Different growth pattern during microbial electrosynthesis using C. ljungdahlii evolutionary adapted on iron

Clostridium ljungdahlii is an acetogen used for syngas fermentation and capable of microbial electrosynthesis. While C. ljungdahlii has potential for industrial application because of its broad spectrum of metabolites and substrates, including CO ₂ and CO, the efficiency of extracellular electron uptake in a bioelectrochemical system is low. Therefore, C. ljungdahlii was evolutionary adapted on iron as sole electron source with the aim to improve extracellular electron uptake. Over 38 batches, 95% of the culture was replaced with fresh medium biweekly to retain iron-attached C. ljungdahlii , leading to improved acetate production rates with each cycle. Eight isolated strains were tested on fructose, H ₂ and CO ₂ , and iron to screen evolved mutants with desired mutations. Compared to the wild-type, growth on fructose was similar and growth on H ₂ and CO ₂ was, surprisingly, worse, with only minor differences between isolates. The isolated mutants produced acetate at a rate of 0.14 ± 0.01 mM/d on iron, while the wild-type strain produced 0.75 ± 0.14 mM/d. Whole genome sequencing of isolated mutants revealed 16 mutations, of which seven mutations were found in all isolates. Mostly, membrane proteins were mutated. In a BES reactor, acetate production ceased after day 1. The optical density (OD) of isolate #8 stopped increasing after day 2, reaching 0.122 ± 0.005, followed by the production of formate and ethanol. The wild-type strain continued to grow until day 4, reaching an OD of 0.177 ± 0.003. These results may indicate that C. ljungdahlii slows down growth and produces ethanol as an energy reserve as an evolutionary strategy for survival in an electron-limited environment.