Lipid and total fatty acid productivity in photoautotrophic fresh water microalgae: screening studies towards biodiesel production

Microalgae are considered as a promising feedstock for biomass production. The selection of the most suitable species is based on several key parameters such as lipid and fatty acid productivity. In the present study, the growth of different microalgae strains was examined in freshwater media for photoautotrophs suited for large-scale applications to identify the most suitable medium for each species. In the optimal medium, Scenedesmus obliquus showed the highest biomass productivity measured as increase of cell dry weight (0.25 g cellu dry weight (CDW) L^?1 day^?1), while Botryococcus braunii showed the highest lipid and total fatty acid content (430 and 270 mg g^?1 CDW, respectively) among the tested species. Regarding lipid and total fatty acid productivity, S. obliquus was the most lipid and total fatty acid productive strain with 41 and 18 mg L^?1 day^?1 during the exponential phase, respectively. Additionally, the proportion of saturated and monounsaturated fatty acids increased with duration of the incubation in S. obliquus, while polyunsaturated fatty acids decreased. These results nominate S. obliquus as a promising microalga in order to serve as a feedstock for renewable energy production.     

Biomass, lipid content, and fatty acid composition of freshwater Chlamydomonas mexicana and Scenedesmus obliquus grown under salt stress

Two freshwater microalgae including Chlamydomonas mexicana and Scenedesmus obliquus were grown on Bold Basal Medium (BBM) with different levels of salinity up to 100 mM NaCl. The dry biomass and lipid content of microalgae were improved as the concentration of NaCl increased from 0 to 25 mM. Highest dry weight (0.8 and 0.65 g/L) and lipid content (37 and 34 %) of C. mexicana and S. obliquus, respectively, were obtained in BBM amended with 25 mM NaCl. The fatty acid composition of the investigated species was also improved by the increased NaCl concentration. At 50 mM, NaCl palmitic acid (35 %) and linoleic acid (41 %) were the dominant fatty acids in C. mexicana, while oleic acid (41 %) and α-linolenic acid (20 %) were the major fractions found in S. obliquus.     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

Productivity and biochemical composition of <Emphasis Type="Italic">Tetradesmus obliquus</Emphasis> and <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>: effects of different cultivation approaches

The present work evaluated biomass productivity, carbon dioxide fixation rate, and biochemical composition of two microalgal species, Phaeodactylum tricornutum (Bacillariophyta) and Tetradesmus obliquus (Chlorophyta), cultivated indoors in high-technology photobioreactors (HT-PBR) and outdoors both in pilot ponds and low-technology photobioreactors in a greenhouse in southern Italy. Microalgae were grown in standard media, under nitrogen starvation, and in two liquid digestates obtained from anaerobic digestion of agro-zootechnical and vegetable biomass. P. tricornutum, cultivated in semi-continuous mode in indoor HT-PBRs with standard medium, showed a biomass productivity of 21.0?±?2.3 g m^?2 d^?1. Applying nitrogen starvation, the lipid productivity increased from 2.3 up to 4.5?±?0.5 g m^?2 d^?1, with a 24 % decrease of biomass productivity. For T. obliquus, a biomass productivity of 9.1?±?0.9 g m^?2 d^?1 in indoor HT-PBR was obtained using standard medium. Applying liquid digestates as fertilizers in open ponds, T. obliquus gave a biomass productivity (10.8?±?2.0 g m^?2 d^?1) not statistically different from complete medium such as P. tricornutum (6.5?±?2.2 g m^?2 d^?1). The biochemical data showed that the fatty acid composition of the microalgal biomass was affected by the different cultivation conditions for both microalgae. In conclusion, it was found that the microalgal productivity in standard medium was about doubled in HT-PBR compared to open ponds for P. tricornutum and was about 20 % higher for T. obliquus.     

