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

Abstract

High-temperature (v60³C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 ( þ 2.5)U1010 cells gdw 31 in a freeze-dried 74³C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 ( þ 1.0)U10 8 CFU gdw 31 . A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. 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. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50³C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the 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. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237^2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms. ß

Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation

“…The traditional technology of Toscano cigar tobacco fermentation is based on a semicontinuous process whose physicalchemical profile suggests a model resembling that of the wellknown solid waste composting process (11,23,39,51,52), being characterized by intrinsic variation of chemical and physical parameters. Major sources of variability are the local heterogeneity of the bulk, the seasonal fluctuation of temperature, and inconstant shuffling times, all reflecting the small-scale, semiartisanal, nonstandardized nature of the tobacco fermentation process.…”

Microbial Anaerobic Digestion (Bio-Digesters) as an Approach to the Decontamination of Animal Wastes in Pollution Control and the Generation of Renewable Energy

“…Most studies have utilized culture-based methods [2,4,5,6,7] that are known to only capture a small portion of the microbial diversity found in environmental samples [8]. While a few studies have employed cultivation-independent approaches, a comprehensive perspective on compost microbial dynamics is still lacking because these studies have focused on only one of the three compost phases [9,10,11,12,13,14] or have used fingerprinting techniques that offer limited taxonomic resolution [15,16,17,18,19,20,21]. The few available high-throughput sequencing studies have focused primarily on bacteria [22,23,24,25] even though fungi likely play an important role in the compost process and may enhance the quality of compost.…”

Relationship between Microbial C, Microbial N and Microbial DNA Extracts during Municipal Solid Waste Composting Process

“…One such challenge is the dynamic nature of the process, characterized by rapid changes in microbial population, temperature and oxygen gradients, and the availability of nutrients for microorganisms. The analysis of nucleic acids extracted from environmental samples allows researchers to study natural microbial communities without the need for cultivation (Peters et al, 2000;Dees and Ghiorse, 2001). Although there have been many published studies on methods for the extraction DNA from environmental samples, very few have focused upon the extraction of DNA from compost.…”