Production of lipids and formation and mobilization of lipid bodies in Chlorella vulgaris

We investigated the formation of lipid bodies in the microalga Chlorella vulgaris CCALA 256 under lipid-induction conditions in autotrophically grown cultures. We found that cell division ceased after depletion of nitrates from the growth medium within the first days of cultivation. The growth of non-dividing cells subsequently led to the rapid accumulation of lipids. We describe in detail both biogenesis and mobilization of lipid bodies using fluorescence and transmission electron microscopy. Small lipid bodies fused very soon after their creation in the cytosol, forming, eventually, one huge lipid body. In stationary growth phase, lipids were present in the form of a large lipid body occupying most of the cell volume. After replenishment of nitrogen, lipid content decreased rapidly and, within 24 hours, the large lipid body was fragmented into smaller ones. This mobilization of the cellular lipid store occurred independently of light.     

The effect of nitrogen limitation on lipid productivity and cell composition in Chlorella vulgaris

Chlorella vulgaris accumulates lipid under nitrogen limitation, but at the expense of biomass productivity. Due to this tradeoff, improved lipid productivity may be compromised, despite higher lipid content. To determine the optimal degree of nitrogen limitation for lipid productivity, batch cultures of C. vulgaris were grown at different nitrate concentrations. The growth rate, lipid content, lipid productivity and biochemical and elemental composition of the cultures were monitored for 20 days. A starting nitrate concentration of 170 mg L^?1 provided the optimal tradeoff between biomass and lipid production under the experimental conditions. Volumetric lipid yield (in milligram lipid per liter algal culture) was more than double that under nitrogen-replete conditions. Interpolation of the data indicated that the highest volumetric lipid concentration and lipid productivity would occur at nitrate concentrations of 305 and 241 mg L^?1, respectively. There was a strong correlation between the nitrogen content of the cells and the pigment, protein and lipid content, as well as biomass and lipid productivity. Knowledge of the relationships between cell nitrogen content, growth, and cell composition assists in the prediction of the nitrogen regime required for optimal productivity in batch or continuous culture. In addition to enhancing lipid productivity, nitrogen limitation improves the lipid profile for biodiesel production and reduces the requirement for nitrogen fertilizers, resulting in cost and energy savings and a reduction in the environmental burden of the process.     

The effect of degree and timing of nitrogen limitation on lipid productivity in Chlorella vulgaris

Improvements in lipid productivity would enhance the economic feasibility of microalgal biodiesel. In order to optimise lipid productivity, both the growth rate and lipid content of algal cells must be maximised. The lipid content of many microalgae can be enhanced through nitrogen limitation, but at the expense of biomass productivity. This suggests that a two-stage nitrogen supply strategy might improve lipid productivity. Two different nitrogen supply strategies were investigated for their effect on lipid productivity in Chlorella vulgaris. The first was an initial nitrogen-replete stage, designed to optimise biomass productivity, followed by nitrogen limitation to enhance lipid content (two-stage batch) and the second was an initial nitrogen-limited stage, designed to maximise lipid content, followed by addition of nitrogen to enhance biomass concentration (fed-batch). Volumetric lipid yield in nitrogen-limited two-stage batch and fed-batch was compared with that achieved in nitrogen-replete and nitrogen-limited batch culture. In a previous work, maximum lipid productivity in batch culture was found at an intermediate level of nitrogen limitation (starting nitrate concentration of 170 mg L^?1). Overall lipid productivity was not improved by using fed-batch or two-stage culture strategies, although these strategies showed higher volumetric lipid concentrations than nitrogen-replete batch culture. The dilution of cultures prior to nitrogen deprivation led to increased lipid accumulation, indicating that the availability of light influenced the rate of lipid accumulation. However, dilution did not lead to increased lipid productivity due to the resulting lower biomass concentration.     