Impacts of nitrogen and phosphorus starvation on the physiology of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The importance of algae-derived biofuels has been highlighted by the current problems associated with fossil fuels. Considerable past research has shown that limiting nutrients such as nitrogen and phosphorus increases the cellular lipid content in microalgae. However, limiting the supply of nutrients results in decreased biomass, which in turn decreases the overall lipid productivity of cultures. Therefore, nutrient limitation has been a subject of dispute as to whether it will benefit biofuel production on an industrial scale. Our research explores the physiological changes a cell undergoes when exposed to nitrogen and phosphorus limitations, both individually and in combination, and also examines the biotechnological aspects of manipulating N and P in order to increase cellular lipids, by analyzing the lipid production. We show that nitrogen starvation and also nitrogen plus phosphorus starvation combined have a more profound effect on the physiology and macromolecular pools of Chlamydomonas reinhardtii than does phosphorus starvation alone. The photosynthetic performance of C. reinhardtii underwent drastic changes under nitrogen starvation, but remained relatively unaffected under phosphorus starvation. The neutral lipid concentration per cell was at least 2.4-fold higher in all the nutrient-starved groups than the nutrient-replete controls, but the protein level per cell was lower in the nitrogen-starved groups. Overall, nitrogen starvation has a more dramatic effect on the physiology and neutral lipids and protein levels of C. reinhardtii than phosphorus starvation. However, the level of total lipids per volume of culture obtained was similar among nutrient-replete and all of the nutrient-starved groups. We conclude that combined nitrogen and phosphorus starvation does not likely benefit biofuel production in terms of enhanced lipid or biomass production.     

Cultivation of a new microalga,Micractinium reisseri,in municipal wastewater for nutrient removal,biomass, lipid,and fatty acid production

Coupling of advanced wastewater treatment with microalgae cultivation for low-cost lipid production was demonstrated in this study. The microalgal species Micractinium reisseri and Scenedesmus obliquus were isolated from municipal wastewater mixed with agricultural drainage. M. reisseri was selected based on the growth rate and cultivated in municipal wastewater (influent, secondary and tertiary effluents) which varied in nutrient concentration. M. reisseri showed an optimal specific growth rate (μ_opt) of 1.15, 1.04, and 1.01 1/day for the influent and the secondary and tertiary effluents, respectively. Secondary effluent supported the highest phosphorus removal (94%) and saturated fatty acid content (40%). The highest lipid content (40%), unsaturated fatty acid content, including monounsaturated and polyunsaturated fatty acids (66%), and nitrogen removal (80%) were observed for tertiary effluent. Fatty acids accumulating in the microalgal biomass (M. reisseri) were mainly composed of palmitic acid, oleic acid, linoleic acid, and a-linolenic acid. Cultivation of M. reisseri using municipal wastewater served a dual function of nutrient removal and biofuel feedstock generation.     

Microalga <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> as a potential source for biodiesel production

Biodiesel from microalgae seems to be the only renewable biofuel that has the potential to completely replace the petroleum-derived transport fuels. Therefore, improving lipid content of microalgal strains could be a cost-effective second generation feedstock for biodiesel production. Lipid accumulation in Scenedesmus obliquus was studied under various culture conditions. The most significant increase in lipid reached 43% of dry cell weight (dcw), which was recorded under N-deficiency (against 12.7% under control condition). Under P-deficiency and thiosulphate supplementation the lipid content also increased up to 30% (dcw). Application of response surface methodology in combination with central composite rotary design (CCRD) resulted in a lipid yield of 61.3% (against 58.3% obtained experimentally) at 0.04, 0.03, and 1.0 g l⁻¹ of nitrate, phosphate, and sodium thiosulphate, respectively for time culture of 8 days. Scenedesmus cells pre-grown in glucose (1.5%)-supplemented N 11 medium when subjected to the above optimized condition, the lipid accumulation was boosted up to 2.16 g l⁻¹, the value ~40-fold higher with respect to the control condition. The presence of palmitate and oleate as the major constituents makes S. obliquus biomass a suitable feedstock for biodiesel production.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.     

Microalgae as a raw material for biofuels production

Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO₂, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulina maxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.     

Effect of organic carbon,C:N ratio and light on the growth and lipid productivity of microalgae/cyanobacteria coculture

Current culture methods based on monocultures under phototrophic regimes are prone to contamination, predation, and collapse. Native cultures of multiple species are adapted to the local conditions and are more robust against contamination and predation. Growth, lipid and biomass productivity of a Louisiana native coculture of microalgae (Chlorella vulgaris) and cyanobacteria (Leptolyngbya sp.) in heterotrophic and mixotrophic regimes were investigated. Dextrose and sodium acetate at C:N ratios of 15:1 and 30:1 under heterotrophic (dark) and mixotrophic (400 μmol m^?2 s^?1) regimes were compared with autotrophic controls. The carbon source and C:N ratio impacted growth and biomass productivity. Mixotrophic cultures with sodium acetate (C:N 15:1) resulted in the highest mean biomass productivity (156 g m^?3 d^?1) and neutral lipid productivity (24.07 g m^?3 d^?1). The maximum net specific growth rate (U) was higher (0.97 d^?1) in mixotrophic cultures with dextrose (C:N 15:1) but could not be sustained resulting in lower total biomass than in mixotrophic cultures with acetate (C:N 15:1), with a U of 0.67 d^?1. The ability of the Louisiana coculture to use organic carbon for biomass and lipid production makes it a viable feedstock for biofuels and bioproducts.     

