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Algae are a promising feedstock for the sustainable production of feed, fuels, and chemicals. Especially in arid regions such as the Arabian Peninsula, algae could play a significant role in enhancing food security, economic diversification, and decarbonization. Within this context, the regional potential of algae commercialization is discussed, exploring opportunities and challenges across technical, societal, and political aspects. Climate, availability of process inputs, and funding opportunities are identified as essential strengths that increase the global competitiveness of regional algae production. Implementation challenges include climate change, securing human resources, and the vital transitioning from research to commercial scales. With balanced management, however, the region's efforts could be the push that is necessary for algal technologies to take off globally.

Multiple models have been developed in the field to simulate growth and product accumulation of microalgal cultures. These models heavily depend on the accurate estimation of growth parameters. In this paper growth parameters are presented for three industrially relevant microalgae species: Nannochloropsis sp., Neochloris oleoabundans, and Picochlorum sp. (BPE23). Dedicated growth experiments were done in photobioreactors to determine the maximal biomass yield on light and maintenance rate, while oxygen evolution experiments were performed to estimate the maximal specific growth rate. Picochlorum sp. exhibited the highest specific growth rate of 4.98 ± 0.24 day−1 and the lowest specific maintenance rate of 0.079 day−1, whereas N. oleoabundans showed the highest biomass yield on light of 1.78 gx·molph−1. The measured growth parameters were used in a simple kinetic growth model for verification. When simulating growth under light conditions as found at Bonaire (12 °N, 68° W), Picochlorum sp. displayed the highest areal biomass productivity of 32.2 g.m−2·day−1 and photosynthetic efficiency of 2.8%. The presented growth parameters show to be accurate compared to experimental data and can be used for model calibration by scientists and industrial communities in the field.

Microalgae can produce industrially relevantmetabolites using atmospheric CO2 and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughputgenome engineering could accelerate the generation oftailored strains with improved traits. To this end, we developed a genome editing strategy based on Cas12a ribonucleoprotein (RNPs) and homology-directed repair (HDR) to generate scarlessand markerless mutants of the microalga Nannochloropsis oceanica. We also developed an episomal plasmid-based Cas12a system forefficiently introducing indels at the target site. Additionally, weexploited the ability of Cas12a to process an associated CRISPRarray to perform multiplexed genome engineering. We efficientlytargeted three sites in the host genome in a singletransformation,thereby making a major step toward high-throughput genome engineering in microalgae. Furthermore, a CRISPR interference(CRISPRi) tool based on Cas9 and Cas12a was developed for effective downregulation of target genes. We observed up to 85%reduction in the transcript levels upon performing CRISPRi with dCas9 in N. oceanica. Overall, these developments substantiallyaccelerate genome engineering efforts in N. oceanica and potentially provide a general toolbox for improving othermicroalgal strains.

Microalgae are considered an efficient accumulator and promising source of Se for feed additive purposes. This study aimed at investigating, for the first time, the effect of phosphorus limitation on Se accumulation and uptake efficiency in N. oceanica. A range of phosphorus concentrations (0–2470 µM) were tested in either the presence or absence of sodium selenite (0, 5, 30 µM). Se accumulation was increased up to 16-fold and Se uptake efficiency was increased up to 3.6-fold under phosphorus growth-limiting concentrations. N. oceanica was then cultivated in a 1.8 L flat-panel photobioreactor in batch operation under two phosphorus growth-limiting concentrations (250 and 750 µM) where the accumulation of Se in the microalgal biomass, as well as its presence in the spent medium were analysed. This study is the first to investigate the effect of phosphorus limitation for increasing Se accumulation in microalgae, and to prevent the release of Se in wastewater.

The major bottleneck in commercializing biofuels and other commodities produced by microalgae is the high cost associated with phototrophic cultivation. Improving microalgal productivities could be a solution to this problem. Synthetic biology methods have recently been used to engineer the downstream production pathways in several microalgal strains. However, engineering upstream photosynthetic and carbon fixation metabolism to enhance growth, productivity, and yield has barely been explored in microalgae. We describe strategies to improve the generation of reducing power from light, as well as to improve the assimilation of CO2by either the native Calvin cycle or synthetic alternatives. Overall, we are optimistic that recent technological advances will prompt long-awaited breakthroughs in microalgal research.

