Neutral Lipid Production Research Articles

Growth and Physiological State of Microalgae Heterosigma akashiwo (Raphidophyceae) during Exposure to Cadmium, Lead, and Nickel

The effect of heavy metals cadmium, lead, and nickel on the growth and physiological state of raphidophyte algae Heterosigma akashiwo MBRU_HAK-SR11 (Y. Hada) Y. Hada ex Y. Hara, M. Chihara during 7 days of experiments has been assessed. It was found that cadmium and nickel at concentrations of 10 and 20 µg/L stimulated H. akashiwo growth, while lead inhibited it at these concentrations. Chlorophyll a and carotenoids content increased with the addition of 10 μg/L of cadmium and 20 μg/L of nickel, and the content of carotenoids was higher than that in the control with the addition of 20 μg/L of cadmium. With the introduction of lead, an increase in the level of chlorophyll a and a decrease in the content of carotenoids were observed. The content of ROS increased with the introduction of cadmium and lead and decreased with the introduction of nickel. Cadmium had an effect on the production of neutral lipids: their content increased and decreased by the end of the experiment. Nickel stimulated the accumulation of neutral lipids H. akashiwo, while lead had no effect on their content. Metals had the least effect on forward and side light scattering and fluorescence of chlorophyll a. The absence of pronounced changes in direct and lateral light scattering indirectly indicates that the algae cells did not change morphologically under toxic exposure. Thus, cadmium, lead, and nickel at concentrations of 10–20 µg/L changed physiological processes in algae.

Proteomes reveal metabolic capabilities of Yarrowia lipolytica for biological upcycling of polyethylene into high-value chemicals.

Sustainable processes for biological upcycling of plastic wastes in a circular bioeconomy are needed to promote decarbonization and reduce environmental pollution due to increased plastic consumption, incineration, and landfill storage. Strain characterization and proteomic analysis revealed the robust metabolic capabilities of Yarrowia lipolytica to upcycle polyethylene into high-value chemicals. Significant proteome reallocation toward energy and lipid metabolisms was required for robust growth on hydrocarbons with n-hexadecane as the preferential substrate. However, an apparent over-investment in these same categories to utilize complex depolymerized plastic (DP) oil came at the expense of protein biosynthesis, limiting cell growth. Taken together, this study elucidates how Y. lipolytica activates its metabolism to utilize DP oil and establishes Y. lipolytica as a promising host for the upcycling of plastic wastes.

Metabolic Changes and Antioxidant Response in Ustilago maydis Grown in Acetate.

Ustilago maydis is an important model to study intermediary and mitochondrial metabolism, among other processes. U. maydis can grow, at very different rates, on glucose, lactate, glycerol, and ethanol as carbon sources. Under nitrogen starvation and glucose as the only carbon source, this fungus synthesizes and accumulates neutral lipids in the form of lipid droplets (LD). In this work, we studied the accumulation of triacylglycerols in cells cultured in a medium containing acetate, a direct precursor of the acetyl-CoA required for the synthesis of fatty acids. The metabolic adaptation of cells to acetate was studied by measuring the activities of key enzymes involved in glycolysis, gluconeogenesis, and the pentose phosphate pathways. Since growth on acetate induces oxidative stress, the activities of some antioxidant enzymes were also assayed. The results show that cells grown in acetate plus nitrate did not increase the amount of LD, but increased the activities of glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase, suggesting a higher production of reactive oxygen species in cells growing in acetate. The phosphofructokinase-1 (PFK1) was the enzyme with the lowest specific activity in the glycolytic pathway, suggesting that PFK1 controls the flux of glycolysis. As expected, the activity of the phosphoenolpyruvate carboxykinase, a gluconeogenic enzyme, was present only in the acetate condition. In summary, in the presence of acetate as the only carbon source, U. maydis synthesized fatty acids, which were directed into the production of phospholipids and neutral lipids for biomass generation, but without any excessive accumulation of LD.

