Successful Cultivation of Predominant Freshwater Bacterioplankton from Lake Soyang

초록

Freshwater ecosystem has a unique planktonic bacterial community distinct from those of other habitats such as soil and marine environments. Although recent cultivation-independent molecular analyses have revealed a great diversity of freshwater bacteria, ecophysiological characteristics of abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. In an effort to cultivate the uncultured yet major bacterial populations present in freshwater, we have performed high-throughput dilution-to-extinction culturing (HTC) for water samples collected from Lake Soyang in different depths and seasons. As a result of the HTC experiments, we have obtained diverse groups of bacterioplankton that have not been previously cultured, distributed in the Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Acidobacteria, and Verrucomicrobia. Particularly, among these isolates, we focus more on the successful cultivation of the actinobacterial acI lineage, the most abundant freshwater bacterial clade. So far, despite of rich genomic information derived from 20 complete genomes, stable pure cultures of the acI lineage have not been possible. After various efforts, we could maintain two pure acI cultures by supplementing the growth media with catalase, the key enzyme scavenging hydrogen peroxide. The acI cultures showed vigorous growth with a maximum cell density of ca. 5 × 107 cells/ml under the conditions of catalase. Surprisingly, the two strains displayed different phenotypic characteristics and distinct preference for amino acids and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest that uncultured bacteria can be facilitated by a simple catalase-supplement method and indicate that assuming physiological characteristic by genome-based annotation might be incomplete to understand the physiology in nature.