مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Limiting nutrient medium increases the efficiency of lipid micro-algae to produce lipid
Limiting nutrient medium increases the efficiency of lipid micro-algae to produce lipid
Fereshteh Fouladi,1,*Fatemeh Ehsani Besheli,2
1. Department of Chemical Engineering, Amirkabir University of Technology 2. Department of Chemical Engineering, Amirkabir University of Technology
Introduction: Fossil fuels are the main source of the most important greenhouse gas, carbon dioxide, and its high rate of consumption sets out a great challenge concerning worldwide sustainability. Among the numerous research initiatives developing bio regenerative technologies for CO2 mitigation and renewable alternatives to fossil fuels, the use of photosynthetic microorganisms has been proposed[1,2]. In this regard, biodiesel from agricultural crops (first generation biofuel system) is a renewable fuel that is attracting the most attention. However, the production of biofuels from microalgae is still too expensive to accomplish market requirements [3,4].
In this study, cell growth and lipid production of a marinemicroalga C. vulgaris was investigated at various nitrogen and phosphate concentrations in four different OD cell dilution rate . A detailed examination of biomass composition, including lipid, carbohydrate ,and lipid content, was performed throughout the culture pro-cess. Fatty acid profiling in the microalgal lipid was also analyzedto evaluate the feasibility of this microalga for biodiesel production.
Methods: 2.1. Pre culture and media composition
C. vulgaris p12 (obtaind from the culture loboratory Amir kabir) was cultivated in 400 mL shake flasks to produce a starter culture. Illumination was provided by four fluorescent lamps (Sylvania Standard F100 W) on one side of the photobioreactos, at an irradiance level of 70 lmol m_2 s_1. The original growth medium (OGM) based on chemical components present in the microalgal biomass [2] had the following composition (gr/lit):0.36KNO3, 0.017NaH2PO4,0.065Na2HPO4,0.01 MgSO4_7H2O, 0.047CaCl2, 1.7FeNa-C10H12O8N2, 0.107MnCl2_4H2O, 0.03H3BO3, 0.006ZnSO4_7H2O, 0.008CuSO4_5H2O, 0.002 CoSO4_7H2O, 0.006(NH4)6Mo7O24_4H2O and 35Nacl distilled water.
2.2. Photoautotrophic cultivation
Cells were cultivated photoautotrophically in 4 L of Rudic medium in a bubble-column photobioreactor (ID, 6.5 cm; height, 37 cm) (3) at room temperature (stage I). Filtered air was supplied to the reactor through a 0.2-lm PTFE membrane at a rate of 0.15 vvm with 4% CO2. Continuous illumination was provided at 200 lmol/m2/s light intensity using white fluorescent lights.
2.3. Biomass concentration
Microalgae and culture medium were withdrawn from the photobioreactors throughout the assay. Microalgal density was measured microscopically using an improved Neubauer hemocytometer. The growth rate of microalgae was also measured by cell dry weight. Microalgae were also harvested by centrifugation at 4500 rpm during 30 min, washed with distilled H2O, and dried at 110 _C until constant weight (24 h).
2.4. Lipid extraction and measurement
Total lipids were extracted by the method of with some modifications[3]. During the second stage, 50 mL of suspended sample was harvested by centrifugation at,45 00_g for 15 min and re-suspended in 1.6 mL of distilled water to which 8 mL of chloroform/methanol/water (2/4/1.6, v/v/v) was added. The mixture was sonicated for 1 min at 100W and 20 kHz (VCX 130, Sonics & Materials Inc., CT, USA), and vortexed for 30 s. Additional chloroform (4mL) and water (4mL) were added so that the final ratio of chloroform/methanol/water was 1/1/0.9 (v/v/v), and the solution was again vortexed for 30 s. The solution was centrifuged at 45000_g for 15 min and the bottom phase was transferred into a new tube. The upper layer was extracted again using the same procedure but with a half amount of solvent. The chloroform phases were combined and evaporated for 24 h in a drying oven at 100 _C. The total lipid contents were expressed as the % of dry cell weight (DCW).
Results: 3.1. The effect of nitrate concentration with OD different
Fig.1(a) shows that about 2.5g/L of biomass was obtained after 12 day under completely N-depleted (zero) condition with OD 5 ,3,2,1 cell dilution rate . Fig.1(b) shows that about 0.5g/L of lipid was obtained after 12 day under completely N-depleted (zero) condition with OD 5,3,2,1 cell dilution rate. Fig. 1(c) shows that about 27% of lipid content (productive) was obtained after 12 day under completely N-depleted (zero) conditions with OD 5,3,2,1 cell dilution rate . The maximum lipid content increased slightly, up to 18%, when 3.6 mg/L NO3-N was added initially; however, the incubation time required for reaching a maximum lipid content was attenuated to 14 day when nitrate was present. Lipid contents increased until 12 day in all cases except that of the complete N-depletion. The range of nitrate concentrations used was so low that the nitrogen content was depleted to zero within 24 h (data not shown).
However, lipid productivity (mg L_1 day_1) of C. vulgaris cultured under nitrogen limitation condition increased with increases in initial cell concentrations. The lipid productivity
(6.3 mg L_1 day_1) obtained in group OD5(the most cell initial cell concentration) was the most cell among all the ratio cell . The highest lipid productivity (27.3 mg L_1 day_1) was achieved in OD 5 (the highest initial cell OD550 of 5), though the lipid content in ratio cell OD 1 was lower than that achieved in other groups. This was because cells with higher initial concentration reached relatively higher final dry weight (g L_1). The results of lipid concentrations (mg L_1) were similar to those of lipid productivity (mg L_1 day_1)
3.2. The effect of phosphate and nitrate concentration
Fig.2(a) shows the influence of initial inorganic phosphate content on the change of biomass under N-starvation at 0.36g/L NO3-N . Fig .2(b) shows the influence of initial inorganic phosphate content on the change of lipid under N-starvation at 0.36g/L NO3-N. A phosphate content in the range of 0.017g/L as PO4-P exhibited a maximized lipid at 12 day , whereas a maximum content was reached after 12 day when the phosphate level was above 0.67 g/L .Fig. 2(c) shows the influence of initial inorganic phosphate and content on the change of lipid content (productive) under N-starvation at 0.36 g/L NO3-N. A phosphate content in the range of 0.017g/L as PO4-P exhibited a maximized lipid content at 12 day , whereas a maximum content was reached after 12 day when the phosphate level was above 0.3 g/L/g/L. The total lipid contents reached around 50% using 0.002–0.003 g/L of phosphate. These results are in accordance with those obtained by others . Comparisons of the data presented in . Fig .2(c) indicated that the contribution of P-starvation to lipid accumulation under N-starvation was not as significant as N-starvation.
Conclusion: The growth rate and lipid accumulation of microalgae were strongly related to nitrogen and phosphate concentration. Various levels of cell concentration were used to investigate its effects on lipid and lipid content (poductivite). The maximum lipid productivity obtained was 0.297 g d_1 L_1 when cell concentration was OD 5 . Various feed timing and concentrations of nitrate and phosphate were carried out to investigate the cell growth and lipid accumulation of Chlorella vu. Based on the experimental observations, the high lipid content of 0.66 g g_1 was obtained by cultivation with feeding 0.36 g L_1 nitrate and 0.17 gL_1 phosphates at stationary phase. The result implies that cultures with the appropriate amount and timing of nitrate feed can effectively enhance the lipid production of microalgae.