Unveiling the adaptive evolution of halotolerant aceticlastic methanogenesis: Multi-scale responses and energy...

The high concentration of salt ions in saline organic wastewater poses significant challenges for wastewater treatment technologies, particularly impacting the stability of anaerobic digesters. Aceticlastic methanogenesis is a crucial pathway for converting acetate into methane through methanoarchaea whose metabolism is adversely impacted by salt stress. To address this, long-term adaptive laboratory evolution (ALE) was conducted to cultivate halotolerant aceticlastic methanoarchaea, incorporating metagenomics, metatranscriptomic sequencing, metabolomics, and metabolic modeling to delineate genetic and metabolic responses. The evolved microbiome achieved a substantial increase in methanogenic activity at 5 % sodium chloride, reaching 82.25 % theoretical conversion of acetate to methane, significantly outperforming the original microbiome. This ALE process overcame the natural scarcity of aceticlastic methanogens in hypersaline environments. Key adaptation mechanisms were confirmed at the transcriptional level, primarily involving the upregulation of genes for inorganic ion transport, compatible solute uptake, and de novo biosynthesis. Horizontal gene transfer also contributed significantly through the transfer of osmoregulation genes, particularly those for compatible solute transport, suggesting an energy-efficient adaptation strategy of accumulating rather than synthesizing solutes. Metabolic flux analysis revealed that adjustments in energy distribution under salt stress are driven by the energetic cost of synthesizing compatible solutes, which highlights the importance of solute transporters for energy conservation. This study elucidates the complex interplay between metabolic reprogramming and gene transfer in enhancing microbial resilience under salt stress, thereby deepening our understanding of microbial adaptations in extreme environments and advancing biotechnological approaches for saline wastewater treatment.