High‐Yield Production of Lutein by the Green Microalga Chlorella protothecoides in Heterotrophic Fed‐Batch Culture

The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7‐L fermenter containing 40 g/L glucose and 3.6 g/L urea. In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source. As a result, a maximum cell dry weight concentration of 48 g/L was achieved. This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions. In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed‐batch mode to establish a nitrogen‐limited culture. Such a modification resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the N‐sufficient culture. The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This N‐limited fed‐batch culture was successfully scaled up from 3.7 L to 30 L, and a three‐step cultivation process was developed for the high‐yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L). The maintenance of the culture at a higher temperature (i.e., 32 °C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed‐batch culture (30 L) without such a treatment. The enhancement of lutein production resulted from the combination of nitrogen limitation and high‐temperature stress.