Microalgal Lipid Production Research Articles

Continuous versus batch production of lipids in the microalgae Acutodesmus obliquus

This work provides a novel quantitative comparison of batch versus continuous microalgal lipid production in the wild type and starchless mutant strain of Acutodesmus obliquus. Both strains showed higher TAG yields on light under batch operation compared to continuous nitrogen limitation. The starchless mutant showed 0.20gTAGmolph−1 for batch and 0.12gTAGmolph−1 for continuous operation, while the wildtype only showed 0.16gTAGmolph−1 for batch and 0.08gTAGmolph−1 for continuous operation. Also, higher TAG contents were found under batch starvation (26% of dry weight for the wildtype and 43% of dry weight for starchless mutant) compared to continuous cultivations (16% of dry weight for the wildtype and 33% of dry weight for starchless mutant). Starch acts as the favoured storage metabolite during nitrogen limitation in A. obliquus, whereas TAG is only accumulated after starch reaches a cellular maximum of 40% of dry weight.

Outdoor Cultivation of Marine Diatoms for Year-Round Production of Biofuels.

Biofuel production using microalgae is believed to have the advantage of continuous year-round production over crop plants, which have strong seasonality. However, actual year-round production of microalgal lipids using outdoor mass cultivation has rarely been demonstrated. In our previous study, it was demonstrated that the oleaginous diatom, Fistulifera solaris, was culturable in outdoor bioreactors from spring to autumn, whereas biomass and lipid production in winter failed because F. solaris did not grow below 15 °C. Therefore, another candidate strain that is culturable in winter is required. In this study, a cold-tolerant diatom, Mayamaea sp. JPCC CTDA0820, was selected as a promising candidate for biofuel production in winter. Laboratory-scale characterization revealed that this diatom was culturable at temperatures as low as 10 °C. Subsequently, F. solaris (April–October) and Mayamaea sp. JPCC CTDA0820 (November–March) were cultured in outdoor open-pond bioreactors, wherein year-round production of diatom lipids was successfully demonstrated. The maximal values of areal productivities of biomass and lipids reached to 9.79 and 1.80 g/(m2 day) for F. solaris, and 8.62 and 0.92 g/(m2 day) for Mayamaea sp. JPCC CTDA0820, respectively. With the combined use of these two diatom species, stable year-round production of microalgal lipids became possible.

Raman spectroscopy compatible PDMS droplet microfluidic culture and analysis platform towards on-chip lipidomics.

Lipids produced by microalgae are viewed as a potential renewable alternative to fossil fuels, however, significant improvements in productivity are required for microalgal biofuels to become economically feasible. Here we present a method that allows for the use of Raman spectroscopy with poly(dimethylsiloxane) (PDMS) droplet microfluidic devices, which not only overcomes the high Raman background of PDMS, but also achieves pairing of the high-throughput single-cell resolution advantages of droplet microfluidics with the direct, chemically specific, label-free, and non-destructive nature of Raman spectroscopy. The platform was successfully utilized for in situ characterization of microalgal lipid production over time within droplets, paving the way towards high-throughput microalgal lipidomics assays.

Enhanced microalgal biomass and lipid production from a consortium of indigenous microalgae and bacteria present in municipal wastewater under gradually mixotrophic culture conditions

The goal of this study was to investigate the influences of gradually mixotrophic culture conditions on microalgal biomass and lipid production by a consortium of indigenous microalgae and bacteria present in raw municipal wastewater. Lab-scale photobioreactors containing the consortium were operated in repeated batch mode. Initial cultivation (phase I) was performed using only the municipal wastewater, then 10% and 25% of the reactor volumes were replaced with the effluent from a sewage sludge fermentation system producing volatile fatty acids (SSFV) at the beginnings of phase II and phase III, respectively. The highest biomass productivity (117.1±2.7mg/L/d) was attained during phase II, but the lipid productivity (17.2±0.2mg/L/d) was attained during phase III. The increase in the effluent from the SSFV influenced microalgal diversity with a preference for Chlorella sp., but bacterial diversity increased significantly during phase III.

Detoxification of ammonium to Nannochloropsis oculata and enhancement of lipid production by mixotrophic growth with acetate

In this study, the toxicity of ammonium was removed in the microalga Nannochloropsis oculata by using acetate as a carbon source. Algal biomass and lipid production were significantly enhanced when N. oculata was grew on 0.5–50mM of ammonium and 16–64mM of acetate in mixotrophic conditions. When grown mixotrophically on 1mM of ammonium and 32mM of acetate, the biomass and lipid production reached 543mg/L and 279mg/L respectively, which were 1.5 and 9.4times higher than the levels generated when grown autotrophically on nitrate. This suggests that mixotrophic growth with acetate can be a useful method to enhance microalgal lipid production.

