# Biological Control of Phototrophic Biofilms in a Show Cave: The Case of Nerja Cave
## 1 Introduction  
Traditionally, caves have been considered as extreme environments characterized by low nutrient inputs. Although this may be possible in a few caves, most of the show caves cannot be considered extreme or oligotrophic environments because natural factors are often modified due to different anthropogenic impacts, being one of the most evident the installation of electrical lighting to facilitate visits. In fact, when caves are illuminated by natural or artificial lighting, phototrophic microorganisms become predominant. Under these conditions, complex communities develop on speleothems, walls and ground, mostly composed of cyanobacteria and algae, heterotrophic bacteria and fungi, and other eukaryotic organisms embedded in a matrix of extracellular polymeric substances. In further steps, mosses, ferns and other plants may also colonize caves. Examples of these communities have been studied in artificially illuminated show caves and catacombs, because their unconstrained growth can induce damage on valuable archeological or natural rock surfaces [1; 2; 3].  
The biofilms cause aesthetic, physical and chemical damage [4; 5], subject of great concern in general, and in particular in cases of caves with rock art paintings [6]. In addition, this invasion of foreign microorganisms induces great changes on the autochthonous microbial population, adapted to minimal inputs of C and N, and on the cave ecology.  
A wide array of methods has been proposed to control and/or eliminate phototrophic biofilms in show caves, including physical, chemical and biological procedures. Physical methods comprise:(i) the mechanical removal of biofilms by brushing and subsequent washing with pressurized water; (ii) the reduction of light intensity, and (iii) the use of ultraviolet radiation. Chemical methods include the use of sodium and calcium hypochlorite, hydrogen peroxide or commercial biocides. A detailed discussion of pros and cons were presented by [PERSON] and [PERSON] [7] and [PERSON] et al. [8]. Some of these methods were tested in _Cuewa del Tesoro_, a limestone cave in Rincon de la Victoria, Spain, depicting abundant biofilms coating the cave walls and speleothems [1]. The most effective treatments were sodium hypochlorite and hydrogen peroxide, but for the final cleaning hydrogen peroxide was adopted due to the absence of chemical residues on the rock [1].  
Yet, there is no general consensus on the type of biological methods to be applied in cleaning processes. [PERSON] et al. [2] suggested the use of quorum-sensing molecules and siderophores, whereas [PERSON] and [PERSON] [7] proposed biological antagonists like genetically modified viruses. However, to our knowledge field-based experiments have not been conducted thus far.  
Trophic relationships among components in phototrophic biofilms can be deduced by analyzing their composition; however, they have rarely been investigated in detail. Usually, most reports rely on a list of cyanobacteria and algae on speleothems but without establishing the trophic interactions among the different biofilm components [9; 10; 11; 12]. Biofilms can suffer a loss of cells through different processes: grazing, erosion, abrasion, and sloughing [13]. The most relevant mechanisms to cave biofilms are likely grazing and sloughing, the first by predation of bacteria and protozoa, and the second by periodic loss of large patches of biofilms by drying processes.  
By adopting advanced high throughput sequencing techniques, we aimed at investigating the composition of phototrophic biofilms within the Nerja Cave to highlight the potential role of natural biological control. To achieve this aim, we compared the composition of biofilms showing extensive detachments and/or rounded areas without photosynthetic microorganisms, with homogeneous biofilms on two speleothems, in order to understand the mechanisms involved in the process of detachment.