Biomass production,total protein,chlorophylls, lipids and fatty acids of freshwater green and blue-green algae under different nitrogen regimes

Two green algae (Chlorella vulgaris and Scenedesmus obliquus) and four blue-green algae (Anacystis nidulans, Microcystis aeruginosa, Oscillatoria rubescens and Spirulina platensis) were grown in 81 batch cultures at different nitrogen levels. In all the algae increasing N levels led to an increase in the biomass (from 8 to 450 mg/l), in protein content (from 8 to 54 %) and in chlorophyll. At low N levels, the green algae contained a high percentage of total lipids (45 % of the biomass). More than 70 % of these were neutral lipids such as triacylglycerols (containing mainly 16:0 and 18:1 fatty acids) and trace amounts of hydrocarbons. At high N levels, the percentage of total lipids dropped to about 20 % of the dry weight. In the latter case the predominant lipids were polar lipids containing polyunsaturated C₁₆ and C₁₈ fatty acids. The blue-green algae, however, did not show any significant changes in their fatty acid and lipid compositions, when the nitrogen concentrations in the nutrient medium were varied. Thus the green but not the blue-green algae can be manipulated in mass cultures to yield a biomass with desired fatty acid and lipid compositions. The data may indicate a hitherto unrecognized distinction between prokaryotic and eukaryotic organisms.     

High biomass production and CO<Subscript>2</Subscript> fixation from biogas by <Emphasis Type="Italic">Chlorella</Emphasis> and <Emphasis Type="Italic">Scenedesmus</Emphasis> microalgae using tequila vinasses as culture medium

Tequila vinasses (TVs) generated during Tequila production are brown liquid residues rich in nutrients. The nutrient content of agro-industrial effluents represents an excellent resource to support low-cost biomass production of microalgae; nonetheless, it is crucial to select the suitable microalgal strain to attain the highest biomass production in each residue used. In this study, biomass production, CO₂ fixation from biogas, and cell compound accumulation by Chlorella vulgaris U162, Chlorella sp., Scenedesmus obliquus U169, and Scenedesmus sp. using biodigested and filtered TVs as culture medium were evaluated and compared with the conventional microalgal culture media, C30, BG-11, Bold 3N, and Bristol. The four microalgae evaluated attained the highest biomass production and CO₂ fixation rate cultured in both residues, accumulating mainly carbohydrates and proteins although the most appropriate microalga to be cultured in TVs was Chlorella sp., recording 2.30 g L^?1. Moreover, the nutrient ratio of filtered TVs was ideal to support biomass production while biodigested TVs need to be supplemented with nitrogen. Overall, these results demonstrated that tequila vinasses are an excellent resource to support high and quick biomass production of microalgae, which can be used to obtain biofuels as ethanol, biogas, and supplement food depicting an extra benefit during the appropriate disposal of this residue.     

Detection and imaging of lipids of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> based on confocal Raman microspectroscopy

In this study, confocal Raman microspectroscopy was used to detect lipids in microalgae rapidly and non-destructively. Microalgae cells were cultured under nitrogen deficiency. The accumulation of lipids in Scenedesmus obliquus was observed by Nile red staining, and the total amount of lipids accumulated in the cells was measured by gravimetric method. The signals from different microalgae cells were collected by confocal Raman microspectroscopy to establish a prediction model of intracellular lipid content, and surface scanning signals for drawing pseudo color images of lipids distribution. The images can show the location of pyrenoid and lipid accumulation in cells. Analyze Raman spectrum data and build PCA-LDA model using four different bands (full bands, pigments, lipids, and mixed features). Models of full bands or pigment characteristic bands were capable of identifying S. obliquus cells under different nitrogen stress culture time. The prediction accuracy of model of lipid characteristic bands is relatively low. The correlation between the fatty acid content measured by the gravimetric method and the integral Raman intensity of the oil characteristic peak (1445 cm^?1) measured by Raman spectroscopy was analyzed. There was significant correlation (R ² = 0.83), which means that Raman spectroscopy is applicable to semi-quantitative detection of microalgal lipid content.     