Inhibition of monogalactosyldiacylglycerol synthesis by down-regulation of MGD1 leads to membrane lipid remodeling and enhanced triacylglycerol biosynthesis in Chlamydomonas reinhardtii

BackgroundMembrane lipid remodeling involves regulating the physiochemical modification of cellular membranes against abiotic stress or senescence, and it could be a trigger to increase neutral lipid content. In algae and higher plants, monogalactosyldiacylglycerol (MGDG) constitutes the highest proportion of total membrane lipids and is highly reduced as part of the membrane lipid remodeling response under several abiotic stresses. However, genetic regulation of MGDG synthesis and its influence on lipid synthesis has not been studied in microalgae. For development of an industrial microalgae strain showing high accumulation of triacylglycerol (TAG) by promoting membrane lipid remodeling, MGDG synthase 1 (MGD1) down-regulated mutant of Chlamydomonas reinhardtii (Cr-mgd1) was generated and evaluated for its suitability for biodiesel feedstock.ResultsThe Cr-mgd1 showed a 65% decrease in CrMGD1 gene expression level, 22% reduction in MGDG content, and 1.39 and 5.40 times increase in diacylglyceryltrimethylhomoserines (DGTS) and TAG, respectively. The expression levels of most genes related to the decomposition of MGDG (plastid galactoglycerolipid degradation1) and TAG metabolism (diacylglycerol O-acyltransferase1, phospholipid:diacylglycerol acyltransferase, and major lipid droplet protein) were increased. The imbalance of DGDG/MGDG ratio in Cr-mgd1 caused reduced photosynthetic electron transport, resulting in less light energy utilization and increased reactive oxygen species levels. In addition, endoplasmic reticulum stress was induced by increased DGTS levels. Thus, accelerated TAG accumulation in Cr-mgd1 was stimulated by increased cellular stress as well as lipid remodeling. Under high light (HL) intensity (400 µmol photons/m2/s), TAG productivity in Cr-mgd1–HL (1.99 mg/L/d) was 2.71 times higher than that in wild type (WT–HL). Moreover, under both nitrogen starvation and high light intensity, the lipid (124.55 mg/L/d), TAG (20.03 mg/L/d), and maximum neutral lipid (56.13 mg/L/d) productivity were the highest.ConclusionsBy inducing lipid remodeling through the mgd1 gene expression regulation, the mutant not only showed high neutral lipid content but also reached the maximum neutral lipid productivity through cultivation under high light and nitrogen starvation conditions, thereby possessing improved biomass properties that are the most suitable for high quality biodiesel production. Thus, this mutant may help understand the role of MGD1 in lipid synthesis in Chlamydomonas and may be used to produce high amounts of TAG.

Nutrient Deprivation Coupled with High Light Exposure for Bioactive Chrysolaminarin Production in the Marine Microalga Isochrysis zhangjiangensis.

Chrysolaminarin, a kind of water-soluble bioactive β-glucan produced by certain microalgae, is a potential candidate for food/pharmaceutical applications. This study identified a marine microalga Isochrysis zhangjiangensis, in which chrysolaminarin production was investigated via nutrient (nitrogen, phosphorus, or sulfur) deprivations (-N, -P, or -S conditions) along with an increase in light intensity. A characterization of the antioxidant activities of the chrysolaminarin produced under each condition was also conducted. The results showed that nutrient deprivation caused a significant increase in chrysolaminarin accumulation, though this was accompanied by diminished biomass production and photosynthetic activity. -S was the best strategy to induce chrysolaminarin accumulation. An increase in light intensity from 80 (LL) to 150 (HL) µE·m−2·s−1 further enhanced chrysolaminarin production. Compared with -N, -S caused more suitable stress and reduced carbon allocation toward neutral lipid production, which enabled a higher chrysolaminarin accumulation capacity. The highest chrysolaminarin content and concentration reached 41.7% of dry weight (%DW) and 632.2 mg/L, respectively, under HL-S, with a corresponding productivity of 155.1 mg/L/day achieved, which exceeds most of the photoautotrophic microalgae previously reported. The chrysolaminarin produced under HL-N (Iz-N) had a relatively competitive hydroxyl radical scavenging activity at low concentrations, while the chrysolaminarin produced under HL-S (Iz-S) exhibited an overall better activity, comparable to the commercial yeast β-glucan, demonstrating I. zhangjiangensis as a promising bioactive chrysolaminarin producer from CO2.

