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The use of culture-dependent and culture-independent techniques in microbial biodiversity studies

While the microbiota of some traditional beers had been characterized with classical methods several decades ago, more recent microbial biodiversity studies have been performed using culture-independent community fingerprinting techniques in combination with traditional cultivation methods (Dolci et al., 2010; Laureys and De Vuyst, 2014; Scheirlinck et al., 2008; Van der Meulen et al., 2007; Wouters et al., 2013). Some of these studies have focused on specific microbial groups of those communities, e.g. LAB or yeasts, while others address the entire community (Martens et al., 1991; Shanta Kumara and Verachtert, 1991; Van Oevelen et al., 1977; Verachtert and Iserent- ant, 1995). In recent years, more advanced and high-throughput culture-independent methods, such as bar-coded amplicon sequencing (BAS) and shotgun metagenomics, are rapidly replacing community fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) gene amplicons. The data obtained are considered superior to culture-dependent data. Indeed, methods that involve cultivation of microorganisms are often considered less informative, as some microorganisms can be present in a viable but non-culturable (VBNC) state, while isolation media favour the cultivation of specific microorganisms only (Gorski, 2012; Millet and Lonvaud-Funel, 2000). Moreover, metagenomic techniques are also superior to classical fingerprint-based culture-independent techniques such as PCR-DGGE, because of their ability to detect low-abundance species in the communities (Bokulich and Bamforth, 2013).

However, culture-independent techniques also introduce biases, for instance through differences in DNA extraction efficacy or in PCR-based amplification of target sequences (Hong et al., 2009; Yuan et al., 2012), and potentially detect not only metabolically inactive but also dead cells. Therefore, although these modern culture-independent approaches provide a more in-depth analysis of the microbial communities, they are not without limitations and also do not reveal which species are metabolically most active. Other tools, not only transcriptomics or meta-metabolomics but also the availability of pure cultures of community members will be required to reveal a more complete image of the microbial diversity present in an ecosystem.

Ironically, the potential to examine microbial ecosystems by means of modern metagenomics approaches triggered a renewed interest in the development of new approaches to cultivate a larger fraction of microorganisms that are known through the detection of their DNA only (Nature Reviews: Microbiology Editorial, 2013; Rappe, 2013; Teske, 2010). Based on metagenomics information about the genes and metabolic potential present, new culture media have been developed that target the isolation of specific microbial groups (Bomar et al., 2011). An approach referred to as culturomics (Lagier et al., 2012) is currently gaining momentum in the field of gut microbiome research. Culturomics refers to the high-throughput and miniaturized application of numerous classical media for the isolation of microorganisms and is limited only by the rate of identification of the isolates obtained. For this purpose, sequence-based identification methods are too slow and expensive but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been advocated as an ideal identification technique in culturomics approaches to study microbial diversity (Lagier et al., 2012).

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