Nitrate concentration-shift cultivation to enhance protein content of heterotrophic microalga Chlorella vulgaris: Over-compensation strategy

•Over-compensation of nitrogen can improve protein content of heterotrophic Chlorella.•Convenient nitrate-added culture was adopted to implement concentration shift.•Operating parameters were optimized to maximize protein content by one factor RSM.•Chlorella was a potential single-cell protein source according to amino acid profile. Protein production from microalgae requires both high cell density during cultivation and high protein content in cells. Heterotrophic microalgae can achieve high cell density, and yet are confronted with the problem of low protein content. Based on over-compensation strategy, a new concentration-shift method was proposed to cultivate heterotrophic Chlorella vulgaris, aiming to increase protein content. With a prior starvation period, microalgae utilized more nitrate and accumulated more proteins compared to one-stage cultivation. Considering the convenience of operation, nitrate-added culture was adopted for producing heterotrophic microalgae, rather than sterile centrifugal culture. Operating parameters including nitrate concentration in N-deficient medium, N-starved time and nitrate concentration in N-rich medium were optimized, which were 0.18gl−1, 38h and 2.45gl−1, respectively. Under the optimized conditions, protein content in heterotrophic Chlorella reached 44.3%. Furthermore, the heterotrophic microalga was suggested to be a potential single-cell protein source according to the amino acid composition.