High Cell Density Strategies and Adaptive Evolution for Microbial Propionic Acid Production

Propionic acid (PA) is a naturally occurring short chain fatty acid produced by microbial fermentation of the indigestible foods in the intestine, having a positive effect on the gastrointestinal health. PA inhibits the growth of pathogenic bacteria and molds; hence it is widely used as preservative for food, feed and grains. It is also used as a component in several industrial products like polymers, cosmetics and pharmaceuticals. Its industrial production still depends on petrochemistry, however microbial fermentation as a route for production from renewable resources has attracted attention but is not yet competitive with the existing route. Dairy propionibacteria, used as starters for the maturation of Swiss-type cheeses and also known for their probiotic properties, produce propionic acid from different sugars or glycerol via Wood-Werkman cycle under anaerobic conditions. The fermentation process has a long lag phase and is also subject to product inhibition, resulting in low productivity and yield.This thesis involves studies on high cell density fermentations and development of acid tolerant strain as ways to improve the efficiency of propionic acid fermentation. Studies have been performed using Propionibacterium acidipropionici DSM 4900 (ATCC 25562), a bacterium that is generally regarded as safe (GRAS). Glycerol, a by-product of biodiesel production process, was used as the carbon source for fermentations.Different strategies of high cell density fermentations including sequential batch cultivation, cyclic batch cultivation, cell immobilization and cell retention using membrane filter, were evaluated. Propionate yield and productivity of 93 mol % and 0.53 g/L/h were obtained in cyclic batch while maximum productivity of 1.63 g/L/h was obtained from 90 g/L glycerol during sequential batch cultivations with free cells. P. acidipropionici cells were immobilized on plastic based AnoxKaldnes® and recycled glass based Poraver® carriers, either by polyethyleneimine (PEI) treatment or by biofilm formation. Exposing the cells to stress factors like 30 mM citric acid and 1 M NaCl was beneficial for increasing the Biofilm Forming Capacity index (BFC) and for production of exopolysaccharides (EPS), both indicators of biofilm formation. Fermentations in the immobilized packed-bed bioreactors using the bacteria pre-exposed to citric acid and NaCl gave propionic acid productivities of 0.7 and 0.78 g/L/h, respectively, with Poraver® matrix, and the corresponding values for AnoxKaldnes® were 0.39 and 0.43 g/L/h, respectively. Continuous propionic acid fermentations by cell retention in a ceramic membrane filter, gave productivity of 2.35 g/L/h in a medium with 60 g/L glycerol and 20 g/L yeast extract.A tolerant strain able to grow in a culture medium supplemented with 20 g/L of PA, was obtained by adaptive laboratory evolution. It exhibited 1.4-fold higher growth rate, 3.7-fold higher propionic acid production kinetics, 1.2-fold higher yield and almost 2-fold higher titer in batch cultivations compared to the parental cells in a basal cultivation medium. It was immobilized to PEI-treated Poraver® and used for fermentations in recycled batch mode with increasing glycerol concentration and decreasing pH, respectively, in packed-bed bioreactors. Glycerol up to a concentration of 100 g/L was completely consumed, resulting in 58 g/L propionic acid and product yield of 0.64 mol/mol. Progressive decrease in pH values gave increased propionic acid yield and productivity from 20 g/L glycerol, while maintaining the product titer nearly constant at pH 5.0.The exposure to stress factors for biofilm formation and to high propionic acid concentration during cultivation of P. acidipropionici showed trehalose to play a protective role. Following the expression of the trehalose synthesis-related genes treY and otsA, and by Real Time-qPCR revealed treY expression (but not otsA) to be significantly increased during biofilm formation under the influence of citric acid. Expression of the biofilm-related luxS gene was also detected by fluorescence in situ hybridization (FISH). Expression of treY was increased 82-fold in the tolerant strain obtained by adaptive evolution with respect to the 16S rRNA gene used as a control

Membrane-based continuous fermentation with cell recycling for propionic acid production from glycerol by Acidipropionibacterium acidipropionici

