# Biological Control of Phototrophic Biofilms in a Show Cave: The Case of Nerja Cave
## 4 Discussion  
Phototrophic microorganisms, such as cyanobacteria and algae, are usually found on speleothems, rocks and walls washed by percolation water or in the vicinity of light sources. The development of cyanobacteria and algae, which can be considered as primary colonizers, is usually influenced by the availability of light, water and dissolved mineral elements. The development of a photosynthetic-based biofilm may lead to the formation of a more complex ecosystem, because phototrophic microorganisms release extracellular compounds, which favor the establishment of heterotrophic bacteria. In later phases of the community, fungi, protozoa and metazoans will appear, giving rise to a complex community with important trophic interactions. The formation of plaques or spots where the detachment of phototrophic microorganisms was evident, as found in Nerja Cave, can be assumed on the basis of processes of predation of some microorganisms on others. The samples labeled B and C in the two biofilms analyzed presented detachments indicative of some type of action (lysis, predation, etc.) on the cyanobacteria with respect to the intact biofilm (labeled A). It is interesting to note that while the cyanobacterium _Spirulina_ reached a remarkable abundance in sample 1A, it disappeared in sample 1C (complete detachment) and its concentration decreased six times in sample 1B (intermediary phase) with respect to 1A. This would indicate that the population of _Spirulina_ has been predated by other microorganisms. Other groups and genera of cyanobacteria are minority in sample 1 (_Desertifilum_, _Chroococcidiopsaceae_ and uncultured members of _Cyanophorecea_) and disappeared in the detached area (sample 1C). The cyanobacterial composition of sample 2 is completely different. In this case, the most abundant cyanobacterium was not _Spirulina_, absent in these biofilms, but unidentified members of the class _Cyanophorecea_. Comparing the retrieved sequences with the NCBI database, we found that these unidentified members were related, although with a low percentage of similarity (87%) with _Loriellopsis cavernicola_. In sampling site 2, the detachment or loss of the biofilm occupies large areas, suggesting that the process of predation was much more pronounced. This is compatible with the high abundance of eukaryotes. The richness of _Bacillus_ in sample 1B was remarkable, although it was also present in considerable abundance in all other samples. Members of the phylum _Frimicutes_, and in particular the genus _Bacillus_, are well represented in caves [18; 19]. The presence of _Bacillus_ in Nerja Cave biofilms can be due to: (i) species of the genus _Bacillus_ (_B. cereus_, _B. mycoides_) can lyse different species of cyanobacteria [20; 21; 22; 23], and contributed to their disappearance (detached areas); or (ii) the periodic processes of humidification and desiccation suffered by the phototrophic biofilms. A similar process has been described by [PERSON] et al. [23] for biological soil crusts dominated by cyanobacteria. In this case, the communities are well adapted to survive desiccation and quickly take advantage of rainfall periods, producing a bloom of _Frimicutes_, mainly of the order _Bacillus_ (_Bacillus_ spp.). This does not preclude a lytic activity. The existence of a high number of _Dadabacteria_ sequences in sample 1C is an unprecedented fact and reflects the poor existing knowledge about cave microbial ecology, and the microbial strategies necessary to growth and proliferate in cave conditions. _Dadabacteria_ is a recently proposed candidate phylum, previously known as CSP1-2, and rarely present in environmental samples. These aerobic bacteria have been identified through metagenomic analysis [24]. The genome encodes glycolysis, a complete Krebs cycle and an electron transport chain for oxidative phosphorylation that includes a cytochrome c oxidase and possibly intervention in the nitrogen and sulfur cycles [24].