Effect of organic acids on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans

Abstract

Background: Microbial lipids have drawn increasing attention in recent years as promising raw materials for biodiesel production, and the use of lignocellulosic hydrolysates as carbon sources seems to be a feasible strategy for cost-effective lipid fermentation with oleaginous microorganisms on a large scale. During the hydrolysis of lignocellulosic materials with dilute acid, however, various kinds of inhibitors, especially large amounts of organic acids, will be produced, which substantially decrease the fermentability of lignocellulosic hydrolysates. To overcome the inhibitory effects of organic acids, it is critical to understand their impact on the growth and lipid accumulation of oleaginous microorganisms.

Results: In our present work, we investigated for the first time the effect of ten representative organic acids in lignocellulosic hydrolysates on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans cells. In contrast to previous reports, we found that the toxicity of the organic acids to the cells was not directly related to their hydrophobicity. It is worth noting that most organic acids tested were less toxic than aldehydes to the cells, and some could even stimulate the growth and lipid accumulation at a low concentration. Unlike aldehydes, most binary combinations of organic acids exerted no synergistic inhibitory effects on lipid production. The presence of organic acids decelerated the consumption of glucose, whereas it influenced the utilization of xylose in a different and complicated way. In addition, all the organic acids tested, except furoic acid, inhibited the malic activity of T. fermentans. Furthermore, the inhibition of organic acids on cell growth was dependent more on inoculum size, temperature and initial pH than on lipid content.

Conclusions: This work provides some meaningful information about the effect of organic acid in lignocellulosic hydrolysates on the lipid production of oleaginous yeast, which is helpful for optimization of biomass hydrolysis processes, detoxified pretreatment of hydrolysates and lipid production using lignocellulosic materials.

Figure 1

Effect of initial sugar concentration on the growth and lipid accumulation of Trichosporon fermentans. Triangles, biomass; circles, lipid content; squares, lipid yield; inverted triangles, sugar consumption.

Figure 2

Production of microbial oils on medium without inhibitors by Trichosporon fermentans. (A) Time courses of growth and lipid accumulation. Squares, biomass; circles, lipid content; triangles, lipid yield. (B) Time course of sugar utilization. Squares, glucose; circles, xylose; triangles, total sugars.

Figure 3

Effect of selected organic acids on the growth and lipid accumulation of Trichosporon fermentans. The biomass, lipid content, lipid yield and sugar consumption of T. fermentans on the seventh day in medium without inhibitors were, respectively, 24.0 g/L, 61.7%, 14.8 g/L and 84.3 g/L. Open squares, relative biomass; closed triangles, relative lipid content; closed circles, relative lipid yield; open inverted triangles, relative sugar consumption.

Figure 4

Effect of the selected organic acids on the sugar metabolism of Trichosporon fermentans. (A) and (D) Glucose consumption. (B) and (E) Xylose consumption. (C) and (F) Total sugar consumption. Ac, acetic acid; Fo, formic acid; Le, levulinic acid; Hy, 4-hydroxybenzoic acid; Fu, furoic acid; Ca, caproic acid; Ga, gallic acid; Fe, ferulic acid; Sy, syringic acid; Va, vanillic acid.

Figure 5

Effects of selected organic acids on malic enzyme activity in Trichosporon fermentans. Con, control; Ac, acetic acid; Fo, formic acid; Le, levulinic acid; Hy, 4-hydroxybenzoic acid; Fu, furoic acid; Ca, caproic acid; Ga, gallic acid; Fe, ferulic acid; Sy, syringic acid; Va, vanillic acid.

Figure 6

Effect of inoculum size on the inhibition of organic acids. Each organic acid was tested at the molar concentration of the tested organic acids that caused 50% inhibition of the lipid yield of Trichosporon fermentans. Cultures were incubated initially at pH 6.5, 25°C and 160 rpm for 7 days. The results are expressed relative to controls without organic acids. Biomass, lipid content and lipid yield of cultures in the absence of organic acids with 5%, 10% and 15% inoculum sizes were, respectively, 24.0 g/L, 61.7% and 14.8 g/L; 22.4 g/L, 58.6% and 13.1 g/L; and 21.6 g/L, 54.3% and 11.7 g/L. Ac, acetic acid; Fo, formic acid; Le, levulinic acid; Hy, 4-hydroxybenzoic acid; Fu, furoic acid; Ca, caproic acid; Ga, gallic acid; Fe, ferulic acid; Sy, syringic acid; Va, vanillic acid.

Figure 7

Effect of temperature on the inhibition of organic acids. All organic acids were tested at their respective molar concentrations of the tested organic acids that caused 50% inhibition of the lipid yield. Cultures with 5% inoculum size were incubated at initial pH 6.5 and 160 rpm for 7 days. Results are expressed relative to controls without organic acids. Biomass, lipid content and lipid yield of cultures lacking organic acids at 22°C, 25°C and 28°C were, respectively, 19.9 g/L, 55.8% and 11.1 g/L; 24.0 g/L, 61.7% and 14.8 g/L; and 23.6 g/L, 58.9% and 13.9 g/L. Ac, acetic acid; Fo, formic acid; Le, levulinic acid; Hy, 4-hydroxybenzoic acid; Fu, furoic acid; Ca, caproic acid; Ga, gallic acid; Fe, ferulic acid; Sy, syringic acid; Va, vanillic acid.

Figure 8

Effect of initial pH on the inhibition of organic acids. All organic acids were tested at their respective molar concentrations of the tested organic acids that caused 50% inhibition of the lipid yield. Cultures with 5% inoculum size were incubated at 25°C and 160 rpm for 7 days. Results are expressed relative to controls without organic acids. Biomass, lipid content and lipid yield of cultures lacking organic acids at pH 5.5, pH 6.5 and pH 7.5 were, respectively, 18.4 g/L, 57.2% and 10.5 g/L; 24.0 g/L, 61.7% and 14.8 g/L; and 21.5 g/L, 56.3% and 12.1 g/L. Ac, acetic acid; Fo, formic acid; Le, levulinic acid; Hy, 4-hydroxybenzoic acid; Fu, furoic acid; Ca, caproic acid; Ga, gallic acid; Fe, ferulic acid; Sy, syringic acid; Va, vanillic acid.

Figure 9

Effect of binary combinations on the growth and lipid accumulation of Trichosporon fermentans. (A) Acetic acids (molar concentration of the tested organic acids that caused 25% inhibition of the lipid yield (IC₂₅)). (B) 4-hydroxybenzoic acid (IC₂₅). Ac, acetic acid; Fo, formic acid; Le, levulinic acid; Hy, 4-hydroxybenzoic acid; Fu, furoic acid; Ca, caproic acid; Ga, gallic acid; Fe, ferulic acid; Sy, syringic acid; Va, vanillic acid.