Molecular phylogenetic diversity of Bacillus community and its temporal-spatial distribution during the swine manure of composting

Abstract

In order to obtain the diversity and temporal-spatial distribution of Bacillus community during the swine manure composting, we utilized traditional culture methods and the modern molecular biology techniques of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and -denaturing gradient gel electrophoresis (PCR-DGGE). Bacillus species were firstly isolated from the composting. Based on temperature changes, the temporal-spatial characteristics of total culturable Bacillus were remarkable that the number of the culturable Bacillus detected at the high-temperature stage was the highest in each layer of the pile and that detected in the middle layer was the lowest at each stage of composting respectively. The diversity of cultivated Bacillus species isolated from different composting stages was low. A total of 540 isolates were classified by the RFLP method and partial 16S rDNA sequences. They affiliated to eight species including Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus megaterium, Bacillus licheniformis, Bacillus pumilus, and Bacillus circulans. The predominant species was B. subtilis, and the diversity of culturable Bacillus isolated in the middle-level samples at temperature rising and cooling stages was the highest. The DGGE profile and clone library analysis revealed that the temporal-spatial distribution of Bacillus community was not obvious, species belonging to the Bacillus were dominant (67%) with unculturable bacteria and B. cereus was the second major culturable Bacillus species. This study indicated that a combination of culture and culture-independent approaches could be very useful for monitoring the diversity and temporal-spatial distribution of Bacillus community during the composting process.