Overcoming gelling agent-derived inhibitory factors enables the cultivation of fastidious nitrifiers on solid media

Abstract Nitrification is a key process in the global nitrogen cycle, yet the physiological characterization of nitrifiers remains limited due to their low culturability. Although culture-independent approaches have provided important ecological insights, they do not fully resolve the physiological properties of nitrifying microorganisms. Here, we show that common gelling agents (agar, agarose, and gellan gum) contain toxic substances that inhibit the growth of diverse nitrifying microorganisms as well as some heterotrophic bacteria. These inhibitory substances appear to be generated during autoclaving and act via mechanisms distinct from previously reported hydrogen peroxide. Removal of these inhibitory factors using a simple washing strategy enables colony formation in recalcitrant nitrifiers, including Nitrososphaera (ammonia-oxidizing archaea) and Nitrospira (nitrite-oxidizing bacteria). Application of this approach to environmental samples enabled the isolation of multiple difficult-to-culture nitrifying strains, including ammonia-oxidizing and nitrite-oxidizing microorganisms. Together, these findings demonstrate that the limited culturability of nitrifiers on solid medium is largely driven by gelling agent-derived inhibitory substances and can be overcome by a simple and broadly applicable strategy.