Zhongqiang Wang

Propionic acid production in glycerol/glucose co-fermentation by Propionibacterium freudenreichii subsp. shermanii

Propionibacterium freudenreichii subsp. shermanii can ferment glucose and glycerol to propionic acid with acetic and succinic acids as two by-products. Propionic acid production from glucose was relatively fast (0.19 g/Lh) but gave low product yield (~0.39 g/g) and selectivity (P/A: ~2.6; P/S: ~4.8). In contrast, glycerol with a more reduced state gave a high propionic acid yield (~0.65 g/g) and selectivity (P/A: ~31; P/S: ~11) but low productivity (0.11 g/L h). On the other hand, co-fermentation of glycerol and glucose at an appropriate mass ratio gave both a high yield (0.54-0.65 g/g) and productivity (0.18-0.23 g/L h) with high product selectivity (P/A: ~14; P/S: ~10). The carbon flux distributions in the co-fermentation as affected by the ratio of glycerol/glucose were investigated. Finally, co-fermentation with cassava bagasse hydrolysate and crude glycerol in a fibrous-bed bioreactor was demonstrated, providing an efficient way for economic production of bio-based propionic acid.

Stable high-titer n-butanol production from sucrose and sugarcane juice by Clostridium acetobutylicum JB200 in repeated batch fermentations.

The production of n-butanol, a widely used industrial chemical and promising transportation fuel, from abundant, low-cost substrates, such as sugarcane juice, in acetone-butanol-ethanol (ABE) fermentation was studied with Clostridium acetobutylicum JB200, a mutant with high butanol tolerance and capable of producing high-titer (>20 g/L) n-butanol from glucose. Although JB200 is a favorable host for industrial bio-butanol production, its fermentation performance with sucrose and sugarcane juice as substrates has not been well studied. In this study, the long-term n-butanol production from sucrose by JB200 was evaluated with cells immobilized in a fibrous-bed bioreactor (FBB), showing stable performance with high titer (16-20 g/L), yield (∼ 0.21 g/g sucrose) and productivity (∼ 0.32 g/Lh) for 16 consecutive batches over 800 h. Sugarcane thick juice as low-cost substrate was then tested in 3 consecutive batches, which gave similar n-butanol production, demonstrating that JB200 is a robust and promising strain for industrial ABE fermentation.

Metabolic engineering of Propionibacterium freudenreichii for n-propanol production.

Propionibacteria are widely used in industry for manufacturing of Swiss cheese, vitamin B12, and propionic acid. However, little is known about their genetics and only a few reports are available on the metabolic engineering of propionibacteria aiming at enhancing fermentative production of vitamin B12 and propionic acid. n-Propanol is a common solvent, an intermediate in many industrial applications, and a promising biofuel. To date, no wild-type microorganism is known to produce n-propanol in sufficient quantities for industrial application purposes. In this study, a bifunctional aldehyde/alcohol dehydrogenase (adhE) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. The mutants expressing the adhE gene converted propionyl- coenzyme A, which is the precursor for propionic acid biosynthesis, to n-propanol. The production of n-propanol was limited by NADH availability, which was improved significantly by using glycerol as the carbon source. Interestingly, the improved propanol production was accompanied by a significant increase in propionic acid productivity, indicating a positive effect of n-propanol biosynthesis on propionic acid fermentative production. To our best knowledge, this is the first report on producing n-propanol by metabolically engineered propionibacteria, which offers a novel route to produce n-propanol from renewable feedstock, and possibly a new way to boost propionic acid fermentation.

Engineering Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing propionyl-CoA:Succinate CoA transferase

Propionibacterium freudenreichii subsp. shermanii naturally forms propionic acid as the main fermentation product with acetate and succinate as two major by-products. In this study, overexpressing the native propionyl-CoA:succinate CoA transferase (CoAT) in P. shermanii was investigated to evaluate its effects on propionic acid fermentation with glucose, glycerol, and their mixtures as carbon source. In general, the mutant produced more propionic acid, with up to 10% increase in yield (0.62 vs. 0.56g/g) and 46% increase in productivity (0.41 vs. 0.28g/Lh), depending on the fermentation conditions. The mutant also produced less acetate and succinate, with the ratios of propionate to acetate (P/A) and succinate (P/S) in the final product increased 50% and 23%, respectively, in the co-fermentation of glucose/glycerol. Metabolic flux analysis elucidated that CoAT overexpression diverted more carbon fluxes toward propionic acid, resulting in higher propionic acid purity and a preference for glycerol over glucose as carbon source.