Synergistic effect of multiple stress conditions for improving microalgal lipid production

This increasing consumption of fossil fuels and emission of greenhouse gases causes climate change. Thus, the development of alternative energy sources such as biofuel is necessary. Microalgae have been spotlighted as renewable energy resources because the potential for producing biofuels from CO2. However, the unit cost for the production of algae-based biofuel must be reduced for commercialization compared to the petroleum. The optimization of culture conditions of microalgal strains is crucial to increase lipid production for economically viable biofuels. We rapidly analyzed the combined effect of various stress conditions (nitrogen starvation, temperature, pH, and salt concentration) on the lipid production in various strains using a multiplex microfluidic system, enabling multiple operations from cell culture to lipid extraction of different strains. We found the lipid productivity was enhanced by 25 to 54% under combinations of two stress condition compared to the single stress condition. However, the combination of more than three stress conditions reduced the lipid productivities of all microalgal strains because of more stressful environment to the cells compared to the combinations of two stress conditions. We further validated the synergistic effect of combined stress conditions in flask culture with the increases in lipid productivities by up to 106% compared to the single stress condition. We also observed that fatty acids composition, which influences the quality of algal biofuels, was changed according to the combination of stress conditions. In particular, C. protothecoides can be good candidate for production of high quality of biodiesel, because it has high CN, IV and low CFPP which is suitable for high quality of biodiesel. These results indicate that combination of multiple stress conditions can be efficient strategy for the optimization of microalgal cultivation to produce algal biofuels with high quality and economic feasibility.

Nickel oxide nanoparticle-based method for simultaneous harvesting and disruption of microalgal cells

Microalgae biodiesel is considered one of the most promising renewable fuels. However, the high cost of the downstream process is a major barrier to large-scale microalgal lipid production. In this study, a novel approach based on nickel oxide nanoparticles (NiO NPs) was developed and its effectiveness for simultaneous harvesting and cell disruption in microalgal lipid production was determined. NiO NPs exhibited a microalgal harvesting efficiency of 98.75% in 1min at pH 7. Moreover, after treating with NiO NPs for 96h, the lipid extraction efficiency of microalgae (with 80% water content) reached 91.08% and was 208.37% compared to that without NiO treatment. This approach is simple and does not necessitate drying; furthermore, no equipment with high energy consumption was required.

Effect of nitrogen regime on microalgal lipid production during mixotrophic growth with glycerol

Mixotrophic growth of microalgae to boost lipid production is currently under active investigation. Such a process could be of practical importance if a cheap source of organic carbon, such as waste glycerol from biodiesel production, could be used. Several previous studies have already demonstrated that this carbon source can be used by different indigenous strains of microalgae. In this study it is shown that different nitrogen limitation strategies can be applied to further increase lipid production during growth with glycerol. In one strategy, cultures were grown in nitrogen replete medium and then resuspended in nitrogen free medium. In a second strategy, cultures were grown with different initial concentrations of nitrate. Lipid production by the two microalgal strains used, Chlorella sorokiniana (PCH02) and Chlorella vulgaris (PCH05), was shown to be boosted by strategies of nitrogen limitation, but they responded differently to how nitrogen limitation was imposed.

A Review on the Assessment of Stress Conditions for Simultaneous Production of Microalgal Lipids and Carotenoids.

Microalgal species are potential resource of both biofuels and high-value metabolites, and their production is growth dependent. Growth parameters can be screened for the selection of novel microalgal species that produce molecules of interest. In this context our review confirms that, autotrophic and heterotrophic organisms have demonstrated a dual potential, namely the ability to produce lipids as well as value-added products (particularly carotenoids) under influence of various physico-chemical stresses on microalgae. Some species of microalgae can synthesize, besides some pigments, very-long-chain polyunsaturated fatty acids (VL-PUFA,>20C) such as docosahexaenoic acid and eicosapentaenoic acid, those have significant applications in food and health. Producing value-added by-products in addition to biofuels, fatty acid methyl esters (FAME), and lipids has the potential to improve microalgae-based biorefineries by employing either the autotrophic or the heterotrophic mode, which could be an offshoot of biotechnology. The review considers the potential of microalgae to produce a range of products and indicates future directions for developing suitable criteria for choosing novel isolates through bioprospecting large gene pool of microalga obtained from various habitats and climatic conditions.

