Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

Abstract

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.

FIG. 1

Succession of PCR-amplified products during a composting process as detected by SSCP on a polyacrylamide gel. PCR primers were designed to amplify the hypervariable regions V4 and V5 of eubacterial 16S rRNA genes from directly extracted compost DNA (S, standard DNA). For each day (d), results obtained from two separate composting windrows are shown.

FIG. 2

SSCP patterns of single-stranded PCR products obtained from amplifications targeting the V3 region of actinomycetes. DNA directly extracted from composting material was used as a template. Days of sampling (d) are indicated for each lane.

FIG. 3

Succession of SSCP patterns obtained from single-stranded DNA products amplified by PCR from directly extracted compost DNA. Primers targeted conserved regions to amplify the V8 and V9 regions of the eukaryotic 18S rRNA gene sequence (M, products obtained from DNA extracted from leaves of maize plants). Days of sampling (d) are given above lanes. For each day, parallel samples were obtained from two separate composting windrows. Arrowheads point to products of the prospective clones CP-1 (4 d) and CP-2 (15 d) (see also Table 2).

FIG. 4

Comparison of PCR-SSCP community patterns with single products isolated from profiles and reamplified by PCR. Patterns obtained with primers amplifying the hypervariable regions V4 and V5 of eubacterial 16S rRNA genes from directly extracted compost DNA are shown in lanes A (sample taken after 2 days of composting), B (4 days), C (6 days), D (10 days), and E (15 days). Amplified products of prospective clones are shown in lanes 1 (clone CB-12), 2 (CB-2), 3 (CB-1), 4 (CB-3), 5 (CB-4), 6 (CB-5), 7 (CB-6), 8 (CB-7), 9 (CB-8), 10 (CB-9), 11 (CB-10), and 12 (CB-11). For identification of clones, see Table 2.