Inducing Biofilm Formation in Clostridium kluyveri and Strain Characterization in Clostridium acetobutylicum for Improved Bioconversion.

• Rishav Sharma, Chemical Engineering, University of Delaware
• Samuel Perkins, Chemical Engineering, Oregon state

• Eleftherios Papoutsakis, Chemical & Biomolecular Engineering, University of Delaware

Clostridium species are of growing interest in the renewable energy and fuels field for converting simple carbon substrates into valuable fuels and chemical precursors via anaerobic fermentations, offering a biological alternative to petrochemical production. This work is part of a broader platform coupling electrocatalysis, which converts H₂O and CO₂ into short-chain products (acetate, ethanol, and formate), with anaerobic bioconversion to elongate these products into higher-value medium-chain fatty acids. The key challenge is improving yield and process robustness enough to make this bioconversion step economically competitive at scale. This summer’s work addresses that challenge from two perspectives: using Clostridium kluyveri (Ckl) to grow as a biofilm for more productive, stress-resistant fermentation, and characterizing an engineered Clostridium acetobutylicum (Cac) strain designed to favor acetone production over the cell’s natural sporulation response.

Ckl is an anaerobic chain-elongating bacterium that converts ethanol and acetate, low-value two-carbon (C2) products, into longer-chain butyrate and caproate (C4-C6 medium-chain fatty acids). Because Ckl grows to low cell densities in suspension, this work investigated inducing biofilm formation, since biofilms are associated with greater stress resistance and productivity. Two induction methods were tested: salt-induced osmotic stress, producing visible cells at 400 mM with minimal cost to production, and antibiotic induction using 1 µM thiamphenicol for three days, followed by a media change and further incubation; crystal violet staining confirmed that this treatment successfully induced biofilm formation. A fibrous-packed-bed bioreactor is being investigated as a scalable process.

In parallel, an engineered Cac strain carrying an acetone pathway plasmid was modified to block sporulation, redirecting cellular energy into growth and product formation instead. Clones were evaluated in serum bottle monocultures without rigorous pH control; one clone, adapted to a higher acetate concentration, substantially outgrew the other, reaching a peak OD600 of ~13, though this has not been confirmed by sequencing.