General technical aspects
On natural, rough and heterogenous surfaces, effective and true comparison of process related changes is by observing the same area through the stages of the process. This means that surface imaging of such non-conductive surfaces in original state, after treatment and later after exposure must be possible at sufficiently resolved quality without applying coatings for imaging purposes. Using the variable pressure mode (VP-SEM) at 30–50 Pa in combination with the BSE detector requires settings, that do not deliver the sensitivity required to image thin and organic structures with sufficient quality. A significant disadvantage is the necessity of an aperture between chamber and gun to account for the variable pressure and as such reducing the field of view and signal intensity. Beam deceleration, on the other hand, is performed in high vacuum mode and thus does not require the reducing aperture in the beam path. The weak landing energy by using beam deceleration results in strongly reduced penetration depth and beam spot size, leading to much improved surface sensitivity and surface definition of very thin organic structures such as cell membranes (Fig. 2), while still giving some density contrast of thin, organic materials sitting on top of inorganic substrates (Figs. 3 and 4) using a backscattered electron detector. While a backscattered electron detector generally suffers from very weak signal efficiency at low kV, it is less affected by charging than a secondary electron detector. Combined with the beam deceleration accessory, signal efficiency and contrast of the backscattered image are significantly improved. Thus, the backscattered electron signal delivers an image within reasonable acquisition times (1 to 3 min. per focus level) and a density contrast even at low landing energies, giving the advantage of recognizing, e.g., very thin organic biofilms, bacteria and newly formed microbial calcite on non-coated rock samples (Figs. 4 and 5). Since there is no need for a coating with carbon or gold to achieve stable SEM images with this strategy, it is possible to observe the identical field of view at various stages of interest, be it days, weeks, months or years apart (Figs. 5, 6, and 7). Limitation in the depth of focus is successfully addressed by acquiring multiple images at sequential focal levels and compiling them to a multifocal image.
Imaging stages of artificially induced microbial calcite formation and weathering
The sequential images achieved herewith at the identical location on various test samples give a different insight to the changes induced by the treatment. Imaging at the early stages after treatment and conditioning clearly shows the initial formation of a biofilm of extracellular polymeric substance (EPS), more or less encapsulating large numbers of bacteria emerging from the depth of the porous rock to the surface (Fig. 5a). Imaging the identical location 3 months later upon outdoor weathering (Fig. 5b) unveils complete retreat of the EPS biofilm and bacteria, and the emergence of a newly formed microbial calcite crust. While this crust is reconnecting neighboring grains, it does not–visually-grow to a closed film that could clog the original surface. The open ‘karst’-like structure leads, however, to a considerable increase in surface area. Comparing the situation 13 months later (Fig. 5c) reveals that under the prevalent climatic conditions of the experiment, bacterial activity had ceased and weathering lead to renewed erosion of the microbial calcite at the exposed surface.
A new set of experiments (Figs. 6 and 7) replicating the initial series (Figs. 4 and 5) was run in order to include beam deceleration imaging of the pretreatment state as a reference with identical imaging settings. This was contemplated as necessary since pre-existing organic material or other biological growth (dark grey material in Fig. 6a1 and a2) may be present. The chosen substrate, KBYO nutrient application and procedure were identical. Figure 6 represents such a series with the clean, pretreatment state (a), versus the activated state after the treatment (b) with bacteria dispersed across the surface and initiation of microbial calcite deposition, as well as the situation 5 months later after exposure to outdoor weathering (c). With the intent to apply a consolidating treatment, Fig. 6b clearly demonstrates that the microbial activity does have the potential to reconnect grains with bio-calcite bridges, and that these may survive (Fig. 6c) the somewhat harsher climatic conditions prevalent in northern Switzerland. What these images further tell, however, is that there was no further activity upon completion of the conditioning period in this second series, which never reached the comparatively impressive productivity of the first series (Fig. 5). Putting this into perspective with the heterogeneous nature of the rock surface, the image series in Fig. 7 (compare images a3 versus b3 at arrow) demonstrates that the bacterial activity seems to occur in clusters, with some locations of increased microbial calcite production (b3) and other areas with only marginal activity (b2). At this stage, it is not clear which criteria lead to preferential sites for activity. Comparing Fig. 7b3 with Fig. 5b further unveils that slightly varying conditions may result in different morphologies, which in turn results in variable stability, when it comes to weathering and erosion (Fig. 7c vs. Fig. 5c).
The advantage of imaging identical locations through multiple stages of a process are obvious: There is no speculation on how to translate the situation from one sample to another. An image series that can be superimposed undoubtedly documents changes that have taken place, telling a comprehensible story. Characterizing the results of the presented case study on microbial calcite growth and weathering, it appears that a successful procedure involving KBYO may require prolonged treatment and conditioning to ensure sufficient microbial calcite build-up in this climatic region. As such, the process can be regarded as limited in the sense that it is nutrient and climatic conditioning controlled. Exposing the delicate, high surface morphology of the newly formed microbial calcite to local weathering conditions lead to rapid erosion at exposed surfaces (Figs. 5, 6, and 7). Nevertheless, the rather open structure, combined with bacterial mobility, constitute considerable advantages for potential sub-surface consolidation. In other words, the treatment is likely to cause minimal impediment on diffusive properties of the rock, while potentially reaching greater depths than purely chemical treatments. Direct documentation of the process in a similar way below the surface, i.e., in a depth profile or transect will require yet a different approach. Nevertheless, by training the eye what is being observed at the identical site through the process at the surface may be better translated to observations made on separate sections.