MAGs-centric crack: how long will, spore-positive and most , microsymbionts remain recalcitrant to axenic growth?

Nearly 50 years after the ground-breaking isolation of the primary microsymbiont under axenic conditions, efforts to isolate a substantial number of and strains continue with enduring challenges and complexities. This study aimed to streamline genomic insights through comparative and predictive tools to extract traits crucial for isolating specific in axenic conditions. Pangenome analysis unveiled significant genetic diversity, suggesting untapped potential for cultivation strategies. Shared metabolic strategies in cellular components, central metabolic pathways, and resource acquisition traits offered promising avenues for cultivation. Ecological trait extraction indicated that most uncultured strains exhibit no apparent barriers to axenic growth. Despite ongoing challenges, potential caveats, and errors that could bias predictive analyses, this study provides a nuanced perspective. It highlights potential breakthroughs and guides refined cultivation strategies for these yet-uncultured strains. We advocate for tailored media formulations enriched with simple carbon sources in aerobic environments, with atmospheric nitrogen optionally sufficient to minimize contamination risks. Temperature adjustments should align with strain preferences-28-29°C for and 32-35°C for -while maintaining an alkaline pH. Given potential extended incubation periods (predicted doubling times ranging from 3.26 to 9.60 days, possibly up to 21.98 days), patience and rigorous contamination monitoring are crucial for optimizing cultivation conditions.