Co-culturing of microalgae and bacteria in real wastewaters alters indigenous bacterial communities enhancing effluent bioremediation

Although microalgal–bacterial co-cultures have been largely investigated for nutrient removal, their application in treating real wastewaters is still debatable because effluent systems contain complex bacterial communities that might influence the overall bioremediation. We, therefore, verified the interaction effects of Tetradesmus obliquus IS2–Variovorax paradoxus IS1 co-culture and the native bacterial communities in wastewaters, collected from dairy farm and poultry slaughterhouse, during effluent bioremediation. Co-culturing of the two microbial strains in non-sterile wastewaters alone resulted in significant removal of total N (>92%), PO43−-P (>89%) and COD (>84%) as well as increased production of ROS and neutral lipids, indicating their significant mutualistic interactions with the native bacterial communities for efficient bioremediation of wastewaters. Analysis of 16S rRNA gene-based community data in wastewaters revealed selective enrichment of Firmicutes and Actinobacteria, resulting in enhanced microalgal growth and nutrient removal. The findings of this novel study suggest that microalgal–bacterial co-cultures mediate interspecies interactions for a shift in population dynamics of the indigenous bacteria in wastewaters to facilitate enhanced bioremediation of effluents with sustainable production of biomass and biodiesel.

Supra-Optimal Temperature: An Efficient Approach for Overaccumulation of Starch in the Green Alga Parachlorella kessleri.

Green algae are fast-growing microorganisms that are considered promising for the production of starch and neutral lipids, and the chlorococcal green alga Parachlorella kessleri is a favorable model, as it can produce both starch and neutral lipids. P. kessleri commonly divides into more than two daughter cells by a specific mechanism—multiple fission. Here, we used synchronized cultures of the alga to study the effects of supra-optimal temperature. Synchronized cultures were grown at optimal (30 °C) and supra-optimal (40 °C) temperatures and incident light intensities of 110 and 500 μmol photons m−2 s−1. The time course of cell reproduction (DNA replication, cellular division), growth (total RNA, protein, cell dry matter, cell size), and synthesis of energy reserves (net starch, neutral lipid) was studied. At 40 °C, cell reproduction was arrested, but growth and accumulation of energy reserves continued; this led to the production of giant cells enriched in protein, starch, and neutral lipids. Furthermore, we examined whether the increased temperature could alleviate the effects of deuterated water on Parachlorella kessleri growth and division; results show that supra-optimal temperature can be used in algal biotechnology for the production of protein, (deuterated) starch, and neutral lipids.

Characterization of Cellular Growth, CO2 Assimilation and Neutral Lipid Production for 4 Different Algal Species

Characterization of Cellular Growth, CO2 Assimilation and Neutral Lipid Production for 4 Different Algal Species

A Three‐Pronged Synergistic Approach to Increase the Biofeedstock Viability of Chlorella vulgaris

Over the last several decades, the global temperature has increased at an alarming rate, increasing the need for efficient renewable energy sources. 1 Microalgae are a promising source of biodiesel due to their high rate of lipid accumulation, but are not efficient enough to produce biofuel on a scale matching current fuel needs. 2Chlorella vulgaris is a fast‐growing unicellular green microalga with a high percent of lipids per biomass, making it a promising candidate for biodiesel production. Previous research has used two approaches to increase the accumulation of lipids and photosynthetic activity: the addition of exogenous glycerol or the addition of excess Mn2+. When used separately, the addition of glycerol increases neutral lipid production in C. vulgaris while the addition of Mn2+ increases photosynthetic capacity. 3 Glycerol was found to be directly incorporated into the triacylglycerol (TAG) backbone, which allowed for an increased flux toward TAG synthesis without significantly changing the energy exerted. Manganese was shown to increase the rate of electron transfer through photosystem II, increasing the overall photosynthetic output. While it was assumed that combining these conditions would further optimize the cultures for lipid production, this was not the case: when used simultaneously, they decrease TAG production. To improve lipid productivity, a three‐pronged synergistic approach was taken combining Mn2+‐replete conditions, nitrogen deprivation, and exogenous glycerol. Nitrogen was deprived from the cells, because under nitrogen deplete conditions cells shift away from protein synthesis and towards energy storage compounds that can be used to produce TAGs. Cultures were then monitored for turbidity, chlorophyll content, lipid productivity, biomass, and TAG content. Cells grown using this three‐pronged approach showed an increase in the amount of TAGs produced over time as the glycerol concentrations were increased. This novel approach can be used to increase lipid synthesis to increase biofuel feasibility.Support or Funding Information#NNX15AI01H.D.S. was supported from the Marshall University Department of Chemistry alumni donors to the Marshall University Department of Chemistry’s Summer Undergraduate Research Fellowship.D.R.J.K. and A.L.S. were supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952. A.L.S. was also funded by the NASA West Virginia Space Grant Consortium, Grant #NNX15AK74A and Training Grant

Steryl Ester Formation and Accumulation in Steroid-Degrading Bacteria.

