Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes
S. Peters et al., Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes, APPL ENVIR, 66(3), 2000, pp. 930-936
A cultivation-independent technique for genetic profiling of PCR-amplified
small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversit
y and succession of microbial communities during composting of an organic a
gricultural 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 rRN
A genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA gene
s, Homologous PCR products were converted to single-stranded DNA molecules
by exonuclease digestion and were subsequently electrophoretically separate
d 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 sequenc
ing 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 se
quences were related to the respective gene regions of Candida krusei and C
andida tropicalis, Specific molecular isolates could be attributed to diffe
rent 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 sp
ecies were also detectable in the SSCP community profiles. Our study indica
tes that community SSCP profiles can be highly useful for the monitoring of
bacterial diversity and community successions in a biotechnologically rele
vant process.