## 4 Discussion  
In this study, the antibacterial activity of GrNs on G and Ni substrates against _E.coli_ (Gram-negative) and _S. aureus_ (Gram-positive) was investigated. First, the antibacterial effect of CVD-grown graphene nanosheets on G was examined according to the number of layers (monolayer/1-2 layers/2-3 layers). For CVD-grown graphene nanosheets, as the number of layers of graphene nanosheets decreased, the cell viability (%) of _E. coli_ decreased from 100% to 51.4%. It is concluded that monolayer graphene nanosheets have a more antibacterial effect than multilayer graphene (MLGr) nanosheets. Monolayer graphene has different and unique properties compared to MLGr. It is also known that as the number of layers decreases, graphene shows many interesting and different properties. Furthermore, the antibacterial effect of CVD monolayer graphene nanosheet synthesized onto Ni substrate was studied. In the case of CVD graphene nanosheet synthesized onto Ni substrate, the cell viability (%) of _E. coli_ decreased from 100% to 38.18%. It is concluded that the antibacterial activity of CVD monolayer graphene nanosheet synthesized onto Ni substrate against _E. coli_ was better than that of CVD grown monolayer graphene nanosheet on G substrate. This result is thought to be due to the fact that the graphene grown on the Ni substrate contains many grain boundaries, and the graphene concentration increases at the grain boundaries [44]. It has been shown also that the cell viability (%) of _S. aureus_ was extremely weak (from 100% to 84.3%) for the samples grown on Ni substrate. Therefore, it may be concluded that the monolayer graphene nanosheets grown on the Ni substrate are not effective against Gram-positive _S. aureus_. We conclude that more studies are needed on the effects of transition metal substrates on Gram-positive bacteria. The antibacterial effect of Gr nanowalls grown on G substrates by the PECVD meth-od was also analyzed at 100, 150, and 200 W radio frequency (RF) powers. It was shown that \begin{table}
\begin{tabular}{c c c} \hline \hline
**Treatment** & _E. coli_ & _S. aureus_ \\ \hline Control & 100.00 \(\pm\) 0.00 a & 100.00 \(\pm\) 0 a \\ \hline CVD-1 & & \\ (CVD grown 2D-Monolayer Gr onto glass substrate) & 51.00 \(\pm\) 3 & 46.50 \(\pm\) 9 c \\ \hline CVD-2 & & \\ (CVD grown 2D-1-2 layers Gr onto glass substrate) & 61.50 \(\pm\) 18 & 75.00 \(\pm\) 0 b \\ \hline CVD-3 & & \\ (CVD grown 2D-2-3 layers Gr onto glass substrate) & 90.00 \(\pm\) 25 ab & 55.50 \(\pm\) 4 c \\ \hline PECVD-1 & & \\ (PECVD grown 3D-Gr-100 W onto glass substrate) & 99.50 \(\pm\) 25 a & 75.00 \(\pm\) 6 b \\ \hline PECVD-2 & & \\ (PECVD grown 3D-Gr-150 W onto glass substrate) & 28.50 \(\pm\) 13 c & 25.00 \(\pm\) 6 d \\ \hline PECVD-3 & & \\ (PECVD grown 3D-Gr-200 W onto glass substrate) & 25.50 \(\pm\) 9 c & 9.50 \(\pm\) 2 e \\ \hline CVD-1-Ni & & \\ (CVD grown 2D-Monolayer Gr onto Ni substrate) & 38.00 \(\pm\) 4 c & 84.50 \(\pm\) 5 b \\ \hline Sign & ** & ** \\ \hline \hline \end{tabular} a-e: means with different letters in the same column are significantly different (\(p\) < 0.05); Sign: Significance; \({}^{**}p\) < 0.01.  
\end{table} 
Table 1: Variance analysis of the effect of graphene treatments on the cell viability (%) of _E. coli_ and _S. aureus_. PECVD graphene nanowalls synthesized on G substrates, especially at 200 W, exhibited stronger antibacterial activity against _E. coli_ and _S. aureus_, and the cell viabilities of _E. coli_ and _S. aureus_ decreased from 100% to 25.19% and 100% to 9.02%, respectively. Gr nanowall (3D structure) morphology changes significantly with the presence of RF power. Gr nanowalls become more tightly packed with the increase of RF power. Raman spectroscopy revealed that Gr nanowalls were 3D oriented. It was concluded that 200 W RF power had a significant effect onto the tightly packed structure of these vertically oriented (3D) nanostructures.The high antibacterial activity against the two types of bacteria can reflect its potential in food preservation used as a food preservative.