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis, as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

Nitrogen-fixer enhances lipid yields in algal polycultures

Current algal biomass research still focuses mainly on identifying and growing monocultures that produce high amounts of lipids or other target compounds. However, monocultures might have a lower efficiency of utilizing resources, due to their limited physiological resource use possibilities, compared to algal polycultures. Recent studies showed that species diversity enhances the lipid production of microalgae. To identify the general patterns of enhanced lipid production in diverse microalgal communities it is essential to investigate links between resource use complementarity and the corresponding lipid production.Cyanobacteria and Chlorophyta are highly differentiated in their light spectrum utilization and nutrient requirements, e.g., some Cyanobacteria species can fix atmospheric nitrogen. To investigate whether these two groups are complementary in terms of optimizing biomass and neutral lipid production, we compared the growth of several species of Cyanobacteria and Chlorophyta in monocultures and polycultures consisting of species from both groups. Results show that lipid production increases with increasing functional diversity and that using just two different functional groups of microalgae provides a positive biodiversity effect. Lipid yields were particularly enhanced by the presence of a nitrogen-fixing cyanobacterium. Additionally, lipid yields were decoupled from higher biomass production in diverse communities. In summary, our results show that the enhancement of lipid production due to biodiversity can be achieved through combining just two physiologically different taxonomic groups and including a nitrogen fixer. This may make achieving the benefits of biodiversity in algal mass cultivation more straightforward.

Dynamic Modeling of Microalgae Growth and Lipid Production under Transient Light and Nitrogen Conditions

We developed a new dynamic model to characterize how light and nitrogen regulate the cellular processes of photosynthetic microalgae leading to transient changes in the production of neutral lipids, carbohydrates, and biomass. Our model recapitulated the versatile neutral lipid synthesis pathways via (i) carbon reuse from carbohydrate metabolism under nitrogen sufficiency and (ii) fixed carbon redirection under nitrogen depletion. We also characterized the effects of light adaptation, light inhibition hysteresis, and nitrogen limitation on photosynthetic carbon fixation. The formulated model was calibrated and validated with experimental data of Dunaliella viridis cultivated in a lab-scale photobioreactor (PBR) under various light (low/moderate/high) and nitrogen (sufficient/limited) conditions. We conducted the identifiability, uncertainty, and sensitivity analyses to verify the model reliability using the profile likelihood method, the Markov chain Monte Carlo (MCMC) technique, and the extended Fourier Amplitude Sensitivity Test (eFAST). Our model predictions agreed well with experimental observations and suggested potential model improvement by incorporating a lipid degradation mechanism. The insights from our model-driven analysis helped improve the mechanistic understanding of transient algae growth and bioproducts formation under environmental variations and could be applied to optimize biofuel and biomass production.

Hazardous effects of silver nanoparticles for primary producers in transitional water systems: The case of the seaweed Ulva rigida C. Agardh

The acute toxicity of citrate capped silver nanoparticles (AgNP) and silver nitrate was evaluated on the marine macroalga Ulva rigida C. Agardh (1823). Silver bioaccumulation, ultrastructural chloroplast damages verified by TEM microscopy, inhibition of primary production, neutral lipid production and oxidative stress were observed after 24 h of exposure to AgNP. The toxic effects of silver nitrate in artificial seawater started from a concentration of 0.05 ppm and was more toxic than AgNP that produced effects from a concentration of 0.1 ppm. However only AgNP induced lipid peroxidation in U. rigida. The addition of natural organic and inorganic ligands, represented by transparent exopolymer particles (TEP) and clay, drastically reduced AgNP acute toxicity in a ratio AgNP:ligand of 1:100 and 1:200, respectively. The findings suggest a marked toxicity of Ag on marine macroalgae which however should be mitigated by the high natural ligand concentrations of the transitional environments.

