## 5 Conclusions  
There is a wide variety of methods that can be used to unravel microbial diversity with culture-dependent (CD) or/and culture-independent (CI) approaches. Out of the 50 articles that were included in our review, 12 articles contained both approaches, showing the ever-growing effort of discovering the overall microbiome of the biodeteriorated cultural heritage assets. The trend of utilizing only CD methods has persisted rather constantly through the years, while the usage of CI has gained its popularity in the last 4 years. The microbial biodiversity of CHL is mostly studied in bacteria and fungi, however, phototrophic cyanobacteria and algae are also studied well, and even previously poorly studied archaea are becoming a point of interest in some recent studies on CHL. The authors of this review article believe that it is crucial to keep exploring microbial biodiversity, preferably with a combined CD and CI approach, not only with the intention of uncovering the taxa and microbial communities responsible for the biodeterioration processes, but also with the curiosity that builds up the exiting knowledge of taxonomy, since the efforts of studying the microbial diversity on CHL have even resulted in the discovery and description of three new species in three novel genera, one cyanobacterial and two fungal, which would have otherwise remained unnoticed. The carrying out of studies on the topic of biodeterioration on CHL using molecular approaches is still rather limited to certain countries, even though limestone is widespread worldwide as a common building and artistic material. Therefore, we encourage conservators and biologists worldwide to keep exploring this field on their own cultural heritage assets; moreover, as for future directions, we encourage further studies on microbial biodeterioration to use CD and CI methods hand-in-hand and couple them with carefully monitored environmental and substrate characteristics. With the growing popularity of building predictive models that integrate biology datasets with data mining statistical algorithms to predict future outcomes of certain phenomena, we believe that predictive models could also be used in the field of microbial biodeterioration in the future to provide novel insights and answer questions such as how different combinations of microbial taxa commonly found on CHL alternate their substrate under different environmental factors.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14167429/s1](https://www.mdpi.com/article/10.3390/app14167429/s1)]([https://www.mdpi.com/article/10.3390/app14167429/s1](https://www.mdpi.com/article/10.3390/app14167429/s1)). Table S1: Fungi detected on limestone cultural heritage assets using molecular methods in the literature; Table S2: Bacteria detected on limestone cultural heritage assets using molecular methods in the literature; Table S3: Cyanobacteria detected on limestone cultural heritage assets using molecular methods; Table S4: Algae detected on limestone cultural heritage assets using molecular methods; Table S5: Archaea detected on limestone cultural heritage using molecular methods; Table S6: Culture media for microorganisms used in biodeteriorated culture heritage limestone studies.  
[PERSON], [PERSON] and M.F.M. contributed to the conceptualization, investigation, writing, and editing; software: Microsoft Word Version 2407, Microsoft Excel Version 2407, Microsoft PowerPoint Version 2407, NCBI Taxonomy Browser, Data Wrapper; funding acquisition, H.S. All authors have read and agreed to the published version of the manuscript.  
This research was funded by The EUTOPIA Student Individual Research Project, grant number 803-3/2023, by enabling H.S. the mobility from the home Biotechnical faculty of University of Ljubljana to the host university FCT-Portuguese Foundation for Science and Technology.  
Not applicable.  
Data Availability Statement: Data are contained within the references or in the Supplementary Material.