# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
## 2 Cultivation Methods, the First Assessments of Cave Microbial Diversity
In addition, unsuitable lysis techniques (i.e., chemical, mechanical, enzymatic) can provide a biased view of the microbial community studied by (i) not efficiently lysing all microbial membranes and walls, and/or (ii) leading to DNA degradation for some microorganisms [39; 45; 46; 47]. Indeed, some lysis methods led to a reduced retrieval of Gram-positive bacteria compared with Gram-negative bacteria (due to the cellular composition of their membrane) [39; 47]. Finally, the presence of humic acids and/or salts co-extracted with environmental DNA that can inhibit enzymes used for library construction and/or sequencing is a shortcoming. These issues are even more critical in the case of meta-omic approaches (see Sections 4 and 5 below). In the 2000's, 16S rRNA gene cloning was largely applied to cave studies. One of earliest applications of molecular cloning in a cave was achieved in a sulfidic stream located in Parker Cave (Kentucky, USA), showing that bacterial clones were affiliated to phylotypes known to obtain energy for CO\({}_{2}\) fixation from the oxidation of inorganic compounds [48]. Cloning Sanger sequencing method was also used for the first characterization of the microbial environment of Lascaux Cave, where as many as 696 clones of bacterial 16S rRNA genes were retrieved from 11 samples in different galleries of the cave [49; 50]. Rarefaction analysis on these clones showed 90% clone coverage, indicating that a large part of the bacterial diversity was detected [49]. By focusing on Lascaux's fungal community on altered surfaces (black stains) colonized by arthropods and unaltered surfaces, 607 clones of 18S rRNA genes were also obtained [50]. Among these 607 clones, 8 of the 10 most abundant phylotypes (representing approximately 50% of the clones) corresponded to _Fungi_ known to associate with arthropods, such as _Cladospori classosporioides_, _Geomyces pannorum_, _Lecanicillium psalliidae_, etc. Other studies applied cloning sequencing to explore the microbiology of underground ferromanganese corrosion residues [51] or highly acidic caves [52]. The 16S rRNA analyses evidenced that clones associated with water drips in a highly acidic cave were affiliated to _Thibacilli_, which may obtain energy from the oxidation of sulfur compounds, or _Acidimicrobium ferrooxidans_, a sulfide-oxidizing bacterium [52]. In Lechuguilla Cave, sequence analysis of clones showed the presence of _Hyphomicobium_, _Pedomicrobium_, _Leptospirillum_, _Stentrorophomonas_, and _Pantoa_, which have known iron- and manganese-oxidizing/reducing bacteria among close relatives [51]. While providing the advantage to give long fragments (about 1500 bp for 16S rRNA genes), cloning sequencing is expensive and time consuming. In consequence, new sequencing methods have been developed to replace cloning sequencing techniques, allowing simpler library construction (without cloning), faster sequencing, and generating more data for more in-depth diversity analyses. These methods provide much more information,allowing scientists to document rarer taxa, and also benefit from improvements made in microbial taxonomy and in database quality.