Study On Environmental Regulation And Physiological Mechanism Of Lipid Accumulation In Chlorella Vulgaris

Microalgae are unicellular photosynthetic organisms with high growth rate and high photosynthetic efficiency. Some microalgae can accumulate large number of lipids which can be transferred into biodiesel under certain environmental conditions. Because microalgae cultivation was free from farm land, biodiesel from microalgae was regarded as the most promising alternative energy of the new energy in twenty-first Century.Chlorella has the advantages of rapid growth, easy to cultivate and strong environmental adaptability. Moreover, Chlorella cultivation has been commercialized. In this work, two strains of marine Chlorella (Chlorella sp. HN11 and Chlorella sp. HN08)were used to investigate the lipid induction effects of nitrogen, glucose and H2O2. Then time-variations of some main metabolites were determined under nitrogern starvation or glucose rich conditions. Based on our previous results, C. vulgaris HN11 was selected as materials to study the effect of low- nitrogen and glucose on some key enzymes activity during lipid accumulation to explore the important metabolic steps involved in lipid accumulation.Our results showed that under low- nitrogen conditions, lipid accumulation in the two strains of Chlorella was promoted but the growth was inhibited. However, both growth and lipid content increased with 1000mg/L or lOg/L glucose addition. H2O2 could stimulate lipid accumulation within 4h to 12h, but depressed the algae growth at the same time.2mM H2O2 could result in the death of cells.When exposed to low nitrogen condition, the protein content of C. vulgaris HN11 and C. sp. HN08 was significant decreased (P<0.05), but the content of soluble sugar and starch were significantly increased (P<0.05). When the algae were exposed to low nitrogen condition, the content of starch increased within 4 hours, and then decreased after 24 hours with the increase of the lipid content. However, glucose had little influence on the content of soluble protein and sugar. Starch content increased quickly after 6 hours of glucose addition. However, no significant difference of starch content was observed after 24h of glucose addition. It was inferred that protein and starch might be the carbon donor for the increased lipid under low- nitrogen condition. While under glucose rich condition, the carbon donor of the accumulated lipid might be glucose.Then the impact of low- nitrogen and glucose on the activity of some key enzymes involved in the lipid accumulation were analyzed. Result showed that the activity/content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), copper amine oxidase3(AOC3), semicarbazide-sensitive amine oxidase (SSAO) were significant decreased (P<0.05) under two conditions; but the activity of key enzymes in the pentose phosphate pathway (PPP): glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphaogluconate dehydrogenase (6PGDH), ethanol dehydrogenase (ADH),acetaldehyde dehydrogenase (NAD+), peroxidase (POD) increased under low-nitrogen or glucose rich conditions. The activity of acetyl-CoA carboxylase (ACCase) decreased under low- nitrogen condition and increased under glucose rich condition.Based on our previous and the present results, we speculated that the key metabolic steps involved in lipid accumulation in C. vulgaris HN11 under low nitrogen condition was:(1) PPP pathway became active and produced NADPH and some intermediate metabolites,which providing reduced agent and material for fatty acid synthesis. (2) ADH aand ALDH might be involved in the process of amino acid degradation. The catalytic product"carboxylate" might act as the carbon unit for fatty acid synthesis. In the early stages of low nitrogen, the starch was mainly material; in the late stage(24h), the cell was accumulated lipid. Through the above way, the carbon skeleton of starch and protein was flowed into lipids synthesis.Under glucose induced condition, the key steps of C. HN11 for lipid regulation might be PPP. The activity of key enzymes in the pentose phosphate pathway(G6PDH and 6GPDH) was increased and more NADPH could be produced. The carbon source of lipids synthesis was organic carbon.Under low nitrogen and glucose conditions (high ratio of C/N), the activities of G6PDH, 6GPDH and ADH increased under low nitrogen or rich glucose conditions.Therefore, it could be condluded that those enzymes might be the key enzymes for lipid accumulation in Chlorella vulgaris cells.