# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
## 2 Cultivation Methods, the First Assessments of Cave Microbial Diversity
Since effective cultivation requires the reproduction of both abiotic (i.e., pH, temperature, etc.) and biotic (i.e., synthrophy, symbiosis, etc.) conditions that microorganisms experience in situ [17], new methods of cultivation have emerged, such as the in situ cultivation of natural microorganisms using diffusion chambers or cultivation of soil microorganisms in soil extract agar medium [32; 33]. Microbial cultromics is also used. It is based on the multiplication of culture conditions aimed at facilitating the growth of fastidious species and the use of growth inhibitors against the majority species, allowing the selection of minority species, subsequently identified with MALDI-TOF mass spectrometry [34] or other methods. Such culturonic approaches were applied in Maxwellton Sink Cave (USA) to recover a wider microbial diversity for better screening of bioactive compound production and/or to confirm in situ metabolic strategies [35]. To conclude this section, all microorganisms might be cultivable, at least in theory, but successful isolation of the majority of microorganisms is hard to achieve, both in caves and elsewhere. The most abundant taxa (as indicated by cultivation-independent methods) could be isolated, such as _Proteobacteria_ or _Actinobacteria_ (see Sections 3 and 4), but members of the rare biosphere were only isolated once in a while. Consequently, cultivation methods offered a partial view of microbial diversity occurring in caves. However, cultivation is a powerful tool for delivering reference strains and identifying their metabolic capabilities, providing new genome sequences that assist, for example, in designing better primers and probes for refinement of molecular and microscopy detection methods [17], or providing microbial strains used for their metabolites or enzymes in a biotechnological context. It is also useful to complement findings obtained by sequencing methods (see below). Molecular Cloning, the First Culture-Independent Approach to Investigate the Microbial Ecology of Caves  
Cloning has been widely applied to environmental rRNA genes over previous decades [36]. Briefly, classical cloning is a technique by which environmental genes are inserted into a vector and subsequently introduced into a host such as _Escherichia coli_ cells (via transformation). _E. coli_ are then grown on rich media and cells containing the environmental fragment are screened and selected for Sanger sequencing (Table 1). In detail, clone library construction proceeds as follows: (i) DNA extraction, (ii) amplification of targeted rRNA genes via PCR, (iii) ligation into a plasmid vector, (iv) transformation into competent cells, (v) selection on antibiotic-supplemented agar plate of _E. coli_ cells that contain a plasmid insert, (vi) selection of individual _E. coli_, and (vii) growth of _E. coli_ for replication of environmental gene inserts to provide sufficient copies for sequencing by capillary Sanger technology [37; 38]. Prior to sequencing, environmental DNA must be extracted from cave samples, considering that environmental samples contain DNA in a variety of forms (i.e., free DNA, DNA in cells). Thus, extraction methods must be driven according to the environmental matrix and the targeted microbial species to optimize extraction efficiency [39]. The use of DNA extraction kits is very common, but the presence of ubiquitous contaminants is widely recognized in these kits and varies considerably in composition between different kits and batches of kits [40]. These microbial DNA contaminants can impact estimates of microbial richness and diversity by contributing to incorrect interpretations and identifications, especially in low biomass samples [40; 41; 42; 43]. To avoid this bias, simultaneous sequencing of extraction blanks and negative controls is required for accurate interpretation of microbial diversity results. Contamination is a general challenge for environmental sequencing studies and the identification and removal of sequence contaminants is particularly problematic [44].