Automation of a Nile red staining assay enables high throughput quantification of microalgal lipid production.

BackgroundWithin the context of microalgal lipid production for biofuels and bulk chemical applications, specialized higher throughput devices for small scale parallelized cultivation are expected to boost the time efficiency of phototrophic bioprocess development. However, the increasing number of possible experiments is directly coupled to the demand for lipid quantification protocols that enable reliably measuring large sets of samples within short time and that can deal with the reduced sample volume typically generated at screening scale. To meet these demands, a dye based assay was established using a liquid handling robot to provide reproducible high throughput quantification of lipids with minimized hands-on-time.ResultsLipid production was monitored using the fluorescent dye Nile red with dimethyl sulfoxide as solvent facilitating dye permeation. The staining kinetics of cells at different concentrations and physiological states were investigated to successfully down-scale the assay to 96 well microtiter plates. Gravimetric calibration against a well-established extractive protocol enabled absolute quantification of intracellular lipids improving precision from ±8 to ±2 % on average. Implementation into an automated liquid handling platform allows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to a third compared to manual operation. Moreover, it was shown that automation enhances accuracy and precision compared to manual preparation. It was revealed that established protocols relying on optical density or cell number for biomass adjustion prior to staining may suffer from errors due to significant changes of the cells’ optical and physiological properties during cultivation. Alternatively, the biovolume was used as a measure for biomass concentration so that errors from morphological changes can be excluded.ConclusionsThe newly established assay proved to be applicable for absolute quantification of algal lipids avoiding limitations of currently established protocols, namely biomass adjustment and limited throughput. Automation was shown to improve data reliability, as well as experimental throughput simultaneously minimizing the needed hands-on-time to a third. Thereby, the presented protocol meets the demands for the analysis of samples generated by the upcoming generation of devices for higher throughput phototrophic cultivation and thereby contributes to boosting the time efficiency for setting up algae lipid production processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0433-7) contains supplementary material, which is available to authorized users.

Multiplex microfluidic system integrating sequential operations of microalgal lipid production.

The unit cost for the production of algal biofuel needs to be reduced in order to be a substitute for fossil fuel. To achieve this goal, the development of a novel system is needed for a rapid screening of numerous microalgal species to isolate superior strains with the highest lipid productivity. Here, we developed a PDMS-based multiplex microfluidic system with eight chambers and micropillar arrays to expedite multiple steps for lipid sample preparation from different microalgal strains. We could rapidly and efficiently perform sequential operations from cell culture to lipid extraction of eight different microalgal strains simultaneously on a single device without harvesting and purification steps, which are labor- and energy-intensive, by the simple injection of medium and solvent into the central inlet due to the integrated micropillar arrays connecting the chambers and central inlet. The lipid extraction efficiency using this system was comparable (94.5-102.6%) to the conventional Bligh-Dyer method. We investigated the cell growth and lipid productivity of different strains using the microfluidic device. We observed that each strain has a different lipid accumulation pattern according to stress conditions. These results demonstrate that our multiplex microfluidic approach can provide an efficient analytical tool for the rapid analysis of strain performances (e.g. cell growth and lipid productivities) and the determination of the optimal lipid induction condition for each strain.

Strategies for Lipid Production Improvement in Microalgae as a Biodiesel Feedstock.

In response to the energy crisis, global warming, and climate changes, microalgae have received a great deal of attention as a biofuel feedstock. Due to a high lipid content in microalgal cells, microalgae present as a promising alternative source for the production of biodiesel. Environmental and culturing condition variations can alter lipid production as well as chemical compositions of microalgae. Therefore, application of the strategies to activate lipid accumulation opens the door for lipid overproduction in microalgae. Until now, many original studies regarding the approaches for enhanced microalgal lipid production have been reported in an effort to push forward the production of microalgal biodiesel. However, the current literature demonstrates fragmented information available regarding the strategies for lipid production improvement. From the systematic point of view, the review highlights the main approaches for microalgal lipid accumulation induction to expedite the application of microalgal biodiesel as an alternative to fossil diesel for sustainable environment. Of the several strategies discussed, the one that is most commonly applied is the design of nutrient (e.g., nitrogen, phosphorus, and sulfur) starvation or limitation. Other viable approaches such as light intensity, temperature, carbon dioxide, salinity stress, and metal influence can also achieve enhanced microalgal lipid production.

