Principles of enrichment, isolation, cultivation, and preservation of prokaryotes

Abstract

Currently, a total of 9,409 prokaryotic species are recognized (as of January 2012, validly published names not including homotypic and heterotypic synonyms, comb. nov. and nomina nova; DSMZ 2012; Euzéby 2012). By comparison, the number of small subunit ribosomal RNA (SSU rRNA) gene sequences deposited in public databases keeps increasing exponentially (Pruesse et al. 2007; Yarza et al. 2008) and surmounted the species numbers already some 15 years ago (Fig. 7.1). Meanwhile, a total of 2,492,653 sequences are available of which 2,282,670 are prokaryotic whereas only 33,842 originate from cultured strains (SILVA 2012). In line with these cumulative data, culture-independent analyses of DNA reassociation kinetics and of 16S rRNA gene sequences in individual environmental samples also indicate that prokaryotic diversity is poorly represented by the species cultivated so far. Thus, estimates of bacterial species numbers in just one type of soil reached values of up to 53,000 (Sandaa et al. 1999; Roesch et al. 2007). Furthermore, molecular investigations of 16S rRNA gene sequences in natural bacterial assemblages typically yielded many more sequence types than those recovered by cultivation-based approaches (Fuhrman et al. 1992; Ward et al. 1992; Barns et al. 1994; DeLong et al. 1994; Hiorns et al. 1997; Kuske et al. 1997; Ludwig et al. 1997; Suzuki et al. 1997; Gich et al. 2001; Béjà et al. 2002; Roesch et al. 2007). In light of these findings, the earlier estimates of the fraction of already cultured bacterial species of 12-20% (Wayne et al. 1987; Bull et al. 1992) or even the commonly cited estimate of 1% appears to be far too optimistic. Based on recent estimates of total bacterial species numbers (10 7 -10 9; Dykhuizen 1998; Curtis et al. 2002), the value more likely ranges between 0.1% and 0.001% and may be even lower (compare the higher estimates of bacterial species numbers in Sogin et al. 2006; Harwood and Buckley 2008).