The relationship between Chlorella sp. and zinc oxide nanoparticles: Changes in biochemical, oxygen evolution, and lipid production ability

In this study, the effect of different concentrations (50, 100, and 200 mg L−1) of zinc oxide nanoparticles (ZnO-NP) on the biochemical changes, oxygen evolution, and lipid production in Chlorella sp. were analyzed. The growth rate, oxygen evolution capacity, total carotenoids and protein, starch, and neutral lipid were determined using UV spectrophotometer, oxytherm electrode system, microplate, and fluorescence spectrometer, respectively. Triacylglycerol, polysaccharides, and oligosaccharides were quantified using FT-IR. The results of FT-IR, TGA, and XRD analysis revealed the existence of stretching mode of Zn-O bond (496 cm−1), thermal stability, and crystalline domain size (18.7 nm) of ZnO-NP. Among the three concentrations, increased specific growth rate (0.72-fold) and oxygen evolution capacity (0.96–0.90 nmol/min) without aggregation was obtained in 50 mg L−1 of ZnO-NP treated cells. Conversely, no complete inhibition was observed in the growth and oxygen evolution at higher concentrations. The optimal concentration (50 mg L−1) of ZnO-NP demonstrated that the highest level of total carotenoids (1.23-fold), total protein (1.68-fold), starch (2.26-fold), neutral lipid (6.08-fold), and triacylglycerol (8.21-fold) were recorded on day 2. We conclude that 50 mg L−1 of ZnO-NP treatment enhanced the production of neutral lipids and triacylglycerol without complete inhibition of growth.

Comparative transcriptome analysis of wild type and an oleaginous mutant strain of Desmodesmus sp. reveals a unique reprogramming of lipid metabolism under high light

A mutant generated via ethylmethane sulfonate mutagenesis, Desmodesmus sp. G3, exhibited greater biomass and neutral lipid production over the wild type (WT) strain Desmodesmus sp. G41 in our previous study (Zhang et al., Bioresour Technol 207:268–275, 2016). G3 possessed a higher growth rate and lipid production than WT, with a biomass yield and total lipid content of 1302.86 mg L−1 and 48.58% respectively, which was promoted by 20.50% and 18.84%, compared to WT. Comparative transcriptome analysis was performed to elucidate the mechanism supporting enhanced biomass and lipid production in G3. A total of 1488 differentially expressed genes (DEGs) was identified comparing G3 and WT sequencing datasets, of which 753 and 735 genes were upregulated and downregulated respectively in G3. Pathway enrichment analysis indicated that ‘photosynthesis,’ ‘starch and sucrose metabolism,’ ‘fatty acid elongation,’ and ‘pyruvate metabolism’ were the notable represented DEGs-enriched pathways that might affect cell growth and lipid metabolism. Light harvesting capture was enhanced in G3. To protect PSII from photodamage, light harvesting complex-dependent non-photochemical quenching and state transitions was employed as the photoprotective strategy. De novo fatty acids (FAs) synthesis in chloroplast was downregulated in G3 while mitochondrion localized FAs elongation was enhanced to recycle carbon skeletons for lipid biosynthesis. Pyruvate mechanism was activated in G3 to generate acetyl-CoA through a pyruvate dehydrogenase bypass pathway, as the predominant pathway to provide precursor for lipids biosynthesis.

The influence of bio-optical properties of Emiliania huxleyi and Tetraselmis sp. on biomass and lipid production when exposed to different light spectra and intensities of an adjustable LED array and standard light sources

The effects of bio-optical characteristics on lipid and biomass production rates for Emiliania huxleyi and Tetraselmis sp. were studied under different irradiances and spectra. Biomass and lipid production increased from irradiances of 50–800 μE m−2 s−1 but differed for each spectrum. The highest biomass and neutral lipid production rates for E. huxleyi occurred under a red-blue LED array, which mimicked the absorption spectra of the cell’s chlorophyll a and accessory pigments, especially fucoxanthins. Biomass and neutral lipid production for Tetraselmis sp. were enhanced under broader spectra of cool white and grow lights. Neutral lipids per cell volume were similar for the two species and inversely proportional to chlorophyll a concentrations. High biomass and neutral lipid production rates were associated with high total quantum absorption and low quantum absorption per cell. Generally, quantum efficiencies were highest for high light treatments with high total quantum absorption and high production rates. Using reconstructed quantum absorption by pigments, the bio-optical model showed that photoprotective pigments were dependent on light intensity and spectra. Quantum efficiencies increased when excluding the contribution of photoprotective pigments to total quantum absorption since they did not directly contribute to photosynthesis, but did promote higher production rates.

