Population diversity and accelerated mutation drive anaerobic bacterial adaptation during long-term solvent stress

Experimental evolution can uncover general mechanisms by which microorganisms adapt to environmental stress. Here, we investigate evolutionary trajectories of the anaerobic, lignocellulose-fermenting bacterium Lachnoclostridium phytofermentans ISDg (family Lachnospiraceae) during >2,000 generations of automated, log-phase selection at progressively increasing butanol concentrations. Evolved clones showed large gains in fitness, reaching threefold-faster growth rates in standard medium, and robust growth at concentrations of butanol and other alcohols inhibitory to the parental strain. Longitudinal genome sequencing revealed the succession of genetic changes, including expansion of ISL3 insertion sequences, activation of a cryptic phosphotransferase system, and mutations leading to increased cell size and remodeling of membrane fatty acids. The fixation of a hypermutator genotype by generation 940 preceded a burst of genetic diversity and accelerated adaptation, followed by population destabilization as the mutational load accumulated. These findings provide insights into the mechanisms by which anaerobes acquire tolerance to industrially relevant stresses during adaptive laboratory evolution.IMPORTANCELong-term growth selection under controlled conditions can be applied to dissect complex, multigenic traits such as solvent tolerance. Butanol is an industrial solvent whose toxicity remains a major barrier to its efficient microbial production. We adapted Lachnoclostridium phytofermentans ISDg, a lignocellulose-fermenting, strictly anaerobic bacterium, to increasing butanol concentrations by automated cultivation with alternating periods of constant or variable butanol selection pressure. Evolved strains grew faster and tolerated solvent concentrations that completely inhibited the parental strain, attaining tolerance levels similar to those of native butanol producers. Genome analysis identified both previously recognized and novel adaptive changes, revealing how prolonged solvent tolerance arises through the interplay of mobile genetic elements, cellular restructuring, and altered mutation rates.