Advances and Challenges in Studying Cave Microbial Diversity

Abstract

In the last decade, we have seen significant changes in how we study composition and diversity of microbial communities in various environmental samples. Advances in culture-independent molecular phylogenetic techniques have made studies on microbial communities in diverse environments more attractive and feasible (Muyzer et al. 1993; Pace 1997; Torsvik et al. 1998; Hill et al. 2000; Giraffa and Neviani 2001; Kirk et al. 2004; Barton et al. 2004; Leckie 2005; Barton et al. 2006; Malik et al. 2008; Maukonen and Saarela 2009; Hirsch et al. 2010; and Northup et al. 2011). However, using modern molecular techniques alone to study both known and unknown microbial populations in an environment has its own limitations. Several studies suggest that using a combination of both culture-independent and culture-dependent methods gives a more realistic representation of the indigenous microbial diversity (Hill et al. 2000; Gurtner et al. 2000). For example in a 2000 study Gurtner and colleagues reported using of both classical cultivation techniques and molecular approaches to compare bacterial diversity on two medieval biodeteriorated wall paintings from two churches in Austria and Germany. They obtained 70 microbial sequences of 16S rDNA sequence belonging to several genera of bacteria. The molecular approach evaluated the bacterial community by Denaturing gradient gel electrophoresis (DGGE, one of the genetic fingerprinting tools), construction of 16S rDNA clone libraries, and sequence analysis of those libraries. In the same study, isolation of heterotrophic bacteria from one of the samples using Tripticase Soy (TSB) agar and TSB agar supplemented with 10 % sodium chloride (with 3 weeks of incubation at 28 °C) was also done in parallel to the above-mentioned molecular approach (Heyrman et al. 1999). The isolated strains were then characterized using fatty acid methyl ester (FAME) analysis and major FAME clusters found to belong to the genus Bacillus. Results from these two approaches failed to cross-detect similar microbial flora. In the molecular approach, 70 members of Actinobacteria and Proteobacteria including Actinobiospora, Amycolata, Halomonas, Deleya, Rhizobiam, and Salmonella were identified, while it is important to note that there was no Bacillus detected by the molecular approach. Their findings demonstrate that the combined approach of molecular and culturing techniques may provide a better understanding of the community being evaluated. There are other review and original research works that supported using a combined approach to study microbial diversity and function in a community (Dunbar et al 1999; Torsvik and Øvreås 2002; Crecchio et al 2004). Since no individual approach is completely effective to evaluate the microbial biodiversity of a given environment, this integrated approach may provide a closer representation of the microbial community. In its own context each of these approaches should be used and evaluated accordingly.