Isolation of xylose-utilizing yeasts from oil palm waste for xylitol and ethanol production

Abstract

The energy crisis triggers the use of energy sources that are renewable, such as biomass made from lignocellulosic materials, to produce various chemical compounds for food ingredients and biofuel. The efficient conversion of lignocellulosic biomass into products with added value involves the activity of microorganisms, such as yeasts. For the conversion, microorganisms must be able to use various sugars in lignocellulosic biomass, including pentose sugars, especially xylose. This study aims to isolate xylose-utilizing yeasts and analyze their fermentation activity to produce xylitol and ethanol, as well as their ability to grow in liquid hydrolysate produced from pretreated lignocellulosic biomass. Nineteen yeast isolates could grow on solid and liquid media using solely xylose as a carbon source. All isolates can grow in a xylose medium with incubation at 30 °C, 37 °C, 42 °C, and 45 °C. Six isolates, namely SLI (1), SL3, SL6, SL7, R5, and OPT4B, were chosen based on their considerable growth and high xylose consumption rate in a medium with 50 g/L xylose with incubation at 30 °C for 48 h. Four isolates tested, namely SLI (1), SL6, SL7, and R5, can produce xylitol in media containing xylose carbon sources. The concentration of xylitol produced was determined using high-pressure liquid chromatography (HPLC), and the results ranged from 5.0 to 6.0 g/L. Five isolates tested, namely SLI (1), SL6, SL3, R5, and OPT4B, can produce ethanol. The ethanol content produced was determined using gas chromatography (GC), with concentrations ranging from 0.85 to 1.34 g/L. Three isolates, namely SL1(1), R5, and SL6, were able to produce xylitol and ethanol from xylose as carbon sources and were also able to grow on liquid hydrolyzate from pretreated oil palm trunk waste with the subcritical water method. The three isolates were further analyzed using the 18S rDNA sequence to identify the species and confirm their phylogenetic position. Identification based on DNA sequence analysis revealed that isolates SL1(1) and R5 were Pichia kudriavzevii, while isolate SL6 was Candida xylopsoci. The yeast strains isolated from this study could potentially be used for the bioconversion process of lignocellulosic biomass waste to produce value-added derivative products.

Keywords: Ethanol; Lignocellulosic-biomass; Xylitol; Xylose; Yeast.

Fig. 1

Viable cells profiles of yeast isolates. Cells were precultured overnight in liquid YPD (glucose, 20 g/L) medium in shaking water bath at 100 rpm for 30 °C and then after serial dilution was laid on the YPX (xylose, 20 g/L) agar plate. Data points were obtained from three separately replicate experiments. Differences between replicates were < 10%

Fig. 2

Growth profiles of yeast isolates. Cells were grown in YPX (xylose 50 g/L) media for 72 h at 30 °C and 100 rpm. Three independently replicated experiments generated data points. The variance between repetitions was < 10%

Fig. 3

Cell dry weight profiles of yeast isolates. Cells were grown in YPX (xylose 50 g/L) media for 72 h at 30 °C and 100 rpm. Three independently replicated experiments generated data points. The variance between repetitions was < 10%

Fig. 4

Xylose consumption profiles by yeast isolates. Cells were grown in YPX (xylose 50 g/L) media for 72 h at 30 °C and 100 rpm. Two independently replicated experiments generated data points. The variance between repetitions was < 10%

Fig. 5

Growth profiles of yeast isolates at different temperatures. Cells were grown in YPX (xylose 50 g/L) media at 100 rpm at certain temperatures, i.e., 30, 37, 40, 42, 45 °C. Three independently replicated experiments generated data points. The variance between repetitions was < 10%

Fig. 6

Xylitol production profiles by yeast isolates. Cells were grown in YPX (xylose 50 g/L) media for 72 h at 30 °C and 100 rpm. Two independently replicated experiments generated data points. The variance between repetitions was < 10%

Fig. 7

Ethanol production in YPD (glucose 100 g/L) (A) and YPX (xylose 50 g/L) (B) medium. Cells were cultured in medium at 30 °C, 100 rpm, 72 h. Two independently replicated experiments generated data points. The variance between repetitions was < 10%

Fig. 8

OD profiles of yeast isolates grown in liquid hydrolysate extracted from pretreated oil palm trunk waste. Cells were grown in liquid hydrolysate from pretreated oil palm trunk waste and incubated at 30 °C and 100 rpm. Three independently replicated experiments generated data points. The variance between repetitions was < 10%

Fig. 9

Dendrogram of the phylogenetic relations of the yeast Pichia kudriavzevii SL1(1) with other closely related species and strains, obtained from NCBI GenBank. The number written next to the name is the GenBank accession number of the reference strain

Fig. 10

Dendrogram of the phylogenetic relations of the yeast Pichia kudriavzevii R5 with other closely related species and strains, obtained from NCBI GenBank. The number written next to the name is the GenBank accession number of the reference strain

Fig. 11

Dendrogram of the phylogenetic relations of the yeast Candida xylopsoci SL6 with other closely related species and strains, obtained from NCBI GenBank. The number written next to the name is the GenBank accession number of the reference strain