Can We Approach Theoretical Lipid Yields in Microalgae?

Can we approach theoretical lipid yields in microalgae? Yes: we can substantially reduce the gap between current and theoretical maximum yield. A realistic maximum is approximately 0.5g triacylglycerol (TAG) per mol photons, about five times higher than what is currently achieved in outdoor cultivation. Achieving this realistic maximum will require several breakthroughs. First, outdoor operation typically has low yields, mainly caused by fluctuating insolation. Future adaptive control models will help increase these yields. Additionally, the lipid production capacity of currently used strains needs to increase. Powerful strain-specific molecular toolboxes are being developed, shifting the bottleneck towards understanding metabolism and identifying target genes. Finally, strains and processes should be improved concurrently to fully exploit the potential lipid production from microalgae.

Towards microalgal triglycerides in the commodity markets.

BACKGROUND: Microalgal triglycerides (TAGs) hold great promise as sustainable feedstock for commodity industries. However, to determine research priorities and support business decisions, solid techno-economic studies are essential. Here, we present a techno-economic analysis of two-step TAG production (growth reactors are operated in continuous mode such that multiple batch-operated stress reactors are inoculated and harvested sequentially) for a 100-ha plant in southern Spain using vertically stacked tubular photobioreactors. The base case is established with outdoor pilot-scale data and based on current process technology. RESULTS: For the base case, production costs of 6.7 € per kg of biomass containing 24% TAG (w/w) were found. Several scenarios with reduced production costs were then presented based on the latest biological and technological advances. For instance, much effort should focus on increasing the photosynthetic efficiency during the stress and growth phases, as this is the most influential parameter on production costs (30 and 14% cost reduction from base case). Next, biological and technological solutions should be implemented for a reduction in cooling requirements (10 and 4.5% cost reduction from base case when active cooling is avoided and cooling setpoint is increased, respectively). When implementing all the suggested improvements, production costs can be decreased to 3.3 € per kg of biomass containing 60% TAG (w/w) within the next 8 years. CONCLUSIONS: With our techno-economic analysis, we indicated a roadmap for a substantial cost reduction. However, microalgal TAGs are not yet cost efficient when compared to their present market value. Cost-competiveness strictly relies on the valorization of the whole biomass components and on cheaper PBR designs (e.g. plastic film flat panels). In particular, further research should focus on the development and commercialization of PBRs where active cooling is avoided and stable operating temperatures are maintained by the water basin in which the reactor is placed.

Dynamics of triacylglycerol and EPA production in Phaeodactylum tricornutum under nitrogen starvation at different light intensities.

Lipid production in microalgae is highly dependent on the applied light intensity. However, for the EPA producing model-diatom Phaeodactylum tricornutum, clear consensus on the impact of incident light intensity on lipid productivity is still lacking. This study quantifies the impact of different incident light intensities on the biomass, TAG and EPA yield on light in nitrogen starved batch cultures of P. tricornutum. The maximum biomass concentration and maximum TAG and EPA contents were found to be independent of the applied light intensity. The lipid yield on light was reduced at elevated light intensities (>100 µmol m-2 s-1). The highest TAG yield on light (112 mg TAG molph-1) was found at the lowest light intensity tested (60 µmol m-2 s-1), which is still relatively low to values reported in literature for other algae. Furthermore, mass balance analysis showed that the EPA fraction in TAG may originate from photosynthetic membrane lipids.

Batch and semi-continuous microalgal TAG production in lab-scale and outdoor photobioreactors.

Microalgal triglycerides (TAGs) represent a sustainable feedstock for food, chemical and biofuel industries. The operational strategy (batch, semi-continuous, continuous cultivations) has an impact on the TAG productivity. In this study, semi-continuous (i.e. with fixed harvesting frequency) and batch cultivations were compared on TAG production both at lab-scale and in outdoor cultivations. At lab-scale, the semi-continuous TAG productivity was highest for a cycle time of 2 days (SC1; 0.21 g L-1 day-1) and similar to the maximum obtained with the batch (optimal harvest time; 0.23 g L-1 day-1). Although TAG content was lower for SC1 (22 %) than for the batch (35 %), higher biomass productivities were obtained with SC1. Outdoors, semi-continuous cultivations were subjected to a lower degree of stress (i.e. higher amount of nitrogen present in the system relative to the given irradiance) compared to lab-scale. This yielded low and similar TAG contents (10-13 %) in the different semi-continuous runs that were outdone by the batch on both TAG content (15-25 %) and productivity (batch, 0.97-2.46 g m-2 day-1; semi-continuous, 0.35-0.85 g m-2 day-1). The lab-scale experiments showed that semi-continuous strategies, besides leading to similar TAG productivities compared to the batch, could make TAG production cost effective by valorising also non-TAG compounds. However, optimization of outdoor semi-continuous cultivations is still required. For instance, the nitrogen supply and the harvest frequency should be adjusted on the total irradiance. Additionally, future research should focus on recovery metabolism upon nitrogen resupply.

Microalgal TAG production strategies: why batch beats repeated-batch.

