Objectives Chlorella pyrenoidosa are high in protein contents and rich in all kinds of essential amino acids and various nutritional health care factors, which have been listed as a new resource of food in China in 2012. The paper studied the specific nitrogen stress levels and days for culture of Chlorella pyrenoidosa.
Methods The Chlorella pyrenoidosa was inoculated to the basic BG11 medium for autotrophic cultivation and BG11 medium supplemented with 10 g/L glucose for mixotrophic cultivation, then obtained seed algae cells. The conventional nitrogen supply of 18 mmol/L sodium nitrite in the BG11 basic medium was used as control, mediums with 3, 6 and 9 mmol/L sodium nitrite were prepared as low nitrogen stress treatments. The algae cell biomass was measured by dry weight and the total lipid was quantified by staining method. The protein and starch contents of algae cells were determined by ultraviolet spectrophotometry, and the three-dimensional fluorescence spectra was used to analyze the components and contents of algae extracellular polymeric substances (EPS).
Results 1) The Chlorella pyrenoidosa cells from both autotrophic and mixotrophic cultivation could grow under low nitrogen stress conditions, algae cells from autotrophic cultivation responded more rapidly to the change of low nitrogen stress and started to grow rapidly on the first day after transfer. The highest cell dry weight and lipid content of the algae were harvested in the NaNO3 6 mmol/L treatment groups. After culturing for 4 days, the algae growth reached stable phase, the cells dry weight from autotrophic and mixotrophic cultivation sources was 2.56 and 4.62 g/L, respectively, and the corresponding lipid contents reached to 15.5% and 39.3%, which were all notably higher than their normal N controls. 2) Low N stress could improve the lipid productivity of Chlorella pyrenoidosa. Both the highest lipid productivities of algae cells derived from mixotrophic cultivation were in NaNO3 6 mmol/L treatment, that for the mixotrophic algae cells was 129.56 mg/(L·d) on the 4th day, which was 7.95 times of that for the autotrophic algae cells. 3) The initial contents of intra- and extra-cellular protein and starch (polysaccharide) in cells from mixotrophic group were notably higher than those from autotrophic group. The lipid, protein, starch contents were decreased significantly in the 2nd or 3rd day of culture, then the intra- and extra-cellular protein contents continued to decline, while the lipid and intracellular starch contents began to increase with an apparent inflection points. The change trends of the lipid and intracellular starch content in algal cells were basically synchronous. 4) The EPS components in the Chlorella pyrenoidosa contained the tryptophan-like proteins (TLP) that advantage to algae cells flocculating and some humic substances including humilic acid (HA) and fulvic acid (FA) that disadvantage to flocculation. After culturing for 4 days in NaNO3 6 mmol/L medium, the content of TLP in algea cells from autotrophic cultivation was increased by 40.3% than the control, while the content of TLP, HA and FA in algae cells from mixotrophic cultivation were 83.6%, 74.8% and 54.8% of that in control groups, respectively. The corresponding self-flocculation rate of algae cells from autotrophic and mixotrophic cultivation source were 78.5% and 80.3% respectively, which were notably improved than their corresponding controls.
Conclusions The response to low-nitrogen stress varied between the autotrophic and mixotrophic Chlorella pyrenoidosa cells. The biochemical component contents of algal cells are influenced by the culture medium nitrogen supply levels and culturing days. The low nitrogen stress influences the self-flocculation rate of algae cells remarkably by changing the EPS compositions of Chlorella pyrenoidosa. For the tested Chlorella pyrenoidosa, the suitable nitrogen stress level is NaNO3 6 mmol/L and the cultural time is 4 days for the highest lipid productivity and flocculation performance.
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