Growth, lipid extraction and thermal degradation of the microalga Chlorella vulgaris

The microalga Chlorella vulgaris was cultured in a combined medium obtained by mixing standard Jaworski medium with a solution from the modified Solvay process that contained only NaHCO(3) and NH(4)Cl. Cell number, pH and nitrogen content were monitored throughout growth. Lipids were extracted from lyophilised biomass using CHCl(3)-MeOH. A combination of grinding, microwave treatment and sonication proved to give the best lipid extract yield. Freeze-dried algal biomass was also utilised for thermal degradation studies. The degradation exhibited three distinct regions - primary cell structure breakage paralleled by evaporation of water, followed by two predominant exothermic degradation processes. The latter were modelled using nth order apparent kinetics. The activation energies of the degradation processes were determined to be 120-126kJ/mol and 122-132kJ/mol, respectively. The degradation model may be readily applied to an assortment of thermal algal processes, especially those relating to renewable energy.     

Effect of iron on growth and lipid accumulation in Chlorella vulgaris

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L(-1) FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed.     

Effect of nanoparticle on cellular growth and lipid production in Chlorella vulgaris culture

Magnetic cobalt ferrite/silica nanoparticles (MSNs) and methyl functionalized MSNs (methyl‐MSNs) were used to enhance lipid production in Chlorella vulgaris culture through enhancement of gas‐water mass transfer and increased dissolved concentration of CO₂. Methyl‐MSNs enhanced CO₂–water mass transfer rate better than MSNs, and 0.3 wt% methyl‐MSNs are more effective than 0.1 wt% MSNs. In the cultivation experiment, 0.3 wt% methyl‐MSNs yielded the highest dry cell weight and subsequently, the highest mass transfer rate. However, enhancement of mass transfer rate did not increase lipid content. The volumetric lipid productivity in C. vulgaris culture depends not only on intracellular lipid content but also on the cell mass concentration. Consequently, 0.1 wt% methyl‐MSNs yielded the highest volumetric lipid productivity in C. vulgaris cultivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:929–933, 2018     

Maximizing biomass productivity and cell density of Chlorella vulgaris by using light-emitting diode-based photobioreactor

Green microalgae have recently drawn attention as promising organisms for biofuel production; however, the question is whether they can grow sufficient biomass relative to limiting input factors to be economically feasible. We have explored this question by determining how much biomass the green microalga Chlorella vulgaris can produce in photobioreactors based on highly efficient light-emitting diodes (LEDs). First, growth results were improved under the less expensive light of 660nm LEDs, developing them in the laboratory to meet the performance levels of the traditional but more expensive 680nm LEDs by adaptive laboratory evolution (ALE). We then optimized several other key parameters, including input superficial gas velocity, CO(2) concentration, light distribution, and growth media in reference to nutrient stoichiometry. Biomass density thereby rose to approximately 20g dry-cell-weight (gDCW) per liter (L). Since the light supply was recognized as a limiting factor, illumination was augmented by optimization at systematic level, providing for a biomass productivity of up to 2.11gDCW/L/day, with a light yield of 0.81 gDCW/Einstein. These figures, which represent the best results ever reported, point to new dimensions in the photoautotrophic performance of microalgal cultures.     

EMS-induced mutation followed by quizalofop-screening increased lipid productivity in Chlorella sp.

Bioprocess and Biosystems Engineering - The objective of this study was to enhance biomass and lipid productivity in Chlorella sp. isolate 6-4 by inducing mutagenesis with two growth inhibitors:...     

A two-stage fed-batch heterotrophic culture of Chlorella protothecoides that combined nitrogen depletion with hyperosmotic stress strategy enhanced lipid yield and productivity

In this study, the influences of major nutrients on cell growth and lipid production were investigated in heterotrophic culture of Chlorella protothecoides. The results demonstrated that phosphorus depletion had no effect on lipid accumulation but restricted cell growth; however, nitrogen depletion could enhance lipid accumulation thus benefiting lipid production. Furthermore, the effects of glucose inhibition were comparatively investigated with osmotic stress, showing that the effects of glucose inhibition were similar to the effect of osmotic stress at equivalent osmotic pressures only if the glucose concentration was less than 100 g/L, otherwise the effects of glucose inhibition became much stronger than osmotic stress. Interestingly, it was found that a specific hyperosmotic stress could significantly enhance lipid accumulation, thus providing a new stress strategy for efficient lipid production. Finally, a novel two-stage fed-batch culture consisting of a growth phase and a lipid accumulation phase with nitrogen depletion and hyperosmotic stress was proposed, yielding a final lipid productivity of 177.3 mg/L/h with a very high lipid yield of 207.0 mg/g glucose and lipid content of 39.2% after 180 h culture, which were 1.60, 1.79 and 1.92-fold of those obtained in one-stage fed-batch culture without stress phase, respectively.     