Metabolic engineering of Propionibacterium freudenreichii: effect of expressing phosphoenolpyruvate carboxylase on propionic acid production

Propionic acid is currently produced mainly via petrochemicals, but there is increasing interest in its fermentative production from renewable biomass. However, the current propionic acid fermentation process suffers from low product yield and productivity. In this work, the gene encoding phosphoenolpyruvate carboxylase (PPC) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. PPC catalyzes the conversion of phosphoenolpyruvate to oxaloacetate with the fixation of one CO2. Its expression in P. freudenreichii showed profound effects on propionic acid fermentation. Compared to the wild type, the mutant expressing the ppc gene grew significantly faster, consumed more glycerol, and produced propionate to a higher final titer at a faster rate. The mutant also produced significantly more propionate from glucose under elevated CO2 partial pressure. These effects could be attributed to increased CO2 fixation and resulting changes in the flux distributions in the dicarboxylic acid pathway.

High cell density propionic acid fermentation with an acid tolerant strain of Propionibacterium acidipropionici.

Propionic acid is an important chemical with wide applications and its production via fermentation is of great interest. However, economic production of bio‐based propionic acid requires high product titer, yield, and productivity in the fermentation. A highly efficient and stable high cell density (HCD) fermentation process with cell recycle by centrifugation was developed for propionic acid production from glucose using an acid‐tolerant strain of Propionibacterium acidipropionici, which had a higher specific growth rate, productivity, and acid tolerance compared to the wild type ATCC 4875. The sequential batch HCD fermentation at pH 6.5 produced propionic acid at a high titer of ∼40 g/L and productivity of 2.98 g/L h, with a yield of ∼0.44 g/g. The product yield increased to 0.53–0.62 g/g at a lower pH of 5.0–5.5, which, however, decreased the productivity to 1.28 g/L h. A higher final propionic acid titer of >55 g/L with a productivity of 2.23 g/L h was obtained in fed‐batch HCD fermentation at pH 6.5. A 3‐stage simulated fed‐batch process in serum bottles produced 49.2 g/L propionic acid with a yield of 0.53 g/g and productivity of 0.66 g/L h. These productivities, yields and propionic acid titers were among the highest ever obtained in free‐cell propionic acid fermentation. Biotechnol. Bioeng. 2015;112: 502–511.

Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for xylose fermentation.

Propionibacterium freudenreichii cannot use xylose, the second most abundant sugar in lignocellulosic biomass. Although Propionibacterium acidipropionici can use xylose as a carbon source, it is difficult to genetically modify, impeding further improvement through metabolic engineering. This study identified three xylose catabolic pathway genes encoding for xylose isomerase (xylA), xylose transporter (xylT), and xylulokinase (xylB) in P. acidipropionici and overexpressed them in P. freudenreichii subsp. shermanii via an expression plasmid pKHEM01, enabling the mutant to utilize xylose efficiently even in the presence of glucose without glucose-induced carbon catabolite repression. The mutant showed similar fermentation kinetics with glucose, xylose, and the mixture of glucose and xylose, respectively, as carbon source, and with or without the addition of antibiotic for selection pressure. The engineered P. shermanii thus can provide a novel cell factory for industrial production of propionic acid and other value-added products from lignocellulosic biomass.

Effects of carbon dioxide on cell growth and propionic acid production from glycerol and glucose by Propionibacterium acidipropionici

The effects of CO2 on propionic acid production and cell growth in glycerol or glucose fermentation were investigated in this study. In glycerol fermentation, the volumetric productivity of propionic acid with CO2 supplementation reached 2.94g/L/day, compared to 1.56g/L/day without CO2. The cell growth using glycerol was also significantly enhanced with CO2. In addition, the yield and productivity of succinate, the main intermediate in Wood-Werkman cycle, increased 81% and 280%, respectively; consistent with the increased activities of pyruvate carboxylase and propionyl CoA transferase, two key enzymes in the Wood-Werkman cycle. However, in glucose fermentation CO2 had minimal effect on propionic acid production and cell growth. The carbon flux distributions using glycerol or glucose were also analyzed using a stoichiometric metabolic model. The calculated maintenance coefficient (mATP) increased 100%, which may explain the increase in the productivity of propionic acid in glycerol fermentation with CO2 supplement.

Propionic acid production from soy molasses by Propionibacterium acidipropionici: Fermentation kinetics and economic analysis

Propionic acid (PA) is a specialty chemical; its calcium salt is widely used as food preservative. Soy molasses (SM), a low-value byproduct from soybean refinery, contains sucrose and raffinose-family oligosaccharides (RFO), which are difficult to digest for most animals and industrial microorganisms. The feasibility of using SM for PA production by P. acidipropionici, which has genes encoding enzymes necessary for RFO hydrolysis, was studied. With corn steep liquor as the nitrogen source, stable long-term PA production from SM was demonstrated in sequential batch fermentations, achieving PA productivity of >0.8 g/L h and yield of 0.42 g/g sugar at pH 6.5. Economic analysis showed that calcium propionate as the main component (63.5%) in the product could be produced at US

Propionic Acid Fermentation

1.55/kg for a 3000-MT plant with a capital investment of US