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The rapid expansion of biodiesel production has generated a significant surplus of crude glycerol, necessitating effective valorization strategies. This study aimed to isolate and characterize indigenous yeast strains with exceptional capacity for utilizing crude glycerol as a primary carbon source for microbial biomass production. Eight robust yeast isolates (BK004–BK015), identified as Saccharomyces cerevisiae (99.8–100% sequence identity), were obtained from tree canopy soil at Rajabhat Rajanagarindra University, Thailand. Growth performance assays revealed the significant uniqueness of these BK strains; they exhibited remarkable biomass accumulation on crude glycerol (29.60–29.90 g L-1, fresh weight), which was comparable to their growth on glucose. In stark contrast, the reference strain S. cerevisiae TISTR 5596 showed markedly limited growth on glycerol, yielding only 3.80 g L-1—a nearly 8-fold difference in efficiency. The BK isolates achieved a biomass productivity (Qx) of 0.61–0.62 g L-1 h-1and an observed fresh biomass yield (Yx/s) of 2.97 g g-1, significantly outperforming the reference strain (Qx = 0.08 g L-1 h-1; Y x/s = 0.38g g-1). While these isolates produced approximately 40 g L-1of bioethanol from glucose, bioethanol production from glycerol was not observed under the tested conditions. This limitation is attributed to the intrinsic NADH/NAD+ redox imbalance in S. cerevisiae and the requirement for active respiratory activity to drive glycerol metabolism via the oxidative pathway (GUT pathway). Consequently, glycerol utilization in these indigenous strains is predominantly directed toward biomass synthesis rather than fermentative conversion. Overall, the superior performance of the BK isolates underscores the importance of bioprospecting for 'robust' wild-type strains that possess more efficient metabolic pathways for non-fermentable carbon sources than domesticated laboratory strains, offering a promising platform for sustainable industrial biodiesel byproduct valorization. GRAPHICAL ABSTRACT HIGHLIGHTS Eight indigenous Saccharomyces cerevisiae strains were isolated from soil and identified via 26S rDNA sequencing The isolated strains significantly outperformed the reference strain TISTR 5596 in glycerol utilization. Glycerol metabolism in BK isolates follows oxidative pathways, favoring biomass production over ethanol fermentation. The study highlights the potential for valorizing biodiesel-derived crude glycerol into value-added microbial biomass.