Galacturonic acid inhibits the growth of Saccharomyces cerevisiae on galactose, xylose, and arabinose

Abstract

The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pK(a) value of galacturonic acid (3.51), the addition of 10 g · liter(-1) galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter(-1) galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks.

Fig 1

Impact of galacturonic acid on performance of S. cerevisiae CEN.PK 113-7D during growth on glucose-galactose mixtures in batch fermentations. The result of one representative batch experiment is shown for each condition. Replicate experiments yielded essentially the same results. Fermentation performance is indicated by the CO₂ (percent) in the exhaust gas of anaerobic batch cultures of S. cerevisiae CEN.PK 113-7D, which were flushed with nitrogen gas at a constant rate of 0.5 liters · liter⁻¹ · h⁻¹. (A) Cultures grown at pH 5.0 on a mixture of 10 g · liter⁻¹ glucose and 10 g · liter⁻¹ galactose with either 0 g · liter⁻¹ (●), 5 g · liter⁻¹ (■), or 10 g · liter⁻¹ (▲) galacturonic acid. (B) Cultures grown at pH 3.5 on a mixture of 10 g · liter⁻¹ glucose, 10 g · liter⁻¹ galactose, and either 0 g · liter⁻¹ (●), 2.5 g · liter⁻¹ (○), 5 g · liter⁻¹ (■), 7.5 g · liter⁻¹ (□), or 10 g · liter⁻¹ (▲) galacturonic acid. (C) Cultures grown at pH 3.5 on a mixture of 10 g · liter⁻¹ glucose and 10 g · liter⁻¹ glucuronic acid (△).

Fig 2

Growth and metabolite production in anaerobic batch cultures of S. cerevisiae CEN. PK113-7D cultivated in duplicate at pH 3.5 on a mixture of 10 g · liter⁻¹ glucose (□) and 10 g · liter⁻¹ galactose (▲) in the absence of galacturonic acid (A) and in the presence of 10 g · liter⁻¹ galacturonic acid (B). Ethanol (●), glycerol (○), and biomass dry weight (DW) (■) were formed during these fermentations.

Fig 3

Growth and metabolite production in anaerobic batch cultures of S. cerevisiae IMS0010 grown at pH 3.5 on a mixture of 20 g · liter⁻¹ glucose (□), 10 g · liter⁻¹ xylose (△), and 10 g · liter⁻¹ arabinose (▼) in the absence of galacturonic acid (A and C) and in the presence of 10 g · liter⁻¹ galacturonic acid (B and D). Ethanol (●), glycerol (○), and biomass dry weight (■) were formed during these fermentations. The data are from single-batch cultivations and are representative of duplicate experiments.