungal cells shows the presence of plenty of triangular, hexagonal and spherical gold nanoparticles bound to the surface of the cells (Figure 8a–c); however, the triangular nanoparticles are relatively smaller in size than the spherical and other shapes. The presence of these shapes indicates that many particles are not transformed into triangle (Figure 8b) due to lack of nucleation or under- gone surface reorganization in such a way that they no longer exhibit the ideal triangular nanostructures. A lot of research in green synthesis of nanomaterials are currently focused on the mechanistic approach to define the mechan- ism involved in this process [22-24]. A huge number of microbial strains are screened for their potential of produ- cing metal nanoparticles; however, the mechanism of bio- synthesis has not been established yet. The metabolic convolutions of viable microorganisms are even more com- plicating the process of analysis and identification of active species in the nucleation and growth of metal nanoparticles. Many researchers, however, speculated about the role of secretary enzymes such as NADH-dependent reductase of microbial origin, which may be responsible for the reduc- tion in metal ions for growth and nucleation of nanoparti- cles [25,26]. However, the biochemical mechanism of metal ion reduction and the subsequent NP formation remain unexplored and need further research. Thus, thorough and in-depth understanding of the biochemical mechanisms associated with nanomaterials biosynthesis is needed. The analysis and identification of active constituents with HPLC and other analytical tools are required to solve the mystery of nucleation and growth of metal nanoparticles in the microbial system.