Thereafter, while the culture volume reach 1 L and more,
the lipid content were determined by modified Bligh and Dyer
method [16]. The extraction of microalgae biomass was use
mixed solvent of methanol:chloroform:water (1:2:1) that
found as best solvent. After vigorously vortex the biomass
with the solvent resulting three layers of organic, aqueous
layers and settling biomass containing the water. The
aqueous (contain chloroform) layer were collected and evaporated
to dryness, resulting the crude oil (lipid). Gravimetrically
the dry crude lipid was storage. Total carbohydrate from
microalgae biomasses were determined by phenol-sulfuric
acid assay that measured at l 490 nm [17]. Protein determination
was using Bradford assay [18] that measured by spectrophotometric
at l 595 nm.