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Botryococcus braunii
, a green alga that can produce a variety of secondary metabolites such as lipids, protein, and carotenoid, is considered a potential biodiesel feedstock for industrial commercialization. This study investigated the effects of exogenous plant growth regulators (PGRs) (6-benzylaminopurine (6-BA), abscisic acid (ABA), 2,4-epibrassinolide (EBR), ethephon (ETH), gibberellic acid (GA
3
), 1-naphthaleneacetic acid (NAA), salicylic acid (SA), and spermidine (SPD)) on the growth, protein, carotenoid and lipid biosynthesis, fatty acid composition, and expression of fatty acid biosynthetic genes in
Botryococcus braunii
B12 (
B. braunii
B12). NAA was the most effective inducer of microalgae biomass, which increased approximately 2-fold compared with that of the control. The maximum lipid accumulation and content were found under 15 mg/L GA
3
treatment; the lipid accumulation reached 0.27 g/L, and the lipid content reached 60.25%. Moreover, the soluble protein content reached 28.49 mg/g DCW under 0.05 mg/L 6-BA treatment. Similarly, the highest carotenoid content (108.70 mg/g DCW) was observed under 50 mg/L NAA treatment. Furthermore, all PGRs altered the fatty acid composition of
B. braunii
B12, particularly the C16-C18 content (%). Finally, the effects of eight PGRs on seven lipid-related genes (acyl-acyl carrier protein (
ACP
), biotin carboxylase (
BC
), ω-3 fatty acid desaturase (
FAD
), acyl carrier protein thioesterase (
FATA
), 3-ketoacyl carrier protein synthase gene (
KAS
), malonyl-CoA:ACP transacylase (
MCTK
), and stearoyl-ACP-desaturase (
SAD
)) were studied by qRT-PCR, and an apparent correlation was found between gene expression and lipid synthesis in the microalgae.