Biological Control of Phototrophic Biofilms in a Show Cave: The Case of Nerja Cave
, Yolanda del Rosal 2, Jose Luis Gonzalez-Pimentel 1,3, Bernardo Hermosin 1 and Cesareo Saiz-Jimenez 1,*Round 1
Reviewer 1 Report
Methods Methods for data analysis, calculations and statistical analyses are absent these need to be included.From a methodological point, The lack of reproduction in sampling, and sample size is an issue. You do not have enough reproduction of each type of sample to draw significant conclusions. From the materials and methods it seems that 2 different sites were sampled and each site had the same type of samples collected, why were these not pooled and used for statistical analysis to make meaningful conclusions about the communities for each type of sample? Scientifically, this makes a lot more sense than individually comparing single samples which cannot be subjected to any real statistical tests to draw scientific conclusions.
Table 1. the heading is not informative enough, there needs to be more description as to the nature of the table contents.
Figure 2. This figure is unreadable, please improve quality of image, as well the description is not informative enough. What type of diversity measure are you using? how did you cluster the communities e.g. what is the tree based on what are the similarity measures between each branch. This figure does not have nearly enough information included with it to independently assess what it is showing. Please revise. ** please look at my comments from Figure 2 and extend this to include the other figures. None of the figure titles and descriptions are complete enough, they are not informative enough to interpret the contents of the figure. Please revise.
Furthermore, the use of the term diversity to me means community level diversity, this is community composition, I would recommend changing the titles of these figures to reflect that. Results
The results are far too extensive, there is no need for that much text and description, the authors need to focus on the important similarities and differences between the different communities and samples and not describe every finding in detail as it is repetitive from the figures and not necessary. Table 2. title is misleading, these are relative abundances, not sequence abundances. Please revise. Discussion
The discussion is too long and unfocused. The authors need to condense the discussion to focus on their main objective in this study and discuss how their findings contribute to answering that question. A significant portion of the discussion is just a repetition of the results, or a discussion of results that are not relevant to the overall question posed in the study.
Author Response
Please, see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
The authors of the manuscript “Biological Control of Phototrophic Biofilms in a Show Cave: The Case of Nerja Cave” investigated the potential of biological control for limiting the growth of phototrophic biofilms in show caves. The presence of phototrophic organisms within caves is a collateral effect of the installation of artificial lights in caves open to the public. This represents a threat to the subterranean environment as the growth of these organisms causes aesthetic, physical and chemical impacts on speleothems. In addition, the increased amount of organic matter within the oligotrophic cave environment can have repercussion on the entire trophic food web. Controlling the growth of phototrophic biofilms in show caves is thus a timely topic in the subterranean biology agenda. In particular, the authors of this manuscript investigate an extremely new aspect within this study area, namely the biological control of phototrophic bacteria through predation from other organisms. This methodology is quite innovative compared to the classic physical and chemical methods proposed to remove phototrophic biofilms from speleothems. However, the methodology here presented and the flow of the manuscript presents some pitfalls that should be solved before the paper can be accepted for publication on Applied Sciences.
First, I noticed an extreme disproportion in the text length among the different paragraphs. The Introduction and Methods sections are too short and not informative compared to the extremely (even too much) long Results and Discussion sections. I would suggest to the authors to improve the Introduction and Methods sections by providing information that is now missing while condensing the text in the other sections (see my specific comments for each section below).
Second, I have some concern about the dataset. The authors examined only two speleothems where they identified three treatments but I am doubting whether this could be representative of the entire situation within the cave or whether it can be exported to other realities. In addition this low number of samples hampers the possibility to perform statistical analyses that could have been highlighted significant differences among the three treatments. This would have strengthened the conclusions of the authors that are now extremely speculative. The best option would be to increment the number of samples, but I acknowledge that this is not always so easy both for the sampling effort and the costs related to the genomic analyses. If this is not possible, I would then suggest to the authors to soften their text by saying that this is a first attempt to investigate the potentialities of the biological control of phototrophic biofilms but that it should be confirmed by future studies with larger samples.
Please, see below my specific comments for each section.
ABSTRACT
Well written and intriguing for the reader.
