Packo P. Lamers

The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains

Microalgae-derived lipids are an alternative to vegetable and fossil oils, but lipid content and quality vary among microalgae strains. Selection of a suitable strain for lipid production is therefore of paramount importance. Based on published results for 96 species, nine strains were selected to study their biomass, total fatty acid, and triacylglycerol (TAG) production under nitrogen-sufficient and deficient cultivation conditions. Under nitrogen-deficient conditions, Chlorella vulgaris, Chlorella zofingiensis, Neochloris oleoabundans, and Scenedesmus obliquus, accumulated more than 35% of their dry weight as TAGs. Palmitic and oleic acid were the major fatty acids produced. The main difference between these strains was the amount of biomass that was produced (3.0-7.8-fold increase in dry weight) and the duration that the biomass productivity was retained (2-7 days) after nitrogen depletion. S. obliquus (UTEX 393) and C. zofingiensis (UTEX B32) showed the highest average TAG productivity (322 and 243 mg l(-1)day(-1)).

Carotenoid and Fatty Acid Metabolism in Light-Stressed Dunaliella salina

β‐Carotene is overproduced in the alga Dunaliella salina in response to high light intensities. We have studied the effects of a sudden light increase on carotenoid and fatty acid metabolism using a flat panel photobioreactor that was run in turbidostat mode to ensure a constant light regime throughout the experiments. Upon the shift to an increased light intensity, β‐carotene production commenced immediately. The first 4 h after induction were marked by constant intracellular levels of β‐carotene (2.2 g LCV−1), which resulted from identical increases in the production rates of cell volume and β‐carotene. Following this initial phase, β‐carotene productivity continued to increase while the cell volume productivity dropped. As a result, the intracellular β‐carotene concentration increased reaching a maximum of 17 g LCV−1 after 2 days of light stress. Approximately 1 day before that, the maximum β‐carotene productivity of 30 pg cell−1 day−1 (equivalent to 37 mg LRV−1 day−1) was obtained, which was about one order of magnitude larger than the average productivity reported for a commercial β‐carotene production facility, indicating a vast potential for improvement. Furthermore, by studying the light‐induced changes in both β‐carotene and fatty acid metabolism, it appeared that carotenoid overproduction was associated with oil globule formation and a decrease in the degree of fatty acid unsaturation. Our results indicate that cellular β‐carotene accumulation in D. salina correlates with accumulation of specific fatty acid species (C16:0 and C18:1) rather than with total fatty acid content. Biotechnol. Bioeng. 2010;106: 638–648.

Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus

Microalgae-derived lipids in the form of triacylglycerols (TAGs) are considered an alternative resource for the production of biofuels and food commodities. Large scale production of microalgal TAGs is currently uneconomical. The cost price could be reduced by improving the areal and volumetric TAG productivity. The economic value could be increased by enhancing the TAG quality. To improve these characteristics, the impact of light intensity, and the combined impact of pH and temperature on TAG accumulation were studied for Scenedesmus obliquus UTEX 393 under nitrogen starved conditions. The maximum TAG content was independent of light intensity, but varied between 18% and 40% of dry weight for different combinations of pH and temperature. The highest yield of fatty acids on light (0.263 g/mol photon) was achieved at the lowest light intensity, pH 7 and 27.5 °C.

Simultaneous growth and neutral lipid accumulation in microalgae.

In this paper the hypothesis was tested whether TAG accumulation serves as an energy sink when microalgae are exposed to an energy imbalance caused by nutrient limitation. In our continuous culture system, excess light absorption and growth-limiting nitrogen supply rates were combined, which resulted in accumulation of TAG (from 1.5% to 12.4% w/w) in visible lipid bodies in Neochloris oleoabundans, while cell replication was sustained. A fourfold increase in TAG productivity showed that TAG indeed served as an energy sink. However, the bulk of excess energy was dissipated leading to a significantly reduced biomass productivity and yield of biomass on light. This demonstrates that when aiming at industrial TAG production, sustaining efficient light energy use under nutrient stress is an important trait to look for in potential production organisms.

Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications

The unicellular alga Dunaliella salina is the most interesting cell factory for the commercial production of beta-carotene because this species accumulates carotenoids to high concentrations. Nevertheless, little is known about the underlying mechanisms of carotenoid accumulation. Here, we review the regulatory mechanisms involved in beta-carotene overproduction in D. salina. The potential roles of reactive oxygen species and the plastoquinone redox state in signal sensing are discussed, together with available evidence on transcriptional and (post)translational regulation. Moreover, future directions that might further our knowledge in this area are given. Ultimately, a better understanding of the regulatory mechanisms involved in beta-carotene overproduction will facilitate innovative production of specific carotenoids and other products in D. salina and in related organisms.

Edible oils from microalgae: insights in TAG accumulation.

