Effects of inhibitory compounds derived from lignocellulosic biomass on the growth of the wild-type and evolved oleaginous yeast Rhodosporidium toruloides

The presence of toxic compounds in lignocellulosic hydrolysates is one of the main problems affecting the efficiency of hydrolysate-based fermentation processes. Understanding the effects of biomass-derived inhibitors on the performance of the microbial strain is essential to develop strategies able to result in an improved fermentation performance. In the present study, efforts were done to elucidate the effects of the main biomass-derived inhibitors on the growth of the oleaginous yeast Rhodosporidium toruloides. Furfural, 5-hydroxymethylfurfural (5-HMF), acetic acid, levulinic acid, benzoic acid, p-coumaric acid, ferulic acid, vanillic acid, vanillin and syringaldehyde were the inhibitory compounds investigated. Assays were performed using a wild-type and an evolved strain of R. toruloides. For the individual inhibitors, more than 3 mM benzoic acid completely inhibited the growth of both strains, while acetic acid and levulinic acid were less toxic, presenting IC50 values of 16 and 15 mM, respectively. In a subsequent step, assays were performed in media containing different combinations of inhibitory compounds and a Placket-Burman experimental design was used to identify the most severe inhibitors in mixture, as well as combination of inhibitors, affecting the yeast growth. Results revealed that in mixture, furfural was the most potent inhibitor affecting the growth of R. toruloides, followed by vanillin and 5-HMF. The combination of furfural, 5-HMF, vanillin, vanillic acid and ferulic acid resulted in the most severe inhibition to this yeast. The evolved strain showed an improved ability to tolerate toxic compounds, suggesting that the adaptive laboratory evolution is a potential strategy to obtain oleaginous yeasts with improved ability to grow in biomass hydrolysates.