BackgroundIn comparison with phototrophic growth, heterotrophic conditions can significantly increase growth rates, final cell number and cell mass in microalgae cultures. Neochloris oleoabundans is a microalga of biotechnological interest that accumulates lipids under phototrophic and nitrogen-limited conditions. Heterotrophic flask culture experiments were conducted to identify carbon sources that can be metabolized by N. oleoabundans, and bioreactor batch and fed-batch (nitrate pulse additions) cultures supplemented with glucose were performed to study the cellular composition of the microalgae under balanced and high C/N ratios (glucose/nitrate).ResultsN. oleoabundans was able to grow using glucose and cellobiose as sole carbon sources under strict heterotrophic conditions. Under a balanced C/N ratio of 17 and using bioreactor batch cultures containing 3 g/L glucose, a maximal cell mass of 1.72 g/L was found, with protein being the major cell component (44% w/w). A maximal cell mass of 9.2 g/L was obtained using batch cultures at a C/N ratio of 278. Under these conditions, lipid accumulation was promoted (up to 52% w/w) through N-limitation, resulting in high lipid productivity (528.5 mg/L/day). Fed-batch cultures were performed at a C/N ratio of 278 and with nitrate pulse additions. This condition allowed a maximal cell mass of 14.2 g/L to be achieved and switched the metabolism to carbohydrate synthesis (up to 54% of dry weight), mainly in the form of starch. It was found that transmembrane transport under these conditions was dependent on a proton-motive force, indicating that glucose is transported by a symporter.ConclusionsN. oleoabundans was able to grow under strict heterotrophic culture conditions with glucose or cellobiose as the only carbon source. The glucose used is transported by a symporter system. Batch cultures with a balanced C/N ratio accumulate proteins as the major cellular component; a high C/N ratio significantly increased the dry cell mass and resulted in a high lipid content, and a high cell density was achieved using fed-batch cultures promoting carbohydrate accumulation. These results suggest heterotrophic batch cultures of N. oleoabundans as an alternative for the production of proteins or lipids with simple culture strategies and minimal-mineral media supplemented with glucose.

Summary Understanding the unique features of algal metabolism may be necessary to realize the full potential of algae as feedstock for the production of biofuels and biomaterials. Under nitrogen deprivation, the green alga C. reinhardtii showed substantial triacylglycerol (TAG) accumulation and up‐regulation of a gene, GPD2, encoding a multidomain enzyme with a putative phosphoserine phosphatase (PSP) motif fused to glycerol‐3‐phosphate dehydrogenase (GPD) domains. Canonical GPD enzymes catalyze the synthesis of glycerol‐3‐phosphate (G3P) by reduction of dihydroxyacetone phosphate (DHAP). G3P forms the backbone of TAGs and membrane glycerolipids and it can be dephosphorylated to yield glycerol, an osmotic stabilizer and compatible solute under hypertonic stress. Recombinant Chlamydomonas GPD2 showed both reductase and phosphatase activities in vitro and it can work as a bifunctional enzyme capable of synthesizing glycerol directly from DHAP. In addition, GPD2 and a gene encoding glycerol kinase were up‐regulated in Chlamydomonas cells exposed to high salinity. RNA‐mediated silencing of GPD2 revealed that the multidomain enzyme was required for TAG accumulation under nitrogen deprivation and for glycerol synthesis under high salinity. Moreover, a GPD2‐mCherry fusion protein was found to localize to the chloroplast, supporting the existence of a GPD2‐dependent plastid pathway for the rapid synthesis of glycerol in response to hyperosmotic stress. We hypothesize that the reductase and phosphatase activities of PSP–GPD multidomain enzymes may be modulated by post‐translational modifications/mechanisms, allowing them to synthesize primarily G3P or glycerol depending on environmental conditions and/or metabolic demands in algal species of the core Chlorophytes.

Microalgae are considered photoautotrophic organisms, however several species have been found living in environments where autotrophic metabolism is not viable. Heterotrophic cultivation, i.e. cell growth and propagation with the use of an external carbon source under dark conditions, can be used to study the metabolic aspects of microalgae that are not strictly related to photoautotrophic growth and to obtain high value products. This manuscript reviews studies related to the metabolic aspects of heterotrophic grow of microalga. From the physiological and metabolic perspective, the screening of microalgal strains in different environments and the development of molecular and metabolic engineering tools, will lead to an increase in the number of known microalgae species that growth under strict heterotrophic conditions and the variety of carbon sources used by these microorganisms.

Several microalgal species are capable of growing heterotrophically, exhibiting considerable metabolic versatility and flexibility. As demonstrated in this review, heterotrophic conditions can enhance the biomass concentration by as much as 25-fold compared with phototrophic conditions. Currently, these types of cultivation are economically feasible only for high-value products, including polyunsaturated fatty acids (PUFAs), pigments, antioxidants, polysaccharides, food and aquaculture feed from carbon sources, such as glucose, acetate or glycerol. To make heterotrophic cultivation economically viable for high-volume, low-value commodities, such as biofuels, the use of unconventional carbon sources, such as food and agricultural wastes and wastewater, is recommended. Since microalgae are capable of modifying their metabolism according to varying culture conditions, it is possible to modify, control and therefore maximize the production of target compounds. This manuscript not only offers a review of the most relevant and recent findings in the use of heterotrophic microalgal cultivation for enhanced metabolite production but also provides recommendations for future research on this promising subject.Fatty acids (FAs) are the base structure of almost all membrane lipids, making them an essential biomolecule for the viability of every cell. In all organisms, except for archaea, where the side chains of the membrane lipids are isoprenoids, FAs are organic molecules consisting of an aliphatic carbon chain with a carboxylic J Chem Technol Biotechnol 2017; 92: 925-936 HYDROCARBONSHydrocarbons are composed of a heterogeneous group of molecules of different sizes, shapes and/or lengths and molecular wileyonlinelibrary.com/jctb