Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High‐cell‐density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh‐cell‐density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5‐L bench‐scale and 1,000‐L pilot‐scale fermenters, respectively. The outdoor scale‐up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot‐scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open‐pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large‐scale microalgal inocula production, but ultrahigh‐productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot‐scale.

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high‐value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark‐grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L⁻¹ day⁻¹) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark‐grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high‐light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L⁻¹ day⁻¹ by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark‐grown cells under photo‐induction conditions. Biotechnol. Bioeng. 2016;113: 2088–2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

The lack of lipidome analytical tools has limited our ability to gain new knowledge about lipid metabolism in microalgae, especially for membrane glycerolipids. An electrospray ionization mass spectrometry-based lipidomics method was developed for Nannochloropsis oceanica IMET1, which resolved 41 membrane glycerolipids molecular species belonging to eight classes. Changes in membrane glycerolipids under nitrogen deprivation and high-light (HL) conditions were uncovered. The results showed that the amount of plastidial membrane lipids including monogalactosyldiacylglycerol, phosphatidylglycerol, and the extraplastidic lipids diacylglyceryl-O-4’-(N, N, N,-trimethyl) homoserine and phosphatidylcholine decreased drastically under HL and nitrogen deprivation stresses. Algal cells accumulated considerably more digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerols under stresses. The genes encoding enzymes responsible for biosynthesis, modification and degradation of glycerolipids were identified by mining a time-course global RNA-seq data set. It suggested that reduction in lipid contents under nitrogen deprivation is not attributable to the retarded biosynthesis processes, at least at the gene expression level, as most genes involved in their biosynthesis were unaffected by nitrogen supply, yet several genes were significantly up-regulated. Additionally, a conceptual eicosapentaenoic acid (EPA) biosynthesis network is proposed based on the lipidomic and transcriptomic data, which underlined import of EPA from cytosolic glycerolipids to the plastid for synthesize EPA-containing chloroplast membrane lipids.

Marine cyanobacteria represent a large untapped source of functional glycolipids enriched with polyunsaturated fatty acids (PUFAs) for human health. However, advanced methods for scalable isolation of diverse species containing high-purity PUFA-rich glycolipids will have to be developed and their possible pharmaceutical and nutraceutical functions identified. This paper introduces a novel solid matrix-supported supercritical CO₂ extraction method for scalable isolation of the PUFA γ-linolenic acid (GLA)-enriched glycolipids from the cyanobacterium Arthrospira (Spirulina) platensis, which has been the most widely used among microalgae in the nutraceutical and pharmaceutical industries. Of various porous materials studied, diatomite was the best to facilitate extraction of GLA-rich glycolipids, resulting in an extraction efficiency of 98%. Gamma-linolenic acid made up 35% of total fatty acids (TFAs) in the extracts, which was considerably greater than that obtained with ethanol (26%), Bligh and Dyer (24%), and in situ transesterification (24%) methods, respectively. Lipidomics analysis revealed that GLA was exclusively associated with galactolipids. Pharmaceutical functions of GLA-rich galactolipids were investigated on a zebrafish caudal fin regeneration model. The results suggested that GLA extracted from A. platensis possessed anti-oxidative, anti-inflammatory, and anti-allergic activities, which acted in a concerted manner to promote post-injury regeneration of zebrafish.

The utilization of gibberellic acid-3, high carbon/nitrogen ratio and salinity concentration can effectively enhance astaxanthin biosynthesis in Chromochloris zofingiensis under the heterotrophic conditions, but the underlying mechanisms remained yet to be investigated. The metabolomics analysis revealed that enhancement of the glycolysis, pentose phosphate pathways (PPP), and tricarboxylic acid (TCA) cycle led to astaxanthin accumulation under the induction conditions. The increased fatty acids can significantly increase astaxanthin esterification. The addition of appropriate concentrations of glycine (Gly) and γ-aminobutyric acid (GABA) promoted astaxanthin biosynthesis in C. zofingiensis, as well as benefiting for biomass yield. With the addition of 0.5 mM GABA, the astaxanthin yield increased to 0.35 g·L^-1, which was 1.97-fold higher than that of the control. This study advanced understanding about astaxanthin biosynthesis in heterotrophic microalga, and provided novel strategies for enhanced astaxanthin production in C. zofingiensis.