# Potential Use of Carrageenanans against the Limestone Proliferation of the Cyanobacterium
## 4 Discussion
essandensis_ need to be transferred to a new and fresh culture medium. Although additional studies are needed, this could potentially indicate that cell viability might be compromised upon exposure to these extracts. In our study, we were able to verify that the lithotype used was bioreceptive to the colonization and growth of the cyanobacterium _P. essandensis_, showing that even single species are able to successfully colonize stone surfaces. Visual inspection in the probes without treatment also demonstrated the capacity of _P. essandensis_ to grow epilithically, leading to undesirable aesthetic alterations due to its intense dark-green color. In regard to the colorimetric analysis, results showed that the application of carrageenan did not cause any color alteration to the surface of the stone in control replicas under dry environment (non-experimental). The \(\Delta\)E* value upon exposure to treatment was 1.10, similar to the reference value for stone replicas (Table 1). However, in control replicas under a humid environment, \(\Delta\)E* values in stone probes (in the presence or absence of carrageenanans treatment) were greater than 1. According to literature data, stone treatments should not cause \(\Delta\)E* variations greater than 4 or 5 [41; 42; 43]. We hypothesize that these differences resulted from the presence of water in the experimental conditions. Nonetheless, both \(\Delta\)E* values for non-experimental control probes with and without carrageenans were revealed to be similar, as expected. Other studies in this field have shown some color alterations on stone replicas after application of natural compounds (e.g., [11]). Colorimetric analysis is an important and reliable non-destructive method used in the cultural heritage field for monitoring biofilm growth on stone surfaces [44; 45]. In this regard, our results demonstrated that biofilm formation was enhanced in non-treated stone replicas as compared to carrageenanans-treated samples, highlighting their effectiveness in controlling the proliferation of _P. essandensis_. It is important to note that, ideally, biocides and other compounds should be tested on the same material as the studied monument, possibly colonized with the same naturally developed microbial community, in order to mimic real case scenarios [10; 46]. In addition, their possible interactions on the tested material should also be evaluated prior to application in situ, to avoid any negative effects to the stone surfaces [43]. Our experiments followed these guidelines, as we use stone replicas similar to the ones used in the construction of the Old Cathedral of Coimbra. Although the results presented here were evaluated on a short-term basis (after 4 months of incubation), they suggest that one sole application of the carrageenans extract may be sufficient to retard the proliferation of _P. essandensis_. In fact, preliminary results performed with one replicate for a period of 1 year without any additional biocide application showed that this effect persists (Supplementary Figure S1), which consolidates our theory that these compounds do not require frequent periodic applications to control these organisms. In addition, and even though these extracts were only tested in one organism, we hypothesize that they could also be used as a complement after cleaning, to help in retarding the growth of some microorganisms. Nevertheless, we are aware that the practical use of carrageenans in cultural heritage scenarios would still need further studies and procedures, as the European Biocidal Product Regulation(Regulation 528/2012) implies that the active substances used in biocidal products must undergo legal approval by the European Chemicals Agency (ECHA) before they can be put on the market, to ensure a high level of protection for human health and the environment. Although carrageenanns are generally regarded as safe and are used in a variety of industrial fields (e.g., food, cosmetics, pharmaceutical, etc.