The microalga Nannochloropsis oceanica is considered a promising platform for the sustainable production of high-value lipids and biofuel feedstocks. However, current lipid yields of N. oceanica are too low for economic feasibility. Gaining fundamental insights into the lipid metabolism of N. oceanica could open up various possibilities for the optimization of this species through genetic engineering. Therefore, the aim of this study was to discover novel genes associated with an elevated neutral lipid content. We constructed an insertional mutagenesis library of N. oceanica, selected high lipid mutants by five rounds of fluorescence-activated cell sorting, and identified disrupted genes using a novel implementation of a rapid genotyping procedure. One particularly promising mutant (HLM23) was disrupted in a putative APETALA2-like transcription factor gene. HLM23 showed a 40%-increased neutral lipid content, increased photosynthetic performance, and no growth impairment. Furthermore, transcriptome analysis revealed an upregulation of genes related to plastidial fatty acid biosynthesis, glycolysis and the Calvin-Benson-Bassham cycle in HLM23. Insights gained in this work can be used in future genetic engineering strategies for increased lipid productivity of Nannochloropsis.

As large-scale outdoor production cannot be done in complete containment, cultures are (more) open for bacteria, which may affect the productivity and stability of the algae production process. We investigated the bacterial diversity in two indoor reactors and four pilot-scale outdoor reactors for the production of Nannochloropsis sp. CCAP211/78 spanning four months of operation from July to October. Illumina sequencing of 16S rRNA gene amplicons demonstrated that a wide variety of bacteria were present in all reactor types, with predominance of Bacteroidetes and Alphaproteobacteria. Bacterial communities were significantly different between all reactor types (except between the horizontal tubular reactor and the vertical tubular reactor) and also between runs in each reactor. Bacteria common to the majority of samples included one member of the Saprospiraceae family and one of the NS11-12_marine group (both Bacteroidetes). Hierarchical clustering analysis revealed two phases during the cultivation period separated by a major shift in bacterial community composition in the horizontal tubular reactor, the vertical tubular reactor and the raceway pond with a strong decrease of the Saprospiraceae and NS11-12_marine group that initially dominated the bacterial communities. Furthermore, we observed a less consistent pattern of bacterial taxa appearing in different reactors and runs, most of which belonging to the classes Deltaproteobacteria and Flavobacteriia. In addition, canonical correspondence analysis showed that the bacterial community composition was significantly correlated with the nitrate concentration. This study contributes to our understanding of bacterial diversity and composition in different types of outdoor reactors exposed to a range of dynamic biotic and abiotic factors. Key points• Reactor types had significantly different bacterial communities except HT and VT• The inoculum source and physiochemical factors together affect bacterial community• The bacterial family Saprospiraceae is positively correlated to microalgal growth

Microalgae express high protein levels and can be produced in contained cultivation systems with low water requirements and complete fertilizer use. The production potential is 22-44 tons of protein per hectare per year although the current production scale is small. Techno economic analyses have shown good potential for scale-up and cost reduction. Large-scale production of microalgae in the post-fossil era will rely on the capture of carbon dioxide from the air, or sugars from crops. Microalgal amino acid composition matches well with requirements for food and feed, which, in combination with novel biomass pre-treatment steps, will guarantee high-quality microalgal protein. For a broadening of the microalgae species available as single-cell protein, novel food approval is required.

Medium chain fatty acids (MCFAs) are compounds of considerable commercial interest that have applications in food, feed, and the production of industrially relevant chemicals. Due to their antipathogenic and health-promoting effects, they are further discussed as potential therapeutic agents for disease treatment and infection prevention and control. Domesticated agricultural crops that can grow in temperate climates lack MCFAs, and increased cultivation of tropical MCFA-rich species such as oil palm is associated with deforestation and a decrease in biodiversity. Alternative sources for more sustainably produced MCFAs are non-domesticated plants such as Cuphea spp., as well as oil crops and oleaginous microalgae that can be genetically engineered to accumulate MCFAs by expression of acyl-acyl carrier protein thioesterases (TEs) from MCFA-producing plants. Here, we report the heterologous expression of a TE from Cuphea palustris in the industrially relevant microalga Nannochloropsis oceanica. Using a recently developed gene expression system, we engineered transformant strains that accumulated up to 2.84 and 4.15% of C8:0 and C10:0 in storage lipids, respectively, and we observed no effect on growth. We further show that MCFA accumulation was negatively correlated with total neutral lipid content, suggesting the presence of regulatory mechanisms that limit MCFA accumulation in Nannochloropsis.

