Continuous Cultivation Of The Oleaginous Yeast Rhodosporidium Toruloides Under Nutrient Limitation Conditions

Biodiesel is an environmentally friendly renewable liquid biofuel, which is now produced mainly from vegetable oil and animal fats that are not sustainable for large scale production. Microbial lipids, similar to vegetable oil in terms of fatty acid composition, are an alternative feedstock for biodiesel production and oleochemical industries as well. However, the high cost of microbial lipid production is the major bottleneck. The synthesis of microbial lipids is considered as a partially growth-associated process with inconstant production rates. In this study, a chemostat process was established for lipid production by the oleaginous yeast Rhodosporidium toruloides AS2.1389under nutrient limitation conditions, and models of lipid production were developed.It was found that culture at low dilution rate under nitrogen limitation was beneficial to achieve higher lipid yield and cellular lipid content. The lipid yield and lipid content were0.18±0.03g lipid/g sugar and57.1±4.8%, respectively, at the dilution rate of0.02h-1, but the lipid productivity was only0.05±0.1g/L/h. As the dilution rate increased to0.1h-1, the average lipid productivity improved to0.084g/L/h, while the lipid content dropped to40.7±5.9%. The impact of C/N ratio in the medium on the lipid production was further investigated at this dilution rate, and it was found that higher C/N ratio favored lipid accumulation.The highest lipid content of38.1±1.0%was observed at the C/N ratio of237mol/mol. No significant variations were observed in the fatty acid composition of the lipids produced under N limitation conditions, and more than85%of the total lipids were palmitic acid, stearic acid and oleic acid.The maintenance coefficient of5.7mg glucose/g cell/h and the maximal biomass yield of0.42g cell/g glucose were obtained for this yeast under carbon limitation conditions, which could be used for developing a cascade system for more efficient lipid production. Furthermore, a kinetic model was developed for lipid production under N limitation conditions, and the dilution rate for the highest specific lipid production was predicted to be0.05-0.09h-1. The experimental result of the highest specific lipid production rate was0.058±0.007g/g/h at the dilution rate of0.08h-1, which was in well aggrement with the kinetic model, indicating that the model would be useful for optimizing the lipid production. A correlation on carbon distribution between lipid and lipid-free cell components was also developed, and the maximal lipid yield YXmax and cell mass yield YLmax were predicted to be 0.52C-mol/C-mol and0.51C-mol/C-mol, respectively. Together with the element balance analysis, the critical C/N ratio for switching to lipid production was estimated to be12.1mol/mol. The predictions for lipid yields were95.2-116.7%of those measured experimentally, indicating the model is reliable.It was found that dilution rate had some effects on the composition of microbial lipids, and contents of soluble protein and carotenoids. The content of neutral lipids decreased with the increase of dilution rate, while the contents of glycolipid, sphingolipid and phospholipids were increased. This was supported by FTIR analysis. The asymmetric and symmetric bands at2,923cm-1and2,852cm-1for the-CH2group and the bands at1,745cm-1and1,725cm-1for the C=O group related to triglycerides (TGAs) and fatty acid esters were decreased as the dilution increased. Carotenoids and soluble proteins, major byproducts from the yeast after lipid extraction were analyzed, and the highest carotenoids of0.12mg/g cells was observed at the dilution rate of0.02h-1, but the highest soluble proteins of171.7mg/g cells was detected at the dilution rate of0.2h-1, which provides insights for a co-production of lipids and value-added by-products.Finally, cell recycle was incorporated into the chemostat system to improve lipid productivity, and a much higher lipid productivity of0.86g/L/h was achieved at the dilution rate of0.08h-1, almost7-fold increment compared to that of0.12g/L/h achieved with the chemostat system at the same dilution rate.