Microalgae are a promising future source for sustainable edible oils. To make microalgal oil a cost-effective alternative for common vegetable oils, increasing TAG productivity and TAG content are of high importance. Fulfilling these targets requires proper understanding of lipid metabolism in microalgae. Here, we provide an overview of our current knowledge on the biology of TAG accumulation as well as the latest developments and future directions for increasing oil production in microalgae, considering both metabolic engineering techniques and cultivation strategies.

Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga

Nitrogen availability and light intensity affect β-carotene overproduction in the green alga Dunaliella salina. Following a previous study on high-light stress, we here report on the effect of nitrogen depletion on the growth characteristics and β-carotene as well as fatty acid metabolism of D. salina under a constant light regime in a turbidostat. Upon nitrogen depletion, the biomass yield on absorbed light approximately doubled, due to a transient increase in cell division rate, swelling of the cells and a linear increase of the density of the cells. Simultaneously, β-carotene started to accumulate up to a final intracellular concentration of 14 mg LCV⁻¹ (i.e. 2.7% of AFDW). This β-carotene production accounted for 6% of the increased density of the cells, indicating that other biochemical constituents accumulated as well. Since D. salina accumulates β-carotene in lipid globules, we also determined the fatty acid content and composition of D. salina. The intracellular concentration of the total fatty acid pool did not change significantly during nitrogen starvation, indicating that β-carotene and total fatty acid accumulation were unrelated, similar to what was found previously for high-light treated cells. However, for both high-light and nitrogen stress, β-carotene accumulation negatively correlated with the degree of unsaturation of the total fatty acid pool and, within the individual fatty acids, correlated positively with oleic acid biosynthesis, suggesting that oleic acid may be a key component of the lipid-globule-localized triacylglycerols and thereby in β-carotene accumulation.

Growth of oil accumulating microalga Neochloris oleoabundans under alkaline-saline conditions

The effect of elevated pH and salt concentration on the growth of the freshwater microalga Neochloris oleoabundans was investigated. A study was conducted in 24-well plates on the design of a growth medium and subsequently applied in a photobioreactor. An artificial seawater medium with reduced Ca(2+) and PO(4)(3-) could prevent mineral precipitation at high pH levels. Growth was characterized in this new medium at pH 8.1 and at pH 10.0, with 420 mM of total salts. Specific growth rates of 0.08 h(-1) at pH 8.1 and 0.04 h(-1) at pH 10.0 were obtained under controlled turbidostat cultivation. The effect of nitrogen starvation on lipid accumulation was also investigated. Fatty acids content increased not only with nitrogen limitation but also with a pH increase (up to 35% in the dry biomass). Fluorescence microscopy gave visual proof that N. oleoabundans accumulates oil bodies when growing in saline conditions at high pH.

Phototrophic pigment production with microalgae: biological constraints and opportunities

There is increasing interest in naturally produced colorants, and microalgae represent a bio‐technologically interesting source due to their wide range of colored pigments, including chlorophylls (green), carotenoids (red, orange and yellow), and phycobiliproteins (red and blue). However, the concentration of these pigments, under optimal growth conditions, is often too low to make microalgal‐based pigment production economically feasible. In some Chlorophyta (green algae), specific process conditions such as oversaturating light intensities or a high salt concentration induce the overproduction of secondary carotenoids (β‐carotene in Dunaliella salina (Dunal) Teodoresco and astaxanthin in Haematococcus pluvialis (Flotow)). Overproduction of all other pigments (including lutein, fucoxanthin, and phycocyanin) requires modification in gene expression or enzyme activity, most likely combined with the creation of storage space outside of the photosystems. The success of such modification strategies depends on an adequate understanding of the metabolic pathways and the functional roles of all the pigments involved. In this review, the distribution of commercially interesting pigments across the most common microalgal groups, the roles of these pigments in vivo and their biosynthesis routes are reviewed, and constraints and opportunities for overproduction of both primary and secondary pigments are presented.

A model for customising biomass composition in continuous microalgae production

A kinetic model is presented that describes functional biomass, starch and storage lipid (TAG) synthesis in the microalga Neochloris oleoabundans as a function of nitrogen and light supply rates to a nitrogen-limited turbidostat cultivation system. The model is based on the measured electron distribution in N. oleoabundans, which showed that starch is the primary storage component, whereas TAG was only produced after an excess of electrons was generated, when growth was limited by nitrogen supply. A fixed 8.6% of the excess electrons ended up in TAG, suggesting close metabolic interactions between nitrogen assimilation and TAG accumulation, such as a shared electron pool. The proposed model shows that by manipulating the cultivation conditions in a light or nitrogen limited turbidostat, algal biomass composition can be customised and the volumetric productivities and yields of the major biomass constituents can be changed on demand.