analysis-of-fatty-acid-content-and-composition-in-microalgae

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Wageningen University and Research Center

Using a comparative genomics approach, a copper resistance gene cluster has been identified in multiple archaeal genomes. The cop cluster is predicted to encode a metallochaperone (CopM), a P-type copper-exporting ATPase (CopA) and a novel, archaea-specific transcriptional regulator (CopT) which might control the expression of the cop genes. Sequence analysis revealed that CopT has an N-terminal DNA-binding helix-turn-helix domain and a C-terminal TRASH domain; TRASH is a novel domain which has recently been proposed to be uniquely involved in metal-binding in sensors, transporters and trafficking proteins in prokaryotes. The present study describes the molecular characterization of the cop gene cluster in the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The polycistronic copMA transcript was found to accumulate in response to growth-inhibiting copper concentrations, whereas copT transcript abundance appeared to be constitutive. DNA-binding assays revealed that CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the copMA gene cluster.

Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2.

For the development of optimal perfusion processes, insight into the effect of feed and bleed rate on cell growth, productivity, and metabolism is essential. In the here presented study the effect of the feed and bleed rate on cell metabolism was investigated using metabolic flux analysis. Under all tested feed and bleed rates the biomass concentration as calculated from the nitrogen balance (biomass-nitrogen) increased linearly with an increase in feed rate, as would be expected. However, depending on the size of the feed and bleed rate, this increase was attained in two different ways. At low feed and bleed rates (Region I) the increase was obtained through an increase in viable-cell concentration, while the cellular-nitrogen content remained constant. At high feed and bleed rates (Region II) the increase was attained through an increase in cellular-nitrogen content, while the cell concentration remained constant. Per gram biomass-nitrogen, the specific consumption and production rates of the majority of the nutrients and products were identical in both regions, as were most of the fluxes. The major difference between the two regions was an increased flux from pyruvate to lactate and a decreased flux of pyruvate toward citrate in region II. The decreased in-flux at the level of citrate can either be balanced by a decreased out-flux toward lipid biosynthesis leading to a lower fraction of lipids in the cell, by a decreased out-flux toward the citric acid cycle resulting in a decreased energy generation, or by a combination of these. Finally, the specific productivity increases less than the nitrogen content per cell in region II, which implies that for obtaining maximum production rates it is important to increase the cell density and not only the biomass density.

Effect of feed and bleed rate on hybridoma cells in an acoustic perfusion bioreactor: metabolic analysis.

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.

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

beta-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, beta-carotene production commenced immediately. The first 4 h after induction were marked by constant intracellular levels of beta-carotene (2.2 g LCV(-1)), which resulted from identical increases in the production rates of cell volume and beta-carotene. Following this initial phase, beta-carotene productivity continued to increase while the cell volume productivity dropped. As a result, the intracellular beta-carotene concentration increased reaching a maximum of 17 g LCV(-1) after 2 days of light stress. Approximately 1 day before that, the maximum beta-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 beta-carotene production facility, indicating a vast potential for improvement. Furthermore, by studying the light-induced changes in both beta-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 beta-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.

Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina.

In this study, a metabolic network describing the primary metabolism of Chlamydomonas reinhardtii was constructed. By performing chemostat experiments at different growth rates, energy parameters for maintenance and biomass formation were determined. The chemostats were run at low irradiances resulting in a high biomass yield on light of 1.25 g  mol(-1). The ATP requirement for biomass formation from biopolymers (K(x)) was determined to be 109 mmol g(-1) (18.9 mol mol(-1)) and the maintenance requirement (m(ATP)) was determined to be 2.85 mmol g(-1) h(-1). With these energy requirements included in the metabolic network, the network accurately describes the primary metabolism of C. reinhardtii and can be used for modeling of C. reinhardtii growth and metabolism. Simulations confirmed that cultivating microalgae at low growth rates is unfavorable because of the high maintenance requirements which result in low biomass yields. At high light supply rates, biomass yields will decrease due to light saturation effects. Thus, to optimize biomass yield on light energy in photobioreactors, an optimum between low and high light supply rates should be found. These simulations show that metabolic flux analysis can be used as a tool to gain insight into the metabolism of algae and ultimately can be used for the maximization of algal biomass and product yield. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10811-011-9674-3) contains supplementary material, which is available to authorized users.

Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance.

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.

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

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)).

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

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.

Simultaneous growth and neutral lipid accumulation in microalgae.