INTRODUCTION
I would improve this section by including some pieces of information that are now missing. For instance, from line 23 to 28 I stress more that the installation of artificial lighting is the main responsible of the growth of phototrophic organisms alien to the cave. Then I would specify that they cause physical, chemical aesthetic damage as well as alterations to the trophic chains and that they should then be controlled to avoid this. From line 31 to 34 I would discuss that multiple methods have been proposed to control the growth of phototrophic biofilms before introducing how the biological control can occur. From line 35 to 38 I would specify that these trophic relations among components of the biofilms can be deduced by analyzing their composition before specify that, however, phototrophic biofilms have rarely been investigated in detail. I would then conclude the paragraph by stating the aim of the work, that I think should be rewritten into something like “By adopting advanced high throughout sequencing techniques, we here aimed at investigating the composition of photrophic biofilms within the Nerja show cave (Spain) to highlight the potential role of biological control. To achieve this aim we compared the composition in biofilms showing extensive detachments and/or rounded areas without photosynthetic microorganisms with normally developed biofilms on two speleothems.”
MATERIALS AND METHODS
Also in this part, several piece of information are missing. From line 55 to 58 you should include how you collected the biofilm, how you preserved it until the genomic analysis, where the speleothems are located within the cave (next to the entrance or deep in the cave? Are they close to each other? How far is the closest artificial light?) and when these samples were collected (which year? Were they collected in the same sampling occasion? Did you performed multiple sampling occasions and then pooled the data together?). Moreover, measures some abiotic parameters should also be provided, such as illuminance, temperature and relative humidity on the speleothems, in order to help a better interpretation of results. I would also state here that you compared two different situations as in the sample 2 the detachment or loss of the biofilm occupies larger areas than in sample 1. This emerges only in the discussion but it is an extremely important information in the description of the sampling design.
RESULTS
Several data are presented twice, both in the text and in the figures. I would report in the text only the most important aspects of the results, while the rest can be interpreted by the reader from the pictures. For instance, I would remove lines from 125 to 132 as they do not provide any additional information compared to what can be deduced from Fig. 2. Concerning the following lines, I would only describe in detail the most abundant groups or the most important groups, without focusing on each group. Also, I would clearly distinguish the groups that include phototrophic organisms, to be described in a first part, and the heterotrophic, and thus potential predator groups, to be described later in the text. I would separate them also in the pictures. You can for example surround the phototrophic organisms with green boxes to separate them from the others. This would greatly help the reader that is not expert in the bacterial ecology (for instance, I know that cyanobacteria are phototrophic but I do not know anything about the other groups) in following the description of the results. Lines from 269 to 284 are the most informative ones of this section. I would further improve them by specifying whether there are differences among the three treatments across speleothems, i.e. whether the pairs of samples 1A and 2A, 1B and 2B, and 1C and 2C share some similarities. Please, note that you cannot talk about significant differences as you did at line 269 if you could not perform any statistical test.
DISCUSSION
In the discussion, the authors progressively focus on each bacterial group mixing together their own considerations with literature data without providing a clear organization to the text. I would rewrite this section by providing a better structure, in particular stressing the differences among the three treatments and the two speleothems: which groups are more present in the detached areas that can be indicative of predation processes? Which groups are present in sample 2 and not in sample 1 that can explain the larger detached areas? Of course, while discussing these aspects, considerations about taxonomic peculiarities can also be included, but I would not discuss each group one by one. In addition, I would clearly state at the beginning or at the end that the results here presented are just a preliminary investigation that should be corroborated by further works in the future.
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript by Jurado et al investigates biofilm samples from a show cave in Spain. Samples were taken from different spots, which represent different states of colonization and predation. Results were extensively discussed and compared with literature. Finally it was concluded that natural predators could be used as biological control for biofilms that evolved due to touristic usage of the cave.
The introduction provides relevant background, research design is appropriate, results are clearly presented and extensively discussed and support the conclusion.
In my opinion this manuscript is ready for publication.
Author Response
No comments to this reviewer
Round 2
Reviewer 2 Report
The authors permformed a deep throughout revision of the text accomplishing to all requirements. They also provided a valid response to my main concern that was about the sampling design. I am quite satified with their revision and I would now recommend to accept their manuscript for publication in Applied Sciences.
Author Response
The manuscript has been revised, including English corrections.
The statement suggested by the Editor was included in the manuscript, lines 82-86.
