In the Materials and Methods section, it is stated that after polishing, the specimen was carbon-coated under vacuum (~50 nm thickness) before SEM observation. According to Table 1, this coating was applied on November 4 and 11, 2022. However, the results describe a dynamic change in the number of observed filaments after this date, specifically, an increase from 11 filaments (November 11) to 147 filaments (November 30), and then a decrease to 36 filaments (January 14).
My concern is: If the sample was carbon-coated, how could the microbial population continue to grow or change on the surface? A conductive carbon coat is typically applied to prevent charging during SEM and is considered a sealed, inert layer. If the filaments were on the surface and then coated, they should have been immobilized and fixed. Any subsequent growth or new colonization would presumably be prevented because:
The coat would act as a physical and diffusion barrier to moisture, nutrients, and gas exchange.
New cells or spores would need to settle on top of the carbon coat, which is not consistent with the images showing filaments embedded in or attached to the mineral surface. Could the authors please clarify:
1. Was the carbon coating applied uniformly and continuously over the entire observed surface?
2. If microbes were already present on the surface before coating, how did they remain viable or continue metabolizing under the coating?
3. Is it possible that the coating was incomplete, cracked, or locally absent, allowing continued microbial activity? If so, how was this assessed?