Ammonium ion concentration-dependent production of extracellular polysaccharides from glycerol by an oleaginous yeast: visualization of the producer cells

ABSTRACT In the oleochemical industry, a fat-splitting process is used to generate a methanol-free byproduct, oleochemical industrial waste, that includes crude glycerol. The oleaginous yeast Rhodotorula toruloides is a nonconventional yeast that is well known for its industrial potential as a producer of lipids, carotenoids, and enzymes. Extracellular polysaccharides (EPSs) are of increasing interest due to their many applications. In this study, we found that R. toruloides NBRC 8766 strain produced mannose-rich and high-molecular-weight EPSs with a mannose content (mol%) exceeding 90% and an estimated molecular weight greater than 10,000 k from oleochemical industrial waste crude glycerol or pure glycerol. Components in the industrial waste increased EPS production. The concentration of NH 4 + ions in the culture medium was identified as a key factor in discriminating the cells producing the EPS (EPS-producing cells) from EPS-nonproducing cells. Further investigations demonstrated that a sufficient concentration of NH 4 + ions (38 mM or more) to acidify the medium to a pH of 2.0 or lower is key to EPS production under these conditions. Therefore, it was hypothesized that the R. toruloides NBRC 8766 strain would produce EPS in response to extracellular acidity. Fluorescent and transmission electron microscopic analyses of the two cell types revealed differences; the EPS-producing cells had immature lipid droplets and marked two-layered cell walls, where the outer layer was darker and looked like it was wearing “fuzz,” which was assumed to be the EPSs released from the cells. The EPS-nonproducing cells had clear lipid droplets, one-layered cell walls, and more abundant intracellular polysaccharide granules. IMPORTANCE In this study, we demonstrated that oleochemical industrial waste is a promising source of crude glycerol for EPS production and that R. toruloides is a promising producer of not only lipids but also EPSs from glycerol. Moreover, the identification of the concentration of NH 4 + ions in the culture medium enabled the discrimination of EPS-producing cells from EPS-nonproducing cells. The presence of sufficient NH 4 + ions to acidify the medium was demonstrated to be key to EPS production in these conditions. This suggests that the R. toruloides NBRC 8766 strain produces EPS in response to extracellular acidity. The morphological observations of the EPS-producing and EPS-nonproducing cells provide a further basis for understanding the molecular mechanism underlying the production of mannose-rich and high-molecular weight-EPS, as well as clues for artificially increasing the amounts of EPSs and lipids produced.