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.

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

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.

A model for customising biomass composition in continuous microalgae production.

Many microalgae accumulate carbohydrates simultaneously with triacylglycerol (TAG) upon nitrogen starvation, and these products compete for photosynthetic products and metabolites from the central carbon metabolism. As shown for starchless mutants of the non-oleaginous model alga Chlamydomonas reinhardtii, reduced carbohydrate synthesis can enhance TAG production. However, these mutants still have a lower TAG productivity than wild-type oleaginous microalgae. Recently, several starchless mutants of the oleaginous microalga Scenedesmus obliquus were obtained which showed improved TAG content and productivity.

Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (II) evaluation of TAG yield and productivity in controlled photobioreactors.

The effect of nitrogen replenishment on the kinetics of secondary carotenoids, triacylglycerol (TAG) and primary cell components was studied in nitrogen-starved Chromochloris zofingiensis (Chlorophyta), an oleaginous and carotenogenic microalga. Nitrogen resupplied after a period of starvation was initially consumed at a more than four times higher rate than in an equivalent nitrogen-replete culture. Simultaneously, chlorophylls, primary carotenoids, polar (membrane) lipids and proteins were rapidly produced. After 2 days, the contents of these primary metabolites, as well as the nitrogen consumption rate and the overall biomass production rate, had returned to values equivalent to those of cells grown under nitrogen-replete conditions, indicating that culture recovery required 2 days. Nitrogen resupply was immediately followed by rapid degradation of TAG and starch, suggesting that these metabolites served as carbon and energy source for the recovery process. Also, the secondary carotenoids canthaxanthin and ketolutein were rapidly degraded upon nitrogen resupply, whereas degradation of astaxanthin, the main secondary carotenoid, started only when the cells were fully recovered 2 days after nitrogen resupply. This is the first time that such culture recovery has been described in detail and, moreover, that astaxanthin was found to be not immediately degraded after nitrogen resupply. The observed rapid recovery of C. zofingiensis and the delay in astaxanthin degradation suggest that a repeated batch cultivation may result in a higher secondary carotenoid productivity than a series of classical single batch cultivations.

Dynamics of biomass composition and growth during recovery of nitrogen-starved Chromochloris zofingiensis.

Microalgae are often considered as a promising alternative source of vegetable oils. These oils can be used for food and biofuel applications. Productivities that are projected for large-scale microalgal oil production are, however, often poorly supported by scientific evidence and based on too optimistic assumptions. To facilitate the inclusion of the microalgal physiology in these projections, existing knowledge and novel scientific insights were condensed into a mechanistic model that describes photosynthesis and carbon partitioning during nitrogen starvation. The model is validated using experimental data from both wild-type and a starchless mutant of Scenedesmus obliquus. The model is subsequently used to quantify how reactor design, process design, and strain improvement can improve the oil productivity from 2.1 to up to 10.9gm(-2)day(-1). These projected productivities are used to reflect on commonly assumed oil productivities and it is concluded that the microalgal oil productivity is often overestimated several folds.

Article	Year
Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2. Microbiology (Reading, England)\| PubMed ID: 16804172	2006
Effect of feed and bleed rate on hybridoma cells in an acoustic perfusion bioreactor: metabolic analysis. Biotechnology progress\| PubMed ID: 17439155	2007
Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications. Trends in biotechnology\| PubMed ID: 18752860	2008
Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnology and bioengineering\| PubMed ID: 20229508	2010
Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance. Journal of applied phycology\| PubMed ID: 22427720	2012
Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga. Journal of biotechnology\| PubMed ID: 22750089	2012
The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresource technology\| PubMed ID: 22995162	2012
Simultaneous growth and neutral lipid accumulation in microalgae. Bioresource technology\| PubMed ID: 23500580	2013
Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresource technology\| PubMed ID: 23774290	2013
A model for customising biomass composition in continuous microalgae production. Bioresource technology\| PubMed ID: 23911819	2013
Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (II) evaluation of TAG yield and productivity in controlled photobioreactors. Biotechnology for biofuels\| PubMed ID: 24883102	2014
Dynamics of biomass composition and growth during recovery of nitrogen-starved Chromochloris zofingiensis. Applied microbiology and biotechnology\| PubMed ID: 25370993	2015
Opportunities to improve the areal oil productivity of microalgae. Bioresource technology\| PubMed ID: 25836038	2015

Packo P. Lamers

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