BACKGROUND: For a commercially feasible microalgal triglyceride (TAG) production, high TAG productivities are required. The operational strategy affects TAG productivity but a systematic comparison between different strategies is lacking. For this, physiological responses of Nannochloropsis sp. to nitrogen (N) starvation and N-rich medium replenishment were studied in lab-scale batch and repeated-batch (part of the culture is periodically harvested and N-rich medium is re-supplied) cultivations under continuous light, and condensed into a mechanistic model. RESULTS: The model, which successfully described both strategies, was used to identify potential improvements for both batch and repeated-batch and compare the two strategies on optimized TAG yields on light (amount of TAGs produced per mol of supplied PAR photons). TAG yields on light, for batch, from 0.12 (base case at high light) to 0.49 g molph (-1) (at low light and with improved strain) and, for repeated-batch, from 0.07 (base case at high light) to 0.39 g molph (-1) (at low light with improved strain and optimized repeated-batch settings). The base case yields are in line with the yields observed in current state-of-the-art outdoor TAG production. CONCLUSIONS: For continuous light, an optimized batch process will always result in higher TAG yield on light compared to an optimized repeated-batch process. This is mainly because repeated-batch cycles start with N-starved cells. Their reduced photosynthetic capacity leads to inefficient light use during the regrowth phase which results in lower overall TAG yields compared to a batch process.

Closing Domestic Nutrient Cycles Using Microalgae.

This study demonstrates that microalgae can effectively recover all P and N from anaerobically treated black water (toilet wastewater). Thus, enabling the removal of nutrients from the black water and the generation of a valuable algae product in one step. Screening experiments with green microalgae and cyanobacteria showed that all tested green microalgae species successfully grew on anaerobically treated black water. In a subsequent controlled experiment in flat-panel photobioreactors, Chlorella sorokiniana was able to remove 100% of the phosphorus and nitrogen from the medium. Phosphorus was depleted within 4 days while nitrogen took 12 days to reach depletion. The phosphorus and nitrogen removal rates during the initial linear growth phase were 17 and 122 mg·L(-1)·d(-1), respectively. After this initial phase, the phosphorus was depleted. The nitrogen removal rate continued to decrease in the second phase, resulting in an overall removal rate of 80 mg·L(-1)·d(-1). The biomass concentration at the end of the experiment was 11.5 g·L(-1), with a P content of approximately 1% and a N content of 7.6%. This high algal biomass concentration, together with a relatively short P recovery time, is a promising finding for future post-treatment of black water while gaining valuable algal biomass for further application.

Microalgal triacylglycerides production in outdoor batch-operated tubular PBRs.

BACKGROUND: Microalgal triacylglycerides (TAGs) are a promising sustainable feedstock for the biofuel, chemical and food industry. However, industrial production of microalgal products for commodity markets is not yet economically viable, largely because of low microalgal productivity. The latter is strictly dependent on initial-biomass-specific (IBS) light availability (i.e. ratio of light impinging on reactor ground area divided by initial biomass concentration per ground area). This study investigates the effect of IBS-light availability on batch TAG production for Nannochloropsis sp. cultivated in two outdoor tubular reactors (i.e. vertical and horizontal) at different initial biomass concentrations for the TAG accumulation phase, during two distinct seasons (i.e. high and low light conditions). RESULTS: Increasing IBS-light availability led to both a higher IBS-TAG production rate and TAG content at the end of the batch, whereas biomass yield on light decreased. As a result, an optimum IBS-light availability was determined for the TAG productivity obtained at the end of the batch and several guidelines could be established. The vertical reactor (VR) should be operated at an initial biomass concentration of 1.5 g L(-1) to achieve high TAG productivities (1.9 and 3.2 g m(-2) day(-1) under low and high light, respectively). Instead, the horizontal reactor (HR) should be operated at 2.5 g L(-1) under high light (2.6 g m(-2) day(-1)), and at 1.5 g L(-1) under low light (1.4 g m(-2) day(-1)). CONCLUSIONS: From this study, the great importance of IBS-light availability on TAG production can be deduced. Although maintaining high light availabilities in the reactor is key to reach high TAG contents at the end of the batch, considerable losses in TAG productivity were observed for the two reactors regardless of light condition, when not operated at optimal initial biomass concentrations (15-40% for VR and 30-60% for HR).

Opportunities to improve the areal oil productivity of microalgae.

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 10.9 g m(-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.

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

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.

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

BACKGROUND: 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. RESULTS: The most promising mutant, slm1, is compared in detail to wild-type S. obliquus in controlled photobioreactors. In the slm1 mutant, the maximum TAG content increased to 57 ± 0.2% of dry weight versus 45 ± 1% in the wild type. In the wild type, TAG and starch were accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The starchless mutant did not produce starch and the liberated photosynthetic capacity was directed towards TAG synthesis. This increased the maximum yield of TAG on light by 51%, from 0.144 ± 0.004 in the wild type to 0.217 ± 0.011 g TAG/mol photon in the slm1 mutant. No differences in photosynthetic efficiency between the slm1 mutant and the wild type were observed, indicating that the mutation specifically altered carbon partitioning while leaving the photosynthetic capacity unaffected. CONCLUSIONS: The yield of TAG on light can be improved by 51% by using the slm1 starchless mutant of S. obliquus, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. The photosynthetic performance is not negatively affected in the slm1 and the main difference with the wild type is an improved carbon partitioning towards TAG.

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 (ß-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.

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 microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes. With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of. This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other. The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

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.

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.

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 nitrogen-starved Dunaliella salina, a unicellular green microalga.

Nitrogen availability and light intensity affect ß-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 ß-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, ß-carotene started to accumulate up to a final intracellular concentration of 14 mg LCV⁻¹ (i.e. 2.7% of AFDW). This ß-carotene production accounted for 6% of the increased density of the cells, indicating that other biochemical constituents accumulated as well. Since D. salina accumulates ß-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 ß-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, ß-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 ß-carotene accumulation.

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

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.

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

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.

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.