# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
## 2 Cultivation Methods, the First Assessments of Cave Microbial Diversity
\begin{table}
\begin{tabular}{|c|c|c|c|} \hline
**Cultural Methods** & **Cloning Sequencing** & **Metabarcoding** & **Shotgun Metagenomics** \\ \hline \multicolumn{4}{|c|}{**Principle**} \\ \hline Microbial isolation followed by characterization & DNA extraction, PCR cloning and sequencing of a taxonomic marker & DNA extraction, PCR amplification and sequencing of a taxonomic marker & DNA extraction and PCR-free sequencing of all DNA fragments \\ \hline \multicolumn{4}{|c|}{**Scale**} \\ \hline Typically hundreds to thousands of colonies per study & Typically hundreds to thousands of clones per study & Typically 2000 to 70,000 sequences per marker per sample & Typically 2000 to 70,000 sequences per marker per sample \\ \hline \multicolumn{4}{|c|}{**Advantages and added value**} \\ \hline \multicolumn{4}{|c|}{} & Enables to document the rare \\ Isolates enable also to assess strain physiology & & biosphere, inexpensive, well-established \\ experimentally and to & No particular advantage now & bioinformatics tools, requires \\ perform genomics, & that NGS is available & less DNA than metagenomics, reference databases usually \\  & & well developed & available \\ \hline \multicolumn{4}{|c|}{**Drawbacks and limits**} \\ \hline \multicolumn{4}{|c|}{} & DNA extraction not effective & DNA extraction not effective \\ Requires culturability, tedious & DNA extraction not effective for certain taxa, PCR primers & DNA extraction not effective for certain taxa, PCR primers & for certain taxa, minor taxa \\ and low-throughput & not fully universal, tedious & not fully universal, amplicon \\ and low-throughput, & and low-throughput, & size limits taxonomic \\  & expensive Sanger sequencing & resolution & exhaustive, requires more \\  & & DNA than metabarcoding \\ \hline \end{tabular}
\end{table} 
Table 1: Comparison of methods to document the taxonomic diversity of microorganisms. 150 to 200 cultivation days to produce enough biomass for further analysis of phylogeny, antimicrobial activity screening, or scanning electron microscopy (SEM) [13]. In the absence of inhibitory chemicals such as antibiotics or toxins in the culture medium, the rapid growth of certain microorganisms often inhibits or delays the growth of the others, yielding a narrow range of microorganisms. This can be amplified by several other factors including niche and nutrient competition, production of secondary metabolites, and production of toxins by these fast growers [13; 17]. Some media only support the growth of fast-growing microorganisms while inhibiting slow-growing oligotrophic microorganisms due to nutritional shock [13; 17; 21]. It is important to note that most members of the archaeal domain in caves are still not cultivated [26]. The majority of cultivation studies include a second screening step performed either by microscopy or molecular methods [1; 19]. To go further with two _Streptomyces_ isolates obtained from Iron Curtain Cave (Canada) using two broth media (R2A and Hickey-Tresener), whole genome sequencing and metabolomic analyses showed a high number of biosynthetic gene clusters for secondary metabolites (polyketides and non-ribosomal peptides), thought to contribute to their fitness [27]. Work conducted on Lechuguilla Cave (New Mexico) allowed the identification of antibiotic resistance among the culturable microbiome and especially in _Streptomyces_ spp. and _Brachyacterium paraconglomeratum_[28]. This was consistent with another study of the walls of a pristine karstic cavity named the Yumugi river cave, where cultivated members of the _Pseudomonas_ genus were identified for their antibiotic resistance properties [29]. Using microscopy coupled with Fluorescent In Situ Hybridization (FISH), previous work identified the microbial structures and taxa composing yellow biofilms, especially _Actinomycetota_, in Pindal Cave (Spain) [30]. Others identified more diverse microbial members, such as _Bacteria_ and _Fungi_, as well as _Cyanobacteria_ and Algae (in relation to light exposition) in biofilm samples from Fornelle Cave (Italy) [31].