不同光质对小球藻光自养培养积累油脂的影响

研究了5种光质对小球藻(Chlorella vulgaris)M209256生长和产油的影响。结果表明:蓝光为小球藻的最适生长和产油光质;与其他光质相比,蓝光培养的小球藻生物量和油脂含量均较高,为2.40×107个/mL和28%;红光培养的小球藻生长最慢且油脂含量最低,为1.32×107个/mL和15.13%,表现出明显的"红降"现象。在GCMS分析的基础上,对油脂甲酯化后的十六烷值进行评估,结果发现:蓝光的十六烷值最高;5种光质培养的小球藻所产油脂,甲酯十六烷值均在47以上。因此,小球藻油脂所制备的生物柴油具有较好的燃烧性能。     

Investigation of biomass concentration,lipid production,and cellulose content in Chlorella vulgaris cultures using response surface methodology

The microalgae Chlorella vulgaris produce lipids that after extraction from cells can be converted into biodiesel. However, these lipids cannot be efficiently extracted from cells due to the presence of the microalgae cell wall, which acts as a barrier for lipid removal when traditional extraction methods are employed. Therefore, a microalgae system with high lipid productivity and thinner cell walls could be more suitable for lipid production from microalgae. This study addresses the effect of culture conditions, specifically carbon dioxide and sodium nitrate concentrations, on biomass concentration and the ratio of lipid productivity/cellulose content. Optimization of culture conditions was done by response surface methodology. The empirical model for biomass concentration (R² = 96.0%) led to a predicted maximum of 1123.2 mg dw L^?1 when carbon dioxide and sodium nitrate concentrations were 2.33% (v/v) and 5.77 mM, respectively. For lipid productivity/cellulose content ratio (R² = 95.2%) the maximum predicted value was 0.46 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 when carbon dioxide concentration was 4.02% (v/v) and sodium nitrate concentration was 3.21 mM. A common optimum point for both variables (biomass concentration and lipid productivity/cellulose content ratio) was also found, predicting a biomass concentration of 1119.7 mg dw L^?1 and lipid productivity/cellulose content ratio of 0.44 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 for culture conditions of 3.77% (v/v) carbon dioxide and 4.01 mM sodium nitrate. The models were experimentally validated and results supported their accuracy. This study shows that it is possible to improve lipid productivity/cellulose content by manipulation of culture conditions, which may be applicable to any scale of bioreactors. Biotechnol. Bioeng. 2013; 110: 2114–2122. © 2013 Wiley Periodicals, Inc.     

Lipid productivity and fatty acid composition in Chlorella and Scenepdesmus strains grown in nitrogen-stressed conditions

Thirty Chlorella and 30 Scenedesmus strains grown in nitrogen-stressed conditions (70 mg L^?1 N) were analyzed for biomass accumulation, lipid productivity, protein, and fatty acid (FA) composition. Scenedesmus strains produced more biomass (4.02?±?0.73 g L^?1) after 14 days in culture compared to Chlorella strains (2.57?±?0.12 g L^?1). Protein content decreased and lipid content increased from days 8 to 14 with an increase in triacylglycerol (TAG) accumulation in most strains. By day 14, Scenedesmus strains generally had higher lipid productivity (53.5?±?3.7 mg lipid L^?1 day^?1) than Chlorella strains (35.1?±?2.8 mg lipid L^?1 day^?1) with the lipids consisting mainly of C16–18 TAGs. Scenedesmus strains generally had a more suitable FA profile with higher amounts of saturated fatty acids and monounsaturated fatty acids (MUFAs) and a smaller polyunsaturated fatty acid (PUFA) component. Chlorella strains had a larger PUFA component and smaller MUFA component. The general trend in the FA composition of Chlorella strains was oleic > palmitic > α-linolenic = linoleic > eicosenoic > heptadecenoic > stearic acid. For Scenedesmus strains, the general trend was oleic > palmitic > linoleic > α-linolenic > stearic > eicosenoic > palmitoleic > heptadecenoic acid. The most promising strains with the highest lipid productivity and most suitable FA profiles were Scenedesmus sp. MACC 401, Scenedesmus soli MACC 721, and Scenedesmus ecornis MACC 714. Although Chlorella sp. MACC 519 had lower lipid productivity, the FA profile was good with a lower PUFA component compared to the other Chlorella strains analyzed and a low linolenic acid concentration.     