Tisochrysis lutea is an important microalgal species for fucoxanthin and docosahexaenoic acid (DHA) production with an optimum cultivation temperature of approximately 30 ◦C. The aim of the present work was to develop a winter strain with high productivity at 15 ◦C. The response of the original strain to a decrease in temperature from 30 ◦C to 15 ◦C was investigated in continuous turbidostat experiments. This was followed by adaptation for >180 days at 15 ◦C and 2 rounds of sorting for cells with high chlorophyll fluorescence (top 5%) using fluorescence-activated cell sorting (FACS). For the original strain the productivity of biomass, fucoxanthin, and DHA decreased by 92 %, 98 % and 85 % respectively when decreasing the temperature from 30 ◦C to 15 ◦C. In the sorted cold-adapted ‘winter strain’, biomass, fucoxanthin, and DHA productivities were similar to those at 30 ◦C. In addition, the fucoxanthin concentration increased from 1.11 to 4.24 mg g− 1 dry weight and the polar lipid fraction in total fatty acids increased from 21 % to 55 %. The winter strain showed a robust and stable phenotype after one year of cultivation, expanding the outdoor fucoxanthin and lipid production seasons for this species.

The use of microalgal starch has been studied in biorefinery frameworks to produce bioethanol or bioplastics, however, these products are currently not economically viable. Using starch-rich biomass as an ingredient in food applications is a novel way to create more value while expanding the product portfolio of the microalgal industry. Optimization of starch production in the food-approved species Chlorella vulgaris was the main objective of this study. High-throughput screening of biomass composition in response to multiple stressors was performed with FTIR spectroscopy. Nitrogen starvation was identified as an important factor for starch accumulation. Moreover, further studies were performed to assess the role of light distribution, investigating the role of photon supply rates in flat panel photobioreactors. Starch-rich biomass with up to 30% starch was achieved in cultures with low inoculation density (0.1 g L−1) and high irradiation (1800 µmol m−2 s−1). A final large-scale experiment was performed in 25 L tubular reactors, achieving a maximum of 44% starch in the biomass after 12 h in nitrogen starved conditions.

Cellular agriculture could represent a more sustainable alternative to current food and nutraceutical production processes. Tisochrysis lutea microalgae represents a rich source of antioxidants and omega-3 fatty acids essential for human health. However, current downstream technologies are limiting its use. The present work investigates mild targeted acoustic treatment of Tisochrysis lutea biomass at different growth stages and acoustic frequencies, intensities and treatment times. Significant differences have been observed in terms of the impact of these variables on the cell disruption and energy requirements. Lower frequencies of 20 kHz required a minimum of 4500 J to disrupt 90% of the cells, while only 1000 J at 1146 kHz. Comparing these results with current industry standards such as bead milling, up to six times less energy use has been identified. These mild biomass processing approaches offer a certain tunability which could suit a wide range of microorganisms with only minor adjustments.

Microalgae are used in food and feed, and they are considered a potential feedstock for sustainably produced chemicals and biofuel. However, production of microalgal-derived chemicals is not yet economically feasible. Genetic engineering could bridge the gap to industrial application and facilitate the production of novel products from microalgae. Here, we report the discovery of a novel gene expression system in the oleaginous microalga Nannochloropsis that exploits the highly efficient transcriptional activity of RNA polymerase I and an internal ribosome entry site for translation. We identified the nucleolus as a genomic safe harbor for Pol I transcription and used it to construct transformant strains with consistently strong transgene expression. The new expression system provides an outstanding tool for genetic and metabolic engineering of microalgae and thus will probably make substantial contributions to microalgal research.

An increase in temperature can have a profound effect on the cell cycle and cell division in green algae, whereas growth and the synthesis of energy storage compounds are less influenced. In Chlamydomonas reinhardtii, laboratory experiments have shown that exposure to a supraoptimal temperature (39 °C) causes a complete block of nuclear and cellular division accompanied by an increased accumulation of starch. In this work we explore the potential of supraoptimal temperature as a method to promote starch production in C. reinhardtii in a pilot-scale photobioreactor. The method was successfully applied and resulted in an almost 3-fold increase in the starch content of C. reinhardtii dry matter. Moreover, a maximum starch content at the supraoptimal temperature was reached within 1–2 days, compared with 5 days for the control culture at the optimal temperature (30 °C). Therefore, supraoptimal temperature treatment promotes rapid starch accumulation and suggests a viable alternative to other starch-inducing methods, such as nutrient depletion. Nevertheless, technical challenges, such as bioreactor design and light availability within the culture, still need to be dealt with.

