Application of deep eutectic solvents (DES) in bacterial growth and microbial electron activity enhancement

Abstract

Bacillus subtilis, a Gram-positive, spore-forming bacterium, exhibits a versatile metabolism and biofilm-forming ability, making it a promising candidate for the production of industrially relevant metabolites. Under anoxic or nutrient-limited conditions, B. subtilis can engage in extracellular electron transfer (EET), a mechanism by which bacteria exchange electrons with charged surfaces or minerals through redox mediators. This process can contribute to the bacterium's energy metabolism and has been associated with enhanced production of primary and secondary metabolites in various electroactive microorganisms. However, B. subtilis is considered a weak electricigen, and its electroactivity must be enhanced to optimize its biotechnological potential. This study investigates two key hypotheses regarding the enhancement of B. subtilis electroactivity through biocompatible nutrient additives. The first hypothesis posits that incorporating deep eutectic solvents (DESs), particularly choline chloride (ChCl)-based DESs, into the growth medium at subtoxic concentrations can improve B. subtilis electroactivity by acting as both nutritional supplements and osmoprotectant. The second hypothesis explores the role of osmotic stress regulators, specifically the combination of inorganic salts and ChCl-containing DESs, in promoting biofilm formation and enhancing EET in B. subtilis under static anoxic conditions. To evaluate the first hypothesis, Chapter 4 demonstrates that the addition of ChCl-based DESs (ChCl:glycerol, 1:2 mol/mol) significantly enhances the current output and biofilm formation of B. subtilis at subtoxic concentrations (55–500 mM). This effect is attributed to the nutritional and osmoprotective properties of ChCl, which acts as a precursor to the compatible solute glycine betaine. Electrochemical analyses and high-performance liquid chromatography (HPLC) further reveal that the enhanced electroactivity is linked to increased riboflavin production, a known redox mediator secreted by B. subtilis. The results indicate that low concentrations of DES promote planktonic growth without altering the growth pattern, while higher concentrations induce pseudo-diauxic growth due to the metabolization of choline chloride. Moreover, ChCl at concentrations above 36 mM independently enhances biofilm biomass, whereas glycerol has a minimal impact. The second hypothesis is addressed in Chapter 5, where a low-carbon tryptone-yeast extract medium supplemented with inorganic salts (NaH2PO4 and KH2PO4) is used to induce salt stress. The combination of salts with D-sorbitol/ChCl DES (1:1 mol/mol) mitigates osmotic stress, enhances biofilm formation, and stimulates electroactivity. The results indicate that ChCl acts as both an osmoprotectant precursor and a stimulant of electroactive exopolymeric substances within the biofilm matrix, while sorbitol serves as a secondary carbon source and a carbon pool for exopolysaccharide (EPS) synthesis. Bioelectrochemical experiments reveal that ChCl has a small positive effect on charge output in the presence of salts but does not alleviate osmotic stress on planktonic growth at higher concentrations. Overall, this research highlights the potential of ChCl-based DESs as cost-effective and biocompatible nutrient additives to improve B. subtilis electroactivity and biofilm formation. The findings suggest that the combination of osmotic stress regulators and DESs can optimize bioelectrochemical processes, such as electrofermentation, particularly in media with high ionic strength. The outcomes of this work provide an optimistic approach to enhancing B. subtilis electroactivity and bioproduction of its high-added-value metabolites under EF conditions.