无机碳源对小球藻自养产油脂的影响

旨在研究小球藻利用无机碳自养产油脂,考察了3种无机碳源 (Na2CO3、NaHCO3和CO2) 及其初始浓度对小球藻产油特性的影响。结果表明,小球藻能利用Na2CO3、NaHCO3和CO2产油;经Na2CO3、NaHCO3和CO2培养10 d后,随着每种无机碳源浓度的增加,小球藻产量均先增加后减少。小球藻经3种无机碳源培养后,其培养液pH值上升。最适宜的Na2CO3和NaHCO3添加量均为40 mmol/L,其生物量分别达到0.52 g/L和0.67 g/L,产油量分别达到0.19 g/L和0.22 g/L。在3种无机碳源中,CO2是最佳无机碳源,当CO2浓度为6％时,小球藻生长最快,生物量达2.42 g/L,产油量最高达0.72 g/L;当CO2浓度过低时,无机碳供应不足,油脂产量低;当CO2浓度过高时,培养液pH偏低,小球藻油脂积累受到抑制。Na2CO3和NaHCO3较CO2更有利于小球藻积累不饱和脂肪酸。     

pH值对沼液培养的普通小球藻生长及油含量积累的影响

以50％的沼液为普通小球藻的全营养培养基,考察培养基的起始pH值对小球藻生长及油脂含量的影响,普通小球藻对不同初始pH的沼液中氮、磷的去除情况。设定了2组实验,一组只调节初始接种培养液的pH,分别为6.0、6.5、7.0、7.5、8.0、8.5;另一组将培养液pH分别固定在6.0、6.5、7.0、7.5、8.0、8.5,pH用稀HCl和NaOH进行调节。研究发现在pH 6.5和pH 7.0的偏酸环境有利于小球藻生长,而pH在7.0~8.5的偏碱性条件下有利于小球藻油脂的积累,因此综合小球藻生长和油脂积累2个因素,得到最适合小球藻生长和油脂积累的pH为7.0。培养结束后沼液中氮磷的去除率分别达到了95％和97％,沼液中的总氮由原来的134.91 mg/L降至4.86 mg/L,总磷由10.19 mg/L降到0.32 mg/L。     

Evaluation of high salinity adaptation for lipid bio-accumulation in the green microalga Chlorella vulgaris

Aiming at the reutilizing wastewater for algal growth and biomass production, a saline water rejected from reverse osmosis (RO) facility (salinity 67.59 g L⁻¹) was used to cultivate the pre-adapted green microalga Chlorella vulgaris. The inoculum was prepared by growing cells in modified BG-11 medium, and adaptation was performed by applying a gradual increase in salinity (56.0 g L⁻¹ NaCl and 125 ppm FeSO₄·7H₂O) to the culture in 200 L photobioreactor. Experiments using the adapted alga were performed using original-rejected water (ORW) and treated rejected water (TRW) comparing with the recommended growth medium (BG-11). The initial salinity of ORW was chemically reduced to 39.1 g L⁻¹ to obtain TRW. Vertical photobioreactors (15 L) was used for indoor growth experiments. Growth in BG-11 resulted in 1.23 g L⁻¹, while the next adaptation growth reached 2.14 g L⁻¹ of dry biomass. The dry weights of re-cultivated Chlorella after adaptation were 1.49 and 2.19 g L⁻¹ from ORW and TRW; respectively. The cellular oil content was only 12% when cells grown under control conditions verses to 14.3 and 15.42% with original and treated water, respectively. Induction of stress affected the fatty acid methyl esters (FAMEs) profile and the properties of the resulting biodiesel. The present results indicated that induction of stress by high salinity improves the quality of FAMEs that can be used as a promising biodiesel fuel.