Besides being considered pathogens, viruses are important drivers of evolution and they can shape large ecological and biogeochemical processes, by influencing host fitness, population dynamics, and community structures. Moreover, they are simple systems that can be used and manipulated to be beneficial and useful for biotechnological applications. In this context, microalgae biotechnology is a growing field of research, which investigated the usage of photosynthetic microorganisms for the sustainable production of food, fuel, chemical, and pharmaceutical sectors. Viruses infecting microalgae have become important subject of ecological studies related to marine and aquatic environments only four decades ago when virus-like-particles associated with bloom-forming algae were discovered. These first findings have opened new questions on evolution and identity. To date, 63 viruses that infect eukaryotic microalgae have been isolated and cultured. In this short review we briefly summarize what is known about viruses infecting eukaryotic microalgae, and how acknowledging their importance can shape future research focussed not only on marine ecology and evolutionary biology but also on biotechnological applications related to microalgae cell factories.

Se-enriched ingredients have recently gained interest in the aquaculture industry as feed supplements due to their positive effects on fish health, growth, and potential effects on animal welfare. This study aims to assess which inorganic selenium (Se) species is suitable to produce Se-enriched Nannochloropsis oceanica (N. oceanica) biomass for aquafeed applications. The effective concentration for 50% growth inhibition (EC50) and Se bioaccumulation of the two inorganic forms of Se, sodium selenite (Na2SeO3), and sodium selenate (Na2SeO4), were assessed at different concentrations after twelve days of cultivation. Toxicity results showed that selenate, EC50 = 32.93 μM, had a greater negative effect on cell growth than selenite, EC50 = 163.82 μM. Total intracellular Se was analysed by inductively coupled plasma - optical emission spectrometry (ICP-OES) and high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS), which revealed that selenite was better accumulated by N. oceanica. Further investigation at 30 μM of selenite in the growth medium resulted in Se bioaccumulation with a minor effect on cell growth and reached a Se intracellular content of 0.131 gSe/kgbiomass after 12 days. Thus, 30 μM of selenite was selected for batch pilot-scale cultivation in a 1500 L tubular photobioreactor. Total Se accumulated in the biomass at pilot-scale was in the same order of magnitude compared with flasks (0.104–0.159 gSe/kgbiomass). The results from this pilot-scale study are fundamental for a proof of concept from laboratory to pilot-scale production and they represent a critical bridging step for the potential use of Se-enriched N. oceanica in aquafeed.

Oil production was tested with Neochloris oleoabundans in a 6 m3, horizontal soft sleeve tubular reactor from 22 October to 7 November in Matalascañas, southern Spain. Biomass productivity during the nitrogen replete phase was 7.4 g dw m−2 day−1. Maximum lipid content in the biomass was 39% and average lipid productivity during the nitrogen depletion phase was 2.0 g m−2 day−1. Nitrogen depletion of the cultures was carried out in order to enhance fatty acid formation, using the inverse nitrogen quota in the biomass to predict the fatty acid content. TFA concentration at harvest was 14% DW, compared to a value of 17%, predicted by the inverse nitrogen quota. The overall feasibility of the horizontal tubular technology for microalgal oil production, including mixing energy expenditure, was evaluated.

Microalgae have the potential to become microbial cell factories for lipid production. Their ability to convert sunlight and CO2 into valuable lipid compounds has attracted interest from cosmetic, biofuel, food and feed industries. In order to make microalgae-derived products cost-effective and commercially competitive, enhanced growth rates and lipid productivities are needed, which require optimization of cultivation systems and strain improvement. Advances in genetic tool development and omics technologies have increased our understanding of lipid metabolism, which has opened up possibilities for targeted metabolic engineering. In this review we provide a comprehensive overview on the developments made to genetically engineer microalgal strains over the last 30 years. We focus on the strategies that lead to an increased lipid content and altered fatty acid profile. These include the genetic engineering of the fatty acid synthesis pathway, Kennedy pathway, polyunsaturated fatty acid and triacylglycerol metabolisms and fatty acid catabolism. Moreover, genetic engineering of specific transcription factors, NADPH generation and central carbon metabolism, which lead to increase of lipid accumulation are also reviewed.

