| Owing to the unique advantages in rapid oil accumulation, high oil yield, good oil quality,easy to control metabolic process and genetic modification, microalgae became the admirablefeedstock for biodiesel production. But the commercial application of microalgal biofuels is stillfaced with many technical challenges, including the breeding of oleaginous microalgae strains,high efficient culture system design, large-scale cultivation technology, microalgal cellsharvesting and the improvement of oil refining technology, and so on. This paper aims tooptimize culture conditions of an oleaginous microalga to solve the inherent contradictionbetween biomass and lipid accumulation, to enhance the yield of lipid, to reduce the total cost ofthe production, and to explore suitable cultivation system for large-scale production. It couldprovide the theoretical and technical support to realize the high efficiency, low cost, low energyconsumption of large-scale microalgae production.An oleaginous microalga, Scenedesmus acuminatus was used as experimental strain in thisresearch. The cell morphology and the process of oil accumulation in S. acuminatus duringdifferent growth periods were observed under light microscope. In the early stage, the coenobiaof S. acuminatus almost overlap in a zigzag with4cells, alternate arrangement, each cell unitedby its apex to the subapical or medium portion of adjacent cell, spindle-shaped to arc-like,tapering to acute or sometimes slightly obtuse apices. During logarithmic growth phase, each cellof coenobia carries on horizontal or longitudinal division for proliferation. On the sixth day, cellsbegan to appear a large number of oil droplets, with the extension of incubation time, coenobiadispersed into single cell and gradually become the oval shape, the oil droplets become largerand mutual integration into even bigger oil body, decreased in the number, up to the end ofcultivation, large oil bodies occupied the main part of the cell.The fluorescent lamps were supplied as artificial light source (light intensity:300μmolphotons·m-2·s-1). The enrichment CO2(1%, v/v) of the compressed air was used as stirring gasand carbon source. S. acuminatus was grown with3.0cm column photobioreactor under thedifferent initial supplies of NaNO3(18mM (1.5g·L-1),9.0mM (0.75g·L-1),6.0mM (0.5g·L-1)and3.6mM (0.3g·L-1)) or CO(NH2)2(9.0mM (0.54g·L-1),4.5mM (0.27g·L-1) and3.0mM(0.18g·L-1),1.8mM (0.108g·L-1)) in modified BG-11medium. The biomass, total lipid contents, lipid fractions, fatty acid compositions of S. acuminatus in different growth phase weredetermined. The biomass concentration increased along with the growth phase, total lipidcontents augmented following the culture time and the reducing of nitrogen concentration. Thefastest lipid accumulation was happened in the lowest concentration of initial nitrogen supply.When NaNO3used as nitrogen source, the highest total lipid content was gained at3.6mM,which was62.37%of dry biomass, the highest biomass concnentration and volumetric lipidproductivity were obtained at6.0mM, which were up to9.45g·L-1,0.31g·L-1·d-1, respectively.When CO(NH2)2used as nitrogen source, the maximum biomass concentration ocuured at4.5mM, which is9.75g·L-1, the highest total lipid content appearred at1.8mM, which was61.89%of dry biomass, the highest volumetric lipid productivity obtained at3.0mM, which was0.32g·L-1·d-1. These results showed that the S. acuminatus was an optimal oleaginous microalga,6.0mM of NaNO3and3.0mM of CO(NH2)2were the best culture conditions for obtaining thehighest yield of lipids, namely the highest volumetric lipid productivities were achieved atrelative low concentration of initial nitrogen supplies.After lipid classification, neutral lipid content increased following the decrease of nitrogenconcentration along with the whole period under the two kinds of nitrogen sources, while thechanges of glycolipids and phospholipids contents were opposite. When NaNO3used as nitrogensource, the highest neutral lipid content ocuured at3.6mM, accounting for91%of the total lipidand55.74%of dry biomass, the largest neutral lipid yield appearred at6.0mM, which is0.276g.L-1·d-1. When CO(NH2)2used as nitrogen source, the highest neutral lipid content exerted at1.8mM, accounting for92.56%of total lipid content and55.59%of dry biomass, the largestneutral lipid yield obtained at3.0mM, which is0.278g·L-1·d-1. The microalgal total lipid andneutral lipid productivities reached the highest at the same concentration of nitrogen with almostthe same amount. It showed that lipids accumulation of S. acuminatus was mainly neutral lipid,namely triacylglycerol (TAG).Fatty acid samples of S. acuminatus obtained from different growth phases were analyzedby Gas-Chromatography-Mass Spectrometry (GC-MS). The data showed that the main fatty acidprofiles were palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2), the sum ofthree kinds of fatty acids accounted for76%~86%of total fatty acids(TFA), saturated fatty acidand monounsaturated fatty acid contents increased with the decrease of nitrogen concentration following the whole culture period, which were approximately80%in TFA. The results showedthat the fatty acids of S. acuminatus were an ideal feedstock for biodiesel production.Under the condition of indoor, artificial light was provided by a bank of fluorescent lamps(light intensity is300μmol photons·m-2·s-1), S.acuminatus was cultivated with flat platephotobioreactor (ca.120cm×60cm×4cm) in large volume. The results showed that themassive biomass were obtained at6.0mM NaNO3and4.5mM CO(NH2)2, which reached5.71g·L-1and5.82g·L-1, respectively. The highest total lipid contents were existed at3.6mM NaNO3and1.8mM CO(NH2)2, which acounted for55.13%and62.93%of of dry biomass, respectively.The highest volumetric total lipids productivities achieved at6.0mM NaNO3and1.8mMCO(NH2)2, which were0.16g·L-1·d-1and0.17g·L-1·d-1, respectively. The results indicated thatwhen S. acuminatus was cultivated in flat plate photobioreactor, per cell received light intensityin flat plate photobioreactor was lower than that of per cell accepted in column photobioreactor,thus leading to the biomass decreased with the same external light intensity exposure.When S. acuminatus was conducted to grow with organic carbon and nitrogen sourcesunder dark condition. It was found that this microalga can completely accommodate to performheterotrophically growth. The best organic carbon source for heterotrophic culture of S.acuminatus was glucose, the optimal concentration was50g·L-1; the best organic nitrogen sourcewas yeast extract, and the optimal concentration was2.70g·L-1. Under the optiml concentrationsof glucose and yeast extract in heterotrophic culture, the maximum biomass was up to24.79g·L-1, which was more than2.6times of photoautotrophic mode. The results of biochemicalcomponent analysis showed that, under the heterotrophic conditions, metabolic pathway of S.acuminatus was notablely changed, carbohydrate became the main component of S. acuminatus,which reached58.16%of dry biomass, the highest carbohydrate productivity was1.05g·L-1·d-1;but the total lipid content was sharply reduced, the highest content was only30.93%of drybiomass, while the lipid productivity was still high(0.64g·L-1·d-1), twice as much as inautotrophic cultures. It indicated that heterotrophic culture could promote the biomass andcarbohydrate accumulation of S. acuminatus, in spite of the decrease of lipid content, the lipidproductivity was still higher than autotrophic mode.This study provides an excellent microalgal strain for the biodiesel production. Determining optimal initial nitrogen supply for grwoth and lipid content tradeoff in oleaginous microalga S.acuminatus. The highest total lipids and neutral lipids productivities could obtained in theseconditions. S. acuminatus could also grow in heterotrophic mode. S. acuminatus was a potentialsource for commercial biodiesel production. |
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