Let-them-stick: Increasing biofilm formation by the acetogen Sporomusa ovata through adaptive laboratory evolution

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 cell 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 carriers in bottles with a H2:CO2 headspace and transferring few carriers to bottles 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. 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 used experimental setup likely stimulated planktonic growth due to a low trickling frequency. Only after some medium replacements to remove planktonic cells, higher gas consumption rates were recorded for the evolved culture. Interestingly, the evolved S. ovata had a relevant point mutation in the gene galU, encoding UDP-glucose pyrophosphorylase, 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. IMPORTANCEAcetogenic bacteria are attractive biocatalysts for the conversion of CO2 with H2 into acetate, as in gas fermentation and microbial electrosynthesis. Both biotechnologies could benefit from biofilm formation, since biofilm formation retains the catalytic activity inside continuously operated reactors. In addition, biofilm-based gas fermentation reactor systems are advantageous to improve the gas to liquid mass transfer of H2 and CO2. Moreover, microbial electrosynthesis benefits from biofilms on cathodes to consume H2 as soon as it gets generated. Acetogenic bacteria are however often found to only form thin or sparse biofilms. In the current study, we demonstrated that adaptive laboratory evolution is a useful strategy to increase the biofilm formation capabilities of the acetogen, Sporomusa ovata. The obtained biofilm improved S. ovata is of interest to deepen our fundamental understanding of biofilm formation by acetogens, as well as to assess the importance of biofilm formation for the performance of biofilm-based bioreactors.