Effect of nitrate on lipid production by T. suecica, M. contortum, and C. minutissima

Microalgae are an alternative and sustainable source of lipids that can be used as a feedstock for biodiesel production. Nitrate is a good nitrogen source for many microalgae and affects biomass and lipid yields of microalgae. In this study, the effect of nitrate on cell growth and lipid production and composition in Monoraphidium contortum, Tetraselmis suecica, and Chlorella minutissima was investigated. Nitrate affected the production of biomass and the production and composition of lipids of the three microalgae tested. Increasing the nitrate concentration in the culture medium resulted in increased biomass production and higher biomass productivity. Furthermore, increasing the nitrate concentration resulted in a reduction in lipid content and productivity in M. contortum; however, the opposite effect was observed in T. suecica and C. minutissima cultures. C. minutissima and M. contortum lipids contain high levels of oleic acid, with values ranging from 26 to 45.7% and 36.4 to 40.1%, respectively. The data suggest that because of its high lipid productivity (13.79 mg L^?1 d^?1) and high oleic acid productivity (3.78 mg L^?1 d^?1), Chlorella minutissima is a potential candidate for the production of high quality biodiesel.     

Characterization of cell structural change,growth, lipid accumulation,and pigment profile of a novel oleaginous microalga, <Emphasis Type="Italic">Vischeria stellata</Emphasis> (Eustigmatophyceae), cultured with different initial nitrate supplies

The appropriate microalgal species and the optimal nitrogen supply in culture medium are vital factors in maximizing biomass and metabolite productivities in microalgae. Vischeria stellata is an edaphic unicellular eustigmatophycean microalga. Cytological and ultrastructural characteristics and the effects of different initial nitrate-nitrogen concentrations on growth, lipid accumulation, fatty acid profile, and pigment composition were investigated in the present study. The cell structures of V. stellata changed with the degree of nutrient utilization and growth phase. The initial nitrate concentration for the optimal growth of V. stellata ranged from 6.0 to 9.0 mM. The maximum total lipid (TLs), neutral lipid (NLs), and total fatty acid (TFAs) contents were 55.9, 51.9, and 44.7 % of dry biomass, respectively. The highest volumetric productivity of TLs, NLs, and TFAs reached 0.28, 0.25, and 0.21 g L^?1 day^?1, respectively. V. stellata had a suitable fatty acid profile for biodiesel production, as well as containing eicosapentaenoic acid (EPA) for nutraceutical applications. In addition, the content β-carotene, increased gradually as culture time was prolonged, resulting in its exclusive production at the end of cultivation. V. stellata is a promising microalgal strain for the production of biofuels and bioproducts.     

Outdoor microalgae cultivation in airlift photobioreactor at high irradiance and temperature conditions: effect of batch and fed-batch strategies,photoinhibition, and temperature stress

The microalgae Scenedesmus abundans cultivated in five identical airlift photobioreactors (PBRs) in batch and fed-batch modes at the outdoor tropical condition. The microalgae strain S. abundans was found to tolerate high temperature (35–45 °C) and high light intensity (770–1690 µmol m^− 2 s^− 1). The highest biomass productivities were 152.5–162.5 mg L^− 1 day^− 1 for fed-batch strategy. The biomass productivity was drastically reduced due to photoinhibition effect at a culture temperature of > 45 °C. The lipid compositions showed fatty acids mainly in the form of saturated and monounsaturated fatty acids (> 80%) in all PBRs with Cetane number more than 51. The fed-batch strategies efficiently produced higher biomass and lipid productivities at harsh outdoor conditions. Furthermore, the microalgae also accumulated omega-3 fatty acid (C18:3) up to 14% (w/w) of total fatty acid at given outdoor condition.