The effect of light on biomass and fucoxanthin (Fx) productivities was studied in two microalgae, Tisochrysis lutea and Phaeodactylum tricornutum. High and low biomass concentrations (1.1 and 0.4 g L−1) were tested in outdoor pilot‐scale flat‐panel photobioreactors at semi‐continuous cultivation mode. Fluorescence spectroscopy coupled with chemometric modeling was used to develop prediction models for Fx content and for biomass concentration to be applied for both microalgae species. Prediction models showed high R 2 for cell concentration (.93) and Fx content (.77). Biomass productivity was lower for high biomass concentration than low biomass concentration, for both microalgae (1.1 g L−1: 75.66 and 98.14 mg L−1 d−1, for T. lutea and P. tricornutum, respectively; 0.4 g L−1: 129.9 and 158.47 mg L−1 d−1, T. lutea and P. tricornutum). The same trend was observed in Fx productivity (1.1 g L−1: 1.14 and 1.41 mg L−1 d−1, T. lutea and P. tricornutum; 0.4 g L−1: 2.09 and 1.73 mg L−1 d−1, T. lutea and P. tricornutum). These results show that biomass and Fx productivities can be set by controlling biomass concentration under outdoor conditions and can be predicted using fluorescence spectroscopy. This monitoring tool opens new possibilities for online process control and optimization., Graphical abstract Biomass and fucoxanthin outdoor production and monitoring with brown microalgae

In this paper the preparations are described to develop a production of oil rich microalgal biomass under south European conditions. Ten microalgal species were compared in shake flasks in an incubator for potential for oil production. Potential oil production capacity was assayed as maximum total fatty acid (TFA) concentration and volumetric TFA productivity. TFA concentration ranged from 5 to 40% DW while TFA productivity rate ranged from 0 to 204 mg TFA L−1 day−1. To control the oil enrichment process in the outdoor microalgal batch culture, a quadratic equation was proposed, predicting the TFA concentration based on biomass inverse nitrogen quota. A concentrated substrate was developed to add to sea water, made from natural sea-salt and tap water.

This study investigated the effect of salt presence and removal on the integrity and elemental determination of the marine microalgae Nannochloropsis oceanica. Cells were harvested by centrifugation and washed with ammonium formate five times and subjected to mineral analysis after each washing step. Supernatants from each washing step were also analysed. Ca, Cu, Fe, Mg, Mn, Na, P, K, S, Zn were determined by inductively coupled plasma – optical emission spectrometry (ICP-OES). Results showed that washing microalgal biomass twice is sufficient to eliminate equipment oversaturation, allowing for more accurate elemental analysis, by using matrix matching. Moreover, washing the biomass did not rupture the cells, removed cell culture debris and decreased the concentration of Na, K, Ca present in the leftover growth medium. This study provides a reliable protocol for elemental determination in marine microalgae saving time in sample processing and analysis.

The bioeconomy is a new and essential paradigm for reducing our dependence on natural resources and responding to the environmental threats that the Earth is currently facing. In this regard, microalgae offer almost unlimited possibilities for developing a modern bioeconomy given their metabolic flexibility and high biomass output rates, even when produced under harsh conditions, such as when treating wastewaters or using flue gases. In this article, the microalgal contribution to important economic activities such as the production of food and feed, cosmetics and health-related compounds is reviewed. Moreover, potential contributions of microalgae to emerging sectors are discussed, as in the production of biomaterials, agriculture-related products, biofuels and provision of services such as wastewater treatment and the clean-up of industrial gases. The different microalgal production technologies have also been analyzed to identify the main bottlenecks affecting microalgal use in different applications. Finally, the major challenges facing microalgal biotechnology in enlarging its contribution to the bioeconomy are evaluated, and future trends discussed. info:eu-repo/semantics/publishedVersion

