Adaptive laboratory evolution increased biofilm formation by Sporomusa ovata through a mutation in galU

Acetogenic bacteria are attractive biocatalysts for the conversion of CO₂ with H₂ into acetate, as in gas fermentation. Gas fermentation reactors may benefit from biofilm formation, but attachment by acetogens is often limited. This study indeed found that the acetogen Sporomusa ovata 2663 was mainly planktonic and aimed to increase its biofilm formation through adaptive laboratory evolution. The adaptation strategy consisted of growing S. ovata on plastic (HDPE) carriers in bottles with a H₂:CO₂ headspace and transferring two carriers to a bottle with fresh carriers over eight serial transfers. This procedure resulted in the evolved S. ovata 2663-BF, which had a consistent increased propensity to attach. In heterotrophic growth conditions, four times more cells attached to the bottom of well plates in comparison to the wild type, while the adapted S. ovata produced 1.8 times more extracellular polysaccharides. Moreover, twice as many cells adhered to carriers when grown on H₂:CO₂. This improved attachment, however, did not lead to higher acetate production rates in simple trickle bed reactors, as the experimental setup rather stimulated planktonic growth. Only medium replacement sometimes favored the rate of the adapted strain. Interestingly, the evolved S. ovata had a point mutation in the gene galU, which likely increased the activity of the encoding UDP-glucose pyrophosphorylase, i.e. an enzyme involved in the synthesis of extracellular polysaccharides. Overall, this study demonstrates that cell attachment by S. ovata was increased through adaptive laboratory evolution, offering the prospect of investigating the importance of biofilm formation in biofilm-based gas fermentation reactors.