# The Marine Bacterial Genus _Euzebya_ Is Distributed Worldwide in Terrestrial Environments: A Review
## 7 Attempts to Isolate _Euzeyba_ from Pindal Cave  
Pindal Cave is a shallow limestone cave formed through epigenic processes and located very close to the surface. The cave is 590 m long and due to the geographical location has a humid oceanic climate. The cave has a stable annual temperature (11.6 \({}^{\circ}\)C) with only minor fluctuations throughout the year (<2 \({}^{\circ}\)C/year). This cave is well-ventilated with relatively low annual average values of CO\({}_{2}\) (680 ppm) and radon (950 Bq/m\({}^{3}\)) [36]. In Pindal Cave, pink biofilms primarily develop on the surface of calcite speleothems in areas near the entrance and _Euzeybaceae_ reached a relative abundance of 7-16% (Table 4); the biofilms have a rough surface and are formed by aggregates of cells, mostly rounded, with extensive filaments (Figure 1). Other abundant genera were _Crosiella_ and wp1-P19. The ecological significance of the five top taxa in Pindal Cave was discussed elsewhere [36; 127; 128]. However, attempts to isolate _Euzebya_ using different culture media failed. The following media were used: nutrient agar (NA), B-4 medium [129], GYM _Streptomyces_ medium (DSMZ 65), Dimethylsulfone medium [130], TSA, diluted TSA/1000, and TSA supplemented with NaCl (3%) and MgSO4\(\cdot\)7H2O (2%) (DSMZ 1350) [125]. In all these media, the pH was near 7, and not as markedly alkaline as _Euzebya_ requires (pH 9-10), as denoted by their habitats; in other cases, the absence of relatively high NaCl concentrations likely prevented its isolation. \begin{table}
\begin{tabular}{l c c} \hline
**Genus** & **Sample Bal1** & **Sample Bal4** \\ \hline _Euzebyacae_ & 16 & 7 \\ \hline _Crosiella_ & 8 & 7 \\ \hline wb1-P19 & 5 & 11 \\ \hline PLTA13 & 4 & 5 \\ \hline _Nitrospina_ & 3 & 3 \\ \hline Other genera & 64 & 67 \\ \hline \end{tabular}
\end{table} 
Table 4: NGS relative abundances (%) of the top five taxa in samples from pink biofilms, Pindal Cave, Spain [36]. Figure 1: (**a**) Pink biofilms growing on calcite speleothems in Pindal Cave, Spain (red arrow). (**b–d**) Scanning electron microscopy microphotographs of pink biofilms from Pindal Cave. (**b**) General view of pink biofilms (red arrow). (**c,d**) Bacterial filaments forming the biofilm. A culture-dependent approach revealed 33 isolates representing 19 identified species, as listed in Table 5. The isolates were affiliated with the phyla _Actinomycetota_ (three genera) and _Bacilota_ (five genera). Both phyla are widespread in nature [131; 132; 133; 134] and the genera isolated from pink biofilms were spore-forming bacteria. Remarkably, none of the bacteria isolated were representatives of the top major taxa recovered by NGS (Table 4). In fact, NGS of the pink biofilm revealed only _Streptomyces_ with a relative abundance of 0.01%, which was also isolated on culture media. The results indicated significant differences in the bacterial communities detected in the pink biofilm when applying these two different approaches. The inability of culture-dependent and independent methods to detect the same bacterial species has already been reported [135; 136]. [PERSON] et al. [137] stated that culture-dependent techniques lead to an overestimation of the spore-forming bacteria, as shown in Table 5. They found that the apparent abundance of these bacteria can be explained by the fast growth of spores in the plates. The failure in obtaining _Euzehya_ isolates prompted us to review the ecological niches where _Euzehya_ can be found. The survey revealed that the environmental conditions requested by _Euzehya_ were not met in the culture media used thus far. Culture media reproducing the environmental conditions outlined in this review, e.g., marine agar and SN medium (including 1/10 dilutions of these media), pH 9-10, and sodium chloride concentrations around 3% or more, could allow the isolation of terrestrial _Euzehya_ and other haloalkaliphilic genera. Marine agar and SN medium [138] have been used for the isolation of marine _Euzehya_[14; 15; 16].