Fermentative production of propionic acid: prospects and limitations of microorganisms and substrates.

Propionic acid is an important organic acid with wide industrial applications, especially in the food industry. It is currently produced from petrochemicals via chemical routes. Increasing concerns about greenhouse gas emissions from fossil fuels and a growing consumer preference for bio-based products have led to interest in fermentative production of propionic acid, but it is not yet competitive with chemical production. To improve the economic feasibility and sustainability of bio-propionic acid, fermentation performance in terms of concentration, yield, and productivity must be improved and the cost of raw materials must be reduced. These goals require robust microbial producers and inexpensive renewable feedstocks, so the present review focuses on bacterial producers of propionic acid and promising sources of substrates as carbon sources. Emphasis is placed on assessing the capacity of propionibacteria and the various approaches pursued in an effort to improve their performance through metabolic engineering. A wide range of substrates employed in propionic acid fermentation is analyzed with particular interest in the prospects of inexpensive renewable feedstocks, such as cellulosic biomass and industrial residues, to produce cost-competitive bio-propionic acid. KEY POINTS: • Fermentative propionic acid production emerges as competitor to chemical synthesis. • Various bacteria synthesize propionic acid, but propionibacteria are the best producers. • Biomass substrates hold promise to reduce propionic acid fermentation cost.

Propionic acid production by Propionibacterium freudenreichii using sweet sorghum bagasse hydrolysate.

Propionic acid, a widely used food preservative and intermediate in the manufacture of various chemicals, is currently produced from petroleum-based chemicals, raising concerns about its long-term sustainability. A key way to make propionic acid more sustainable is through fermentation of low-cost renewable and inedible sugar sources, such as lignocellulosic biomass. To this end, we utilized the cellulosic hydrolysate of sweet sorghum bagasse (SSB), a residue from a promising biomass source that can be cultivated around the world, for fermentative propionic acid production using Propionibacterium freudenreichii. In serum bottles, SSB hydrolysate supported a higher propionic acid yield than glucose (0.51 vs. 0.44 g/g, respectively), which can be attributed to the presence of additional nutrients in the hydrolysate enhancing propionic acid biosynthesis and the pH buffering capacity of the hydrolysate. Additionally, SSB hydrolysate supported better cell growth kinetics and higher tolerance to product inhibition by P. freudenreichii. The yield was further improved by co-fermenting glycerol, a renewable byproduct of the biodiesel industry, reaching up to 0.59 g/g, whereas volumetric productivity was enhanced by running the fermentation with high cell density inoculum. In the bioreactor, although the yield was slightly lower than in serum bottles (0.45 g/g), higher final concentration and overall productivity of propionic acid were achieved. Compared to glucose (this study) and hydrolysates from other biomass species (literature), use of SSB hydrolysate as a renewable glucose source resulted in comparable or even higher propionic acid yields. KEY POINTS: • Propionic acid yield and cell growth were higher in SSB hydrolysate than glucose. • The yield was enhanced by co-fermenting SSB hydrolysate and glycerol. • The productivity was enhanced under high cell density fermentation conditions. • SSB hydrolysate is equivalent or superior to other reported hydrolysates.

Prospects of integrating algae technologies into landfill leachate treatment.

Landfilling of municipal waste, an environmental challenge worldwide, results in the continuous formation of significant amounts of leachate, which poses a severe contamination threat to ground and surface water resources. Landfill leachate (LL) is generated by rainwater percolating through disposed waste materials and must be treated effectively before safe discharge into the environment. LL contains numerous pollutants and toxic substances, such as dissolved organic matter, inorganic chemicals, heavy metals, and anthropogenic organic compounds. Currently, LL treatment is carried out by a combination of physical, chemical, and microbial technologies. Microalgae are now viewed as a promising sustainable addition to the repertoire of technologies for treating LL. Photosynthetic algae have been shown to grow in LL under laboratory conditions, while some species have also been employed in larger-scale LL treatments. Treating leachate with algae can contribute to sustainable waste management at existing landfills by remediating low-quality water for recycling and reuse and generating large amounts of algal biomass for cost-effective manufacturing of biofuels and bioproducts. In this review, we will examine LL composition, traditional leachate treatment technologies, LL toxicity to algae, and the potential of employing algae at LL treatment facilities. Emphasis is placed on how algae can be integrated with existing technologies for biological treatment of LL, turning leachate from an environmental liability to an asset that can produce value-added biofuels and bioproducts for the bioeconomy.