Background: Microbial production of propionic acid (PA) from renewable resources is limited by the slow growth of the producer bacteria and product-mediated inhibition. The present study evaluates high cell density continuous PA fermentation from glycerol (Gly) using Acidipropionibacterium acidipropionici DSM 4900 in a membrane-based cell recycling system. A ceramic tubular membrane filter of 0.22 μm pore size was used as the filtering device for cell recycling. The continuous fermentations were run sequentially at dilution rates of 0.05 and 0.025 1/h using varying glycerol concentrations and two different yeast extract concentrations.Results: PA volumetric productivity of 0.98 g/L.h with a product yield of 0.38 gPA/gGly was obtained with 51.40 g/L glycerol at a yeast extract concentration of 10 g/L. Increasing the glycerol and yeast extract concentrations to 64.50 g/L and 20 g/L, respectively, increased in PA productivity, product yield, and concentration to 1.82 g/L.h, 0.79 gPA/gGly, and 38.37 g/L, respectively. However, lowering the dilution rate to 0.025 1/h reduced the production efficiency. The cell density increased from 5.80 to 91.83 gCDW/L throughout the operation, which lasted for a period of 5 months. A tolerant variant of A. acidipropoinici exhibiting growth at a PA concentration of 20 g/L was isolated at the end of the experiment.Conclusions: Applying the current approach for PA fermentation can overcome several limitations for process industrialization

Propionic acid production from glycerol in immobilized cell bioreactor using an acid-tolerant strain of Propionibacterium acidipropionici obtained by adaptive evolution

Propionic acid (PA) production from agro-industrial residues using propionibacteria has gained interest as an alternative to fossil-based process. Microbial production is however characterized by product inhibition, lowering the product titers and productivity. In this study, Propionibacterium acidipropionici DSMZ4900 was subjected to adaptive evolution to tolerate higher acid concentrations. The strain adapted to growth in medium spiked with 20 g/L PA exhibited improved product titer (16.8 vs 8.72 g/L) and productivity (0.52 vs 0.17 g/L·h) with glycerol as carbon source in batch fermentations. It was immobilized on polyethyleneimine coated recycled glass beads Poraver® and used for fermentations in recycle batch mode with increasing glycerol concentration and decreasing pH, respectively. Doubling yeast extract concentration raised PA yield and productivity by >1.5 fold. Glycerol at 100 g/L was completely consumed to give ∼58 g/L PA at yield of 0.64 mol/mol and productivity of 0.28 g/L·h at pH 6.5. Decreasing fermentation pH to 5.0 increased PA productivity to 0.23 g/L·h from 0.14 g/L·h at pH 6.0 with 20 g/L glycerol, while immobilized cells exhibited no growth. The study shows combination of adaptive evolution and immobilization of cells to result in a robust system for PA fermentation at high glycerol concentration and lower pH

Stress induced biofilm formation in Propionibacterium acidipropionici and use in propionic acid production.

Propionibacterium acidipropionici produces propionic acid from different sugars and glycerol; the production can be improved by high cell density fermentations using immobilized cells that help to overcome the limitations of the non-productive lag phase and product inhibition. In this study, the use of stress factors to induce P. acidipropionici to form biofilm and its use as an immobilization procedure in fermentations in bioreactors for producing propionic acid was investigated. Citric acid and sodium chloride increased exopolysaccharide production, biofilm capacity index and trehalose production. Analysis of the expression of trehalose synthesis-related genes otsA and treY by RT-qPCR showed significantly increased expression of only treY during log phase with citric acid, while FISH analysis showed expression of treY and luxS under the influence of both stress factors. The stress factors were then used for development of microbial biofilms as immobilization procedure on Poraver® and AnoxKaldnes® carriers in recycle batch reactors for propionic acid production from 20 g/L glycerol. Highest productivities of 0.7 and 0.78 g/L/h were obtained in Poraver® reactors, and 0.39 and 0.43 g/L/h in AnoxKaldnes® reactors with citric acid and NaCl, respectively