Strain variation in microalgal lipid production during mixotrophic growth with glycerol

Algal cultivation at high latitudes is challenged by the relatively low annual solar flux. One possible scenario to overcome this limitation is the use of mixotrophic growth to potentially boost biomass and lipid production. Here the effect of glycerol addition on the growth and lipid production by twelve indigenous microalgae was examined. The results show that there is considerable strain dependent variation in the maximum growth rate under mixotrophic conditions with the addition of glycerol causing in some cases up to a 2.4-fold increase in growth rate and a up to a 1.9-fold increase in biomass. In addition, glycerol increased total lipid production 40–60% in some strains. These results also show the value in screening culture collections for desired traits independent of strain identification since here one (PCH02) of the five Chlorella strains showed a large increase in lipid with glycerol.

Construction and characteristics of artificial consortia of Scenedesmus obliquus-bacteria for S. obliquus growth and lipid production

In this work, artificial consortia of Scenedesmus obliquus and selected bacterial strains were constructed to investigate the effects of bacteria on microalgal biomass and lipid production, as well as their inter-relationship in a photoautotrophic system. Microalgal growth was enhanced when co-cultivated with single selected bacterial strains, among which Acidovorax facilis represented the largest biomass concentration increase of 24.8%, compared to the axenic culture of S. obliquus. The consortium system also enhanced microalgal lipid content, lipid productivity, and the proportion of saturated fatty acids and oleic acid. Moreover, the selected bacterial strains adhered directly to the S. obliquus cell surface, which was necessary for the establishment of significant interaction. Co-cultivation of S. obliquus and bacterial strains resulted in changes in both the concentration and composition of proteins and polysaccharides dissolved in the culture supernatant and cell-associated (bound) fractions, which partially contributed to the observed differences in microalgal biomass and lipid production.

Nutritional stress effects under different nitrogen sources on the genes in microalga Isochrysis zhangjiangensis and the assistance of Alteromonas macleodii in releasing the stress of amino acid deficiency

The expressions of nine nitrogen assimilation-associated genes, NRT2, NAR1, NIA2, NIR, GLN2, GLSF, GSN1, GDH, and AAT2, in the microalga Isochrysis zhangjiangensis were investigated to unveil the effects of limitations of various nitrogen sources (NaNO3 , NH4 Cl, NaNO2 , and an amino acid mixture) on the microalgae. The results demonstrated that the NRT2, NAR1, GLN2, GSN1, and AAT2 genes were highly expressed in lipid-rich microalgae under inorganic nitrogen-deficient conditions and they decreased after nitrogen resupply. Significant increases in the expressions of NAR1, GLN2, and GLSF were found in nitrate-depleted microalgae, whereas significant increases in the expressions of NRT2, NAR1, GLN2, and GSN1 were found in nitrite-depleted microalgae. Significant increases in the expressions of only NRT2 and GSN1 were found in ammonium-depleted microalgae (P<0.05). Except for the NRT2, other genes were expressed at lower levels under amino acid-deficient conditions compared with amino acid-sufficient controls. The expression of the NIA2 gene decreased in nitrogen-depleted microalgae regardless of the initial nitrogen source. However, the results of fatty acid analyses showed that the features of fatty acid profiles followed a similar mode, in which the percentage compositions of C16:0 and C18:1Δ(9) increased in nitrogen-depleted cells and that of C16:1Δ(9) , C18:3Δ(9,12,15) , C18:4Δ(6,9,12,15) , and C18:5Δ(3,6,9,12,15) decreased, regardless of the type of nitrogen source applied. It was also found that the epiphytic bacterium Alteromonas macleodii played a particularly important role in releasing microalgae from the stress of amino acid deficiency. These findings also provide a foundation for regulating microalgal lipid production through manipulation of the nitrogen assimilation-associated genes.

Microalgal lipid production using the hydrolysates of rice straw pretreated with gamma irradiation and alkali solution.

