16S rRNA gene sequences showing 99% similarity or higher was considered to belong to the same phylotype. Related 16S rRNA gene sequences were placed within tentative taxa (between Phylum and Order) by determining the taxonomic class (using the NCBI taxonomy database) of the closest relative in GenBank of sequences that formed a phylogenetic clade. Sequences that showed no or low (below 70%) relatedness with known bacterial or archaeal phylogenetic groups were listed as unclassified. The distribution of CRA-024781 types present in the clone libraries was determined and used to calculate the Shannon–Weaver index (H = −∑[pi?ln(pi)]), where pi is the relative contribution of clone type i to the whole library (n1/N). Coverage was calculated as 1 − (n1/N), where n1 is the number of clone types that was encountered only once in the library and N is the total number of clones analyzed. The Chao1 estimator of species (here, clone type) richness (Schao1) was calculated as; Sobs + ni2/2n2 ( Röling and Head, 2005).