Heterotrophic production methods for microbial biomass and bioproducts

An animal feed material or human food comprising non-encysted non-viable microalgal cells classified as Haematococcus pluvialis and produced according to a method, the method comprising:

• (a) providing an organic acid as a carbon source into a culture medium;
• providing a viable microalgal cell wherein the microalgal cell is a rapidly growing motile cell that is a facultative heterotroph, and
• wherein the viable microalgal cell is a rapidly growing motile cell type that is recalcitrant to form cysts under stress by selecting a novel phenotype relative to the microalgal strain from which the microalgal cells are produced when growing in the dark in very low nutrient conditions including a carbon source by isolating a motile cell type away from cysts based on the ability to be phototaxic when a light source is provided at a top of a vessel,
• wherein the motile cell type isolated away retains its variant phenotype for numerous generations under heterotrophic conditions with robust growth to produce improved strains that are fast growing motile and are recalcitrant to form cysts under stress;
• (b) culturing the viable microalgal cell in the culture medium in the dark to produce from the viable microalgal cell a microalgal culture comprising microalgal cells and balancing a pH of the culture medium by the addition of the organic acid in a fed-batch manner without introduction of extra salt;
• wherein components of the culture medium are added to the culture medium in a fed-batch manner;
• wherein the culture medium is devoid of nitrates as a nitrogen source to minimize addition of salts and wherein urea is the primary source of nitrogen;
• wherein the culturing of the viable microalgal cell is performed under a condition of carotenogenesis for at least a portion of the culturing step;
• wherein the condition of carotenogenesis comprises the culture medium depleted of nutrients selected from one or more of sulfate, phosphate, or urea, or with ammonium accumulation in excess of 2.5 mM, in the presence of carbon from the organic acid;
• and
• wherein the production of the animal feed material or human food occurs during vegetative growth of the culture;
• (c) isolating the viable microalgal cells from the microalgal culture after the cells reach a density of at least 6 g/L during a period falling within a range of 24 to 144 hours of culturing, and before the cells form cyst cells;
• and
• (d) obtaining a non-viable microalgal biomass comprising the non-encysted Haematococcus pluvialis microalgal cells as the animal feed material or human food;
• whereby the animal feed material or human food comprising the non-encysted Haematococcus pluvialis microalgal cells is obtained;
• wherein the isolated non-encysted microalgal cells contain microalgal fatty acid C18:1ω7 and comprise microalgal fatty acids C16:0, C18:1ω9, C18:2ω6, C18:3ω3, C20:4ω6, and C20:5ω3;
• wherein the isolated non-encysted microalgal cells contain 3% to 9% ash per dry weight algal biomass;
• wherein the microalgal culture has an isoprenoid productivity rate of 1.875 mg/L-hour or higher;
• and
• wherein the isolated non-encysted microalgal cells are red, yellow, orange, achlorophyllic, or lacking pigment, and optionally, are mutagenized or genetically engineered, wherein red is the color of one carotenoid that comprises greater than 85% of the total carotenoids of a mixture of carotenoids, wherein the one carotenoid is a ketocarotenoid.