Effects of alcohol compounds on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans

Abstract

The inhibitors present in dilute acid-treated lignocellulosic hydrolysates would show great effect on the growth and product formation of microorganisms. To understand their inhibitory law and mechanism on oleaginous microorganism could help improving the efficiency of lignocellulose hydrolysis, detoxification, and lipid fermentation. The effects of four representative alcohol compounds present in lignocellulosic hydrolysates, including furfuryl alcohol, vanillyl alcohol, catechol, hydroquinone on the cell growth and lipid accumulation of Trichosporon fermentans were systematically investigated in this work. The toxicity of selected alcohol compounds was well related to their log P value except furfuryl alcohol, whose log P value was the minimum but with the highest toxicity to T. fermentans. The inhibition of all the alcohol compounds on the growth of T. fermentans was more serious than on the lipid synthesis. Also, the growth of T. fermentans was more sensitive to the variation of inoculum size, temperature, and initial pH than lipid synthesis in the presence of alcohol compounds. Initial pH had more profound influence on the lipid fermentation than inoculum size and cultural temperature did. Careful control of fermentation conditions could be helpful for improving lipid yield of T. fermentans in lignocellulosic hydrolysates. Among the four alcohol compounds tested, most alcohol compounds showed inhibition on both sugar consumption and malic enzyme activity of T. fermentans. However, vanillyl alcohol had little influence on the malic enzyme activity. Similarly, all alcohol compounds except vanillyl alcohol exerted damage on the cell membrane of T. fermentans.

Figure 1. Effect of selected alcohol compounds on the growth and lipid accumulation of T. fermentans.

(A) Biomass; (B) lipid content; (C) lipid yield; (D) sugar consumption. The biomass, lipid content, lipid yield, and sugar consumption of T. fermentans were 24.0 g/L, 61.7%, 14.8 g/L, and 84.3 g/L at the 7^th day in the medium without inhibitors. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol.

Figure 2. Effect of inoculum size on the inhibition of alcohol compounds.

Each alcohol compounds was tested at its concentration of IC₅₀. Cultures were incubated at initial pH 6.5, 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 in the absence of organic acids with 5%, 10% and 15% inoculum size were 24.0 g/L, 61.7% and 14.8 g/L; 22.4 g/L, 58.6% and 13.1 g/L; 21.6 g/L, 54.3% and 11.7 g/L, respectively. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol.

Figure 3. Effect of temperature on the inhibition of alcohol compounds.

All alcohol compounds were tested at their respective concentration of IC₅₀. 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 19.9 g/L, 55.8% and 11.1 g/L; 24.0 g/L, 61.7% and 14.8 g/L; 23.6 g/L, 58.9% and 13.9 g/L, respectively. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol.

Figure 4. Effect of initial pH on the inhibition of alcohol compounds.

All alcohol compounds were tested at their respective concentration of IC₅₀. 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 18.4 g/L, 57.2% and 10.5 g/L; 24.0 g/L, 61.7% and 14.8 g/L; 21.5 g/L, 56.3% and 12.1 g/L, respectively. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol.

Figure 5. Effect of the selected alcohol compounds on the sugar metabolism of T. fermentans.

(A) Glucose consumption; (B) Xylose consumption; (C) Total sugars consumption. The residual glucose, xylose, and total sugars in the control medium at the 2^nd, 4^th, 7^th and 10^th day were (g/L) 31.7, 23.7, 55.4; 8.8, 21.3, 30.1; 0, 15.7, 15.7; and 0, 9.9, 9.9, respectively. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol; Con, Control.

Figure 6. Effects of alcohol compounds on the malic enzyme activity of T. fermentans.

Biomass cultivated in the medium with and without inhibitor was harvested and disrupted to get the crude enzyme. Then the malic enzyme activity was measured and compared. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol; Con, Control.

Figure 7. Effects of alcohol compounds on the cell membrane integrity of T. fermentans.

X-axe: signal intensity; Y-axe: number of cells/mL (×1000). Cells of T. fermentans in the medium with and without inhibitor were stained at the same condition and then stained cells were analyzed by flow cytometry. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol; Con, Control.

Figure 8. PI uptake of T. fermentans’s cells in the medium containing different alcohol compounds.

Cells of T. fermentans in the medium with and without inhibitor were stained at the same condition and then stained cells were analyzed by flow cytometry. The PI uptake was calculated by Cell Lab Quanta SC software. Abbreviations: Ca, catechol; Hy, Hydroquinone; Fu, Furfuryl alcohol; Va, Vanillyl alcohol; Con, Control.