Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (greater than or equal to 60 degreesC) synthetic food wast e compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts tot aled 6.4 (+/- 2.5)x 10(10) cells gdw(-1) in a freeze-dried 74 degreesC comp ost sample, while plate counts for thermophilic heterotrophic aerobes avera ged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method w as developed to obtain community DNA and a suite of 165 and 18S rDNA-target ed PCR primers was used to examine the presence of Bacteria, Archaea and fu ngi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fun gal 18S rDNA was only amplified from a separate dairy manure compost that r eached a peak temperature of 50 degreesC. Amplified rDNA restriction analys is (ARDRA) was used to screen isolated thermophilic bacteria and a clone li brary of full-length rDNA fragments. ARDRA screening revealed 14 unique pat terns among 63 isolates, with one pattern accounting for 31 of the isolates . In the clone library, 52 unique patterns were detected among 70 clones, i ndicating high diversity of uncultivated bacteria in hot compost. Phylogene tic analysis revealed that the two most abundant isolates belonged in the g enera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With tile exception of one Lactobacillus-type sequence. the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearotherm ophilus commonly believed to dominate high-temperature compost. Amplified p artial fragments from Arctinobacteria, spanning the V3 variable region (Nee fs ct al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences rela ted to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacter ium, although none of these organisms were detected among the isolates or f ull-length cloned rDNA sequences. All of the thermophilic isolates and sequ enced rDNA fragments examined in this study were from Gram-positive organis ms. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.