RNA interference in the oleaginous yeast Rhodosporidium toruloides.

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development.

Overexpression of the DOF-Type Transcription Factor Enhances Lipid Synthesis in Chlorella vulgaris

In the present study, DNA binding with one finger (DOF)-type transcription factors were screened from the Chlorella vulgaris genome database. The candidate DOF transcription factor was endogenously overexpressed in C. vulgaris to improve neutral lipid production. The protein expression vector contains the heat shock protein 70 and ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit promoters and self-cleaving 2A peptide to reduce the transgene-silencing effect of C. vulgaris. A total of 74 phleomycin-resistant transformants were obtained. Under nitrogen-deficient conditions, the transformant CvDOF#3 showed approximately 1.5-fold higher neutral lipid content per cell compared to the original strain and also showed a His-tagged DOF candidate protein expression of 0.6%. Microscopic observations revealed that CvDOF#3 cells were larger in size. However, the observed differences in average cell diameter between CvDOF#3 and control cells were not statistically significant. These results indicated that the protein expression vector harboring the dual promoters and the 2A peptide, when used in combination with enzymatic cell wall degradation and glass bead transformation, could be useful for transgene and protein expression in C. vulgaris. Further experiment is necessary to confirm the expression efficiency of the HSP70 and RBCS dual promoter and 2A peptide strategy after construction of homologous recombination system in C. vulgaris. Our findings suggested that the overexpression of the endogenous DOF-type transcription factor can be used for improving the lipid content in C. vulgaris.

Using agro-industrial wastes for mixotrophic growth and lipids production by the green microalga Chlorella sorokiniana

There has been growing interest in the use of microalgae for the production of biofuels, but production costs continue to be too high to compete with fossil fuel prices. One of the main limitations for photobioreactor productivity is light shielding, especially at high cell densities. The growth of the green microalga Chlorella sorokiniana, a robust industrial species, has been evaluated under different trophic conditions with traditional carbon sources, such as glucose and sucrose, and alternative low cost carbon sources, such as carob pod extract, industrial glycerol and acetate-rich oxidized wine waste lees. The mixotrophic cultivation of this microalga with wine waste lees alleviated the problems of light shielding observed in photoautotrophic cultures, improving specific growth rate (0.052 h-1) compared with the other organic sources. The fed-batch mixotrophic culture of Chlorella sorokiniana in a 2 L stirred tank reactor, with optimized nutritional conditions, 100 mM of acetate coming from the oxidized wine waste lees and 30 mM of ammonium, produced an algal biomass concentration of 11 g L-1 with a lipid content of 38 % (w/w). This fed-batch strategy has been found to be a very effective means to enhance the biomass and neutral lipid productivity.

Isolation of lipid-rich marine microalgae by flow cytometric screening with Nile Red staining

Microalgae provide an optimal lipid source for commercial applications. The development a rapid, easy, and efficient screening procedure to isolate lipid-rich microalgae is becoming an extraordinarily active field of study. An automated flow cytometric cell-sorting technique in combination with Nile Red (NR) staining was used to isolate lipid-rich microalgae strains from a field sample. Cell sorting was based on the two-dimensional distribution of chlorophyll autofluorescence against forward scatter and NR fluorescence at 576 nm against that at 620 nm, corresponding to the optimal emission wavelength of NR in neutral and in polar lipid fractions, respectively. Of the 24 strains isolated using this method, 13 marine microalgae strains were successfully grown in culture medium and characterized for biomass production and cellular content of neutral and polar lipids based on NR fluorescence after 12 days of cultivation. The production of neutral lipids was higher in two strains (strains 9 and 12) compared to the marine diatom Phaeodactylum tricornutum, which is well known for its high lipid productivity. Three strains (strains 7, 8, and 9) exhibited a higher productivity of polar lipids than observed for P. tricornutum. These results indicate that our proposed method, an automated isolation technique coupled with the use of a specific cell stain such as NR, is efficient for selecting and isolating lipid-rich microalgae strains from the natural environment.