Process Development for Enhanced 2,3-Butanediol Production by Paenibacillus polymyxa DSM 365

Abstract

While chiral 2,3-Butanediol (2,3-BD) is currently receiving remarkable attention because of its numerous industrial applications in the synthetic rubber, bioplastics, cosmetics, and flavor industries, 2,3-BD-mediated feedback inhibition of Paenibacillus polymyxa DSM 365 limits the accumulation of higher concentrations of 2,3-BD in the bioreactor during fermentation. The Box-Behnken design, Plackett-Burman design (PBD), and response surface methodology were employed to evaluate the impacts of seven factors including tryptone, yeast extract, ammonium acetate, ammonium sulfate, glycerol concentrations, temperature, and inoculum size on 2,3-butanediol (2,3-BD) production by Paenibacillus polymyxa DSM 365. Results showed that three factors; tryptone, temperature, and inoculum size significantly influence 2,3-BD production (p < 0.05) by P. polymyxa. The optimal levels of tryptone, inoculum size, and temperature as determined by the Box-Behnken design and response surface methodology were 3.5 g/L, 9.5%, and 35 • C, respectively. The optimized process was validated in batch and fed-batch fermentations in a 5-L Bioflo 3000 Bioreactor, and 51.10 and 68.54 g/L 2,3-BD were obtained, respectively. Interestingly, the production of exopolysaccharides (EPS), an undesirable co-product, was reduced by 19% when compared to the control. These results underscore an interplay between medium components and fermentation conditions, leading to increased 2,3-BD production and decreased EPS production by P. polymyxa. Collectively, our findings demonstrate both increased 2,3-BD titer, a fundamental prerequisite to the potential commercialization of fermentative 2,3-BD production using renewable feedstocks, and reduced flux of carbons towards undesirable EPS production.

Molecular inactivation of exopolysaccharide biosynthesis inPaenibacillus polymyxaDSM 365 for enhanced 2,3-butanediol production

“…To extract genomic DNA (gDNA), P. polymyxa cells were grown in a previously described pre-culture medium (7) to cell OD 600nm of 0.7. The cells were harvested by centrifugation at 10,000 × g and 4°C for 10 min and then, suspended in Tris-HCl-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).…”

“…The actively growing P. polymyxa levansucrase null mutant (10%, v/v) was inoculated into the fermentation medium containing 100 g/L sucrose or glucose supplemented with 35 μg/ml erythromycin. The pre-culture and fermentation medium components used in this study have been previously described (7). The fermentation medium was further supplemented with 0 - 0.4 g/L CaCl 2 .…”

“…To determine the stability of the levansucrase null mutant, the growth curve was first obtained. Cells were grown to exponential phase (10 h) in the pre-culture medium (7), then harvested and washed twice with sterile distilled water by centrifugation at 5,000 × g for 3 min. The cell pellet was reconstituted to several dilutions and the optical densities (OD 600nm ) were measured against sterile distilled water as blank.…”

“…These co-products compete with 2,3-BD for substrates and pyruvate resulting in decreased 2,3-BD yield (5, 6). Several studies have focused on the manipulation of fermentation medium and conditions as means of reducing the accumulation of competing co-products during 2,3-BD fermentation (7, 8, 9, 10, 11). Although, significant progress has been made, accumulation of competing co-products remains a significant challenge to large-scale production of 2,3-BD.…”

Production of Bio-Derived Fuels and Chemicals

“…Recognizing the importance of chiral compounds in the biotechnology industry, our group [11] contributed an article in which process development for enhanced 2,3-butanediol (2,3-BD) production by non-pathogenic bacterium, Paenibacillus polymyxa DSM 365, was emphasized. Indeed, while our group was able to increase the concentration of 2-3-BD from 47 g/L (un-optimized) to 68.5 g/L (optimized) under fed-batch fermentation condition, the results underscore an interaction between medium components and fermentation conditions, which tends to influence 2,3-BD and undesirable exopolysaccharides (EPS) production [11]. Although butanol is an achiral compound, it is an important chemical with many applications in the production of solvents, butyl acetates, butylamines, plasticizers, amino resins, etc.…”