Biochemical conversion of sweet sorghum bagasse to succinic acid.

Succinic acid, an important intermediate in the manufacture of plastics and other commodity and specialty chemicals, is currently made primarily from petroleum. We attempted to biosynthesize succinic acid through microbial fermentation of cellulosic sugars derived from the bagasse of sweet sorghum, a renewable feedstock that can grow in a wide range of climates around the world. We investigated pretreating sweet sorghum bagasse (SSB) with concentrated phosphoric acid at mild conditions (40-85°C) at various residence times and biomass concentrations. We then subjected the pretreated SSB to enzymatic hydrolysis with a commercial cellulase to release glucose. The highest glucose yield was obtained when SSB was pretreated at 50°C for 43 min at 130 g/L biomass concentration on dry basis. Fermentation was carried out with Actinobacillus succinogenes 130Z, which readily converted 29.2 g/L of cellulosic glucose to 17.8 g/L of succinic acid in a 3.5-L bioreactor sparged with CO2 at a rate of 0.5 vvm, thus reducing the carbon footprint of the process. Overall, we demonstrated, for the first time, the use of SSB for production of succinic acid using practices that lower energy use, future equipment cost, waste generation, and carbon footprint.

Characterization of the GH16 and GH17 laminarinases from Vibrio breoganii 1C10.

Laminarin is an abundant glucose polymer used as an energy reserve by micro- and macroalgae. Bacteria digest and consume laminarin with laminarinases. Their genomes frequently contain multiple homologs; however, the biological role for this replication remains unclear. We investigated the four laminarinases of glycoside hydrolase families GH16 and GH17 from the marine bacterium Vibrio breoganii 1C10, which can use laminarin as its sole carbon source. All four laminarinases employ an endolytic mechanism and specifically cleave the ß-1,3-glycosidic bond. Two primarily produce low-molecular weight laminarin oligomers (DP 3-4) whereas the others primarily produce high-molecular weight oligomers (DP > 8), which suggests that these enzymes sequentially degrade laminarin. The results from this work provide an overview of the laminarinases from a single marine bacterium and also provide insights regarding how multiple laminarinases are used to degrade laminarin.

Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose.

Catabolite repression refers to the process where the metabolism of one sugar represses the genes involved in metabolizing another sugar. While glucose provides the canonical example, many other sugars are also known to induce catabolite repression. However, less is known about the mechanism for catabolite repression by these non-glucose sugars. In this work, we investigated the mechanism of catabolite repression in the bacterium Escherichia coli during growth on lactose, L-arabinose, and D-xylose. The metabolism of these sugars is regulated in a hierarchical manner, where lactose is the preferred sugar, followed by L-arabinose, and then D-xylose. Previously, the preferential utilization of L-arabinose over D-xylose was found to result from transcriptional crosstalk. However, others have proposed that cAMP governs the hierarchical regulation of many non-glucose sugars. We investigated whether lactose-induced repression of L-arabinose and D-xylose gene expression is due to transcriptional crosstalk or cAMP. Our results demonstrate that it is due to cAMP and not transcriptional crosstalk. In addition, we found that repression is reciprocal, where both L-arabinose and D-xylose also repress the lactose gene expression, albeit to a lesser extent and also through a mechanism involving cAMP. Collectively, the results further our understanding of metabolism during growth on multiple sugars.

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.

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.