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?
In response to my earlier query about carbon-coated surfaces permitting microbial proliferation, the authors might suggest that coating was incomplete or that filaments grew through defects. Yet no evidence of coating cracks or unevenness is provided in the SEM images or methods.
More critically:
If the 50 nm carbon layer was continuous, any filament growth after coating would have to occur on top of the carbon, which should appear as raised, non-embedded structures in secondary-electron images, but the presented micrographs (e.g., Fig 1b–g) consistently show filaments appearing integrated with the substrate surface, not atop a surface layer.
Could the authors clarify:
How was coating uniformity verified? Were cross-sectional FIB-SEM or EDS line scans performed to confirm continuous coverage, especially in cavities where filaments were observed?
If coating was uniform, how can the observed post-coating increase in filament counts be reconciled with the expected barrier properties of carbon films? Is it possible that many of the “filaments” are instead electron-beam-induced hydrocarbon contamination artifacts, which can accumulate during prolonged SEM sessions, especially in carbon-rich regions?
Were control experiments performed in which a similar carbon-coated abiotic substrate was exposed to the same lab environment and SEM schedule to rule out non-biological filament formation?
Given that carbon coating is routine in meteoritics precisely to prevent contamination and charging, the reported dynamic biological activity post-coating challenges standard sample-preparation assumptions. Without direct evidence of coating permeability or defect mapping, the interpretation of “growing microbes” remains problematic.