     

The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp

We examined responses of batch cultures of the marine microalga Nannochloropsis sp. to combined alterations in salinity (13, 27, and 40 g/l NaCl) and light intensity (170 and 700 μmol photons/m²·s). Major growth parameters and lipid productivity (based on total fatty acid determination) were determined in nitrogen-replete and nitrogen-depleted cultures of an initial biomass of 0.8 and 1.4 g/l, respectively. On the nitrogen-replete medium, increases in light intensity and salinity increased the cellular content of dry weight and lipids due to enhanced formation of triacylglycerols (TAG). Maximum average productivity of ca. 410 mg TFA/l/d were obtained at 700 μmol photons/m²·s and 40 g/l NaCl within 7 days. Under stressful conditions, content of the major LC-PUFA, eicosapentaenoic acid (EPA), was significantly reduced while TAG reached 25% of biomass. In contrast, lower salinity tended to improve major growth parameters, consistent with less variation in EPA contents. Combined higher salinity and light intensity was detrimental to lipid productivity under nitrogen starvation; biomass TFA content, and lipid productivity amounted for only 33% of DW and ca. 200 mg TFA/l/day, respectively. The highest biomass TFA content (ca. 47% DW) and average lipid productivity of ca. 360 mg TFA/l/day were achieved at 13 g/l NaCl and 700 μmol photons/m²·s. Our data further support selecting Nannochloropsis as promising microalgae for biodiesel production. Moreover, appropriate cultivation regimes may render Nannochloropsis microalgae to produce simultaneously major valuable components, EPA, and TAG, while sustaining relatively high biomass growth rates.     

Mixotrophic cultivation of <Emphasis Type="Italic">Chlorococcum</Emphasis> sp. under non-controlled conditions using a digestate from pig manure within a biorefinery

A number of business opportunities may arise from microalgae and wastewater treatment becoming an integrated system, as biofuels and high-added value products could be obtained simultaneously. This study, performed under controlled and non-controlled conditions, aimed at cultivating Chlorococcum sp. using a digestate from pig manure as culture medium and assessing its growth and biochemical composition for further applications. Under controlled conditions, cultures of Chlorococcum sp. were established testing various digestate dilutions (v/v). It was found that all tested dilutions (up to 8% v/v) promoted a higher biomass density, compared to the control culture in modified Bold’s Basal Medium (modified BBM). Under non-controlled conditions, it was found that the biomass productivity using the digestate diluted 5.6% v/v (23.4 mg L^?1 day^?1) was statistically similar to the one obtained using modified BBM (26.4 mg L^?1 day^?1). The volatile fatty acids contained in the digestate might have allowed mixotrophic growth for Chlorococcum sp. The intracellular lipid content in Chlorococcum sp. remained constant throughout the experiments in both, treatment and control cultures, while carbohydrates increased from 20 to 45% of the cell dry weight in the treatment and from 20 to 42% in the control one. It was concluded that conditions of nitrogen starvation and fluctuating irradiance and temperature benefit carbohydrate accumulation in this strain, since intracellular carbohydrate content increased nearly two-fold during this period. Additionally, the obtained biomass has the potential to be used as feedstock for bioethanol production. This system can meet the concept of a microalgae-based biorefinery, in which biofuels and high-added value products are produced from microalgae and wastewater.     

Improvement of lipid production in the marine strains Alexandrium minutum and Heterosigma akashiwo by utilizing abiotic parameters

Two different strains of microalgae, one raphidophyte and one dinoflagellate, were tested under different abiotic conditions with the goal of enhancing lipid production. Whereas aeration was crucial for biomass production, nitrogen deficiency and temperature were found to be the main abiotic parameters inducing the high-level cellular accumulation of neutral lipids. Net neutral lipid production and especially triacylglycerol (TAG) per cell were higher in microalgae (>200% in Alexandrium minutum, and 30% in Heterosigma akashiwo) under treatment conditions (25°C; 330 μM NaNO₃) than under control conditions (20°C; 880 μM NaNO₃). For both algal species, oil production (free fatty acids plus TAG fraction) was also higher under treatment conditions (57 mg L⁻¹ in A. minutum and 323 mg L⁻¹ in H. akashiwo). Despite the increased production and accumulation of lipids in microalgae, the different conditions did not significantly change the fatty acids profiles of the species analyzed. These profiles consisted of saturated fatty acids (SAFA) and polyunsaturated fatty acids (PUFA) in significant proportions. However, during the stationary phase, the concentrations per cell of some PUFAs, especially arachidonic acid (C20:4n6), were higher in treated than in control algae. These results suggest that the adjustment of abiotic parameters is a suitable and one of the cheapest alternatives to obtain sufficient quantities of microalgal biomass, with high oil content and minimal changes in the fatty acid profile of the strains under consideration.     

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.