BackgroundLignocellulosic biomass has long been recognized as a potential sustainable source of sugars for biofuels. However, many physicochemical structural and compositional factors inhibit the enzymatic digestibility of the lignocellulosic biomass. In this study, efficient pretreatment method of rice straw (RS) was developed and the RS hydrolysate was applied in the cultivation of microalgae for lipid production.ResultsGamma ray irradiation (GRI) and alkali solution were used for the pretreatment, and saccharification was carried out with lignocellulolytic enzymes. When RS was pretreated by combined GRI and alkali method, the glucose and xylose saccharification yield after enzymatic hydrolysis increased up to 91.65 and 98.84 %, respectively. The enzymatic hydrolysate from the RS pretreated with the combined method was used to cultivate Chlorella protothecoides for lipid production. The maximum concentrations of biomass and fatty acid methyl ester of cells were 6.51 and 2.95 g/L, respectively. The lipid content of C. protothecoides from RS hydrolysate was comparable to that from glucose, and the lipid composition was similar between different carbon sources.ConclusionThese results demonstrate that the combined pretreatment with gamma irradiation was highly effective in preparing hydrolysate, and the rice straw hydrolysate could be used as an alternative carbon source for microalgal lipid production for biofuel.

Simultaneous nutrient removal and lipid production with Chlorella vulgaris on sterilized and non-sterilized anaerobically pretreated piggery wastewater

Piggery wastewater is a potent nutrient source for microalgal lipid production. Wastewater has been usually sterilized when used for microalgal cultivation. This is uneconomical in large-scale applications. Therefore, lipid productivity of Chlorella vulgaris CY5 using sterilized and non-sterilized diluted anaerobically pretreated piggery wastewater was studied in batch reactors. The maximum average lipid productivity was obtained after 12 days of incubation and it was higher with the sterilized wastewater than with the non-sterilized one (117g/L/d vs. 91.3g/L/d), due to the higher biomass concentration. Because of the unexpected increase of dissolved organic carbon (DOC) in the cultures, second experiment was conducted to characterize the composition of produced DOC in non-sterilized wastewater. Carbohydrate content increased in the liquid phase but decreased in the biomass after nitrogen had been exhausted. After 12 days of incubation, soluble chemical oxygen demand (CODs) was 414±56mg/L, biomass production was 2.8±0.15g/L, and lipid content was 30.3±1.2wt%. Average lipid productivity from day zero to day 12 was 70.5±1.1g/L/d. C. vulgaris removed nutrients from the non-sterilized wastewater and produced oleaginous biomass, although the lipid productivity was higher with sterilized wastewater.

Effect of carbon source on biomass growth and nutrients removal of Scenedesmus obliquus for wastewater advanced treatment and lipid production

The combination of tertiary wastewater treatment and microalgal lipid production is considered to be a promising approach to water eutrophication as well as energy crisis. To intensify wastewater treatment and microalgal biofuel production, the effect of organic and inorganic carbon on algal growth and nutrient removal of Scenedesmus obliquus were examined by varying TOC (total organic carbon) concentrations of 20–120mgL−1 in wastewater and feeding CO2 concentrations in the range of 0.03–15%, respectively. The results showed that the maximal biomass and average lipid productivity were 577.6 and 16.7mgL−1d−1 with 5% CO2 aeration. The total nitrogen, total phosphorus and TOC removal efficiencies were 97.8%, 95.6% and 59.1% respectively within 6days when cultured with real secondary municipal wastewater. This work further showed that S. obliquus could be utilized for simultaneous organic pollutants reduction, N, P removal and lipid accumulation.

Effects of thermodynamically coupled reaction diffusion in microalgae growth and lipid accumulation: Model development and stability analysis

This study investigates and presents the effects of thermodynamically coupled nonisothermal reaction-diffusion processes on microalgae growth, substrate consumption and neutral lipid production in a pond or wastewater treatment plant. The non-stirred chemostat hypothesis and linear nonequilibrium thermodynamics theory are applied to formulate the model equations that account the bulk phase compositions and temperature, resistances to the heat and mass transfers, and cross effects due to the thermodynamic coupling of heat and mass flow in the presence of chemical reaction. Nondimensional forms of the model equations are numerically solved. Bulk phase concentrations and temperatures, external resistances to heat and substrate transfers, and thermodynamic coupling may generate substantial number of new parameters that control the evolution and stability in microalgal growth and lipid production that are important for biofuels. Instabilities due to perturbations in nutrient concentrations may lead to spatial structures where the wavenumber plays important role in reaction diffusion systems.

Sequential generation of hydrogen and lipids from starch by combination of dark fermentation and microalgal cultivation

Dark fermentative hydrogen production and microalgal lipid production was successfully combined to enhance the energy conversion from starch with simultaneous treatment of volatile fatty acids in the effluent.