Prokaryotic Diversity Of A Non-sulfide, Low-salt Cold Spring Sediment Of Shawan County, China

The prokaryotic diversity of a non-sulfide, low-salt cold spring sediment was investigated by constructing bacterial and archaeal clone libraries of the 16S rRNA gene. 241 bacterial clones were screened, which could be grouped into 86 ribotypes, based on restriction fragment length polymorphism (RFLP) analysis. These were divided into 11 phyla (Actinobacteria, Acidobacteria, Bacteroidetes, Chlorobi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Proteobacteria, Planctomycetes and Verrucomicrobia). Of these, Acidobacteria and Proteobacteria were the most dominant, representing 48% and 25% of the total bacteria clone library, respectively. Nearly 20% of clones were related to nitrogen-fixing and nitrite- oxidizing bacteria. For the archaeal clone library, 121 positive clones were screened and 22 ribotypes were determined. BLAST analysis indicated that all ribotypes were affiliated with the phylum Crenarchaeota. Phylogenetic analysis classified them into two subgroups of Soil-Freshwater-subsurface group, and Marine group I and represented 50% of total clones, respectively. Of them, ribotypes with the potential to assimilate nitrate accounted for 40% of the total archaeal clones. In addition, 20% of ribotypes were related to the cold-loving Crenarchaeota. These findings show that bacteria in spring sediments are more diverse than are archaea; in addition, populations involved in nitrogen cycling are most common and might have an important role in the maintenance of the microbial community in this cold and oligotrophic environment.In order to further surveying the composition and diversity of the actinobacterial population in the Shawan cold spring sediment, the cultivated and uncultivatied actinobacteria were investigated by culturing and constructing an actinobacterial- specific 16S rRNA gene clone library. For the clone library, 156 clones that grouped into 30 ribotypes were screened by RFLP analysis, and divided into three subclasses: Acidimicrobidae (22.7%), Actinobacteridae (65.2%) and unknown affiliation (12.1%). Of these, the Actinobacteridae was the most dominant and diverse, and could be divided into seven genera [Actinoplanes (3.8%), Arthrobacter (18.5%), Blastococcus (4.7%), Mycobacterium (15.3%), Lechevalieria (5.3%), Streptomyces (6.4%) and Actinomadura (0.7%)] and one suborder [Micromonosporineae (14.7%)]. A diverse uncultured actinobacterial community grouped within the Acidimicrobidae and only a fraction of the sequences belonged to genus Acidimicrobium. The 150 isolates collected from 11 culture media, were classified into ten genera of actinobacteria (Acidimicrobium, Arthrobacter, Georgenia, Leifsonia, Micrococcus, Micromonospora, Pseudonocardia, Rhodococcus, Streptomyces and Williamsia). Of these, species of the genera Streptomyces and Arthrobacter were most common. The dual approach of culturing and molecular analysis showed that the spring sediment harbors a large number of cultivable actinobacteria. Additionally, some of the currently uncultured species that grouped within subclasses Acidimicrobidae, Actinobacteridae or had unknown affiliations were only distantly related to putative cultures. This suggests that novel species or lineages might exist in the environment and could be used as guide to develop appropriate methods to culture these bacteria for screening for novel useful metabolites.