Fucoxanthin is a xanthophyll carotenoid with high market value. Currently, seaweeds are the primary source for fucoxanthin industrial production. However, marine microalgae reach 5 to 10 times higher concentrations (2.24 to 26.6 mg g-1 DW) and are considered a promising feedstock. In this work, two marine microalgae were produced at industrial scale to evaluate biomass and fucoxanthin production: Phaeodactylum tricornutum for autumn/winter and Tisochrysis lutea for spring/summer. Both strains were grown in 15 m3 tubular flow-through photobioreactors; for 170 consecutive days of semi-continuous cultivation regime. The average volumetric biomass productivities of P. tricornutum and T. lutea were 0.11 and 0.09 g DW L-1 day-1. P. tricornutum reached higher maximum biomass concentration (2.87 g DW L-1) than T. lutea (1.47 g DW L-1). P. tricornutum fucoxanthin content ranged between 0.2 and 0.7% DW, while T. lutea between 0.2 and 0.6% DW. The fucoxanthin content was correlated with the irradiation (MJ m-2) and biomass concentration in the photobioreactor (g L-1). This is the first work in literature reporting a long-term industrial production of T. lutea. Overall, we showed possible scenarios for fucoxanthin production from microalgae, increasing the window to supply the industry with steady production throughout the year. info:eu-repo/semantics/publishedVersion

This work aimed to select a Tisochrysis lutea phenotype with higher biomass and fucoxanthin productivities using fluorescence-activated cell sorting (FACS). A novel phenotype was obtained after 2 rounds of selection, based on high-fucoxanthin fluorescence. The resulting phenotype forms cell aggregates, has no flagella, and was stable after 15 months. Optimal temperature (30 ◦C) and light (300 μmol m􀀀 2 s􀀀 1) were obtained at laboratory scale, identical to the original strain. The biomass productivity was higher than the original strain: 1.9× at laboratory scale (0.4 L), and 4.5× under outdoor conditions (190 L). Moreover, compared to the original strain, the productivity of fucoxanthin increased 1.6–3.1× and docosahexaenoic acid 1.5–1.9×. These are the highest ever reported outdoor productivities, obtained with a robust new phenotype from a T. lutea monoculture isolated with FACS without genetic manipulation. The resulting phenotype shows high potential for industrial production.

This work aimed to investigate the accumulation of fucoxanthin and lipids in Tisochrysis lutea during growth (N+) and nitrogen-starvation (N−) and to correlate these products with single-cell emissions using fluorescence-activated cell sorting (FACS). Fucoxanthin content decreased 52.94% from N+ to N− in batch cultivation; increased 40.53% as dilution rate changed from 0.16 to 0.55 d−1 in continuous cultivation. Total lipids (N−) were constant (~250 mg/g), but the abundance of neutral lipids increased from 4.87% to 40.63%. Nile red can stain both polar and neutral lipids. However, in vivo, this differentiation is limited due to an overlapping of signals between 600 and 660 nm, caused by neutral lipids concentrations above 3.48% (W/W). Chlorophyll autofluorescence (720 nm) was reported for the first time as a proxy for fucoxanthin (R2 = 0.90) and polar lipids (R2 = 0.98). FACS can be used in high throughput quantification of pigments and lipids and to select and sort cells with high-fucoxanthin/lipids. Paid Open Access

Control of temperature is a major challenge for industrial microalgae production in photobioreactors outdoors. Strains with tolerance for high temperatures can reduce the cost of production as active temperature control is not required. In this study, marine photoautotrophic microorganisms were isolated to reduce the need for control of high temperature. Twenty-two samples were taken from different saline waters on the Caribbean island Bonaire. During strain enrichment, a temperature of 40 °C was used as selective pressure and strains with the highest growth rate were selected. We isolated and identified 59 strains, after which 5 were selected for characterization on growth rate and biomass composition. Picochlorum sp. and Leptolyngbya sp. showed optimal growth at 40 °C and 35 °C with a growth rate of 0.12 h−1 during daytime, respectively. The strains contain 62.1% and 68.2% of protein and have varying fatty acid compositions suitable for application as edible oil and biofuel.

Rhodomonas sp., is an important microalga for aquaculture feed applications and gained increased research interest over the past few years. While efforts to optimise cultivation of the strain have been studied in detail under laboratory conditions, Rhodomonas sp. has never been grown in photobioreactors at large scale under outdoor light conditions. To study the industrial potential of this strain, we cultivated Rhodomonas sp. in three identical tubular photobioreactors with 200 l working volume each, located in a greenhouse using sunlight conditions only. Growth experiments were performed from February with winter light conditions (50 mol m−2 d−1) in July, representing all sunlight conditions in the Netherlands. All nutrients were supplied in surplus and temperature and pH were maintained at optimum values for growth of Rhodomonas sp., based on lab data. The total light per reactor was calculated using a ray-tracing analysis to allow calculations based on the light reaching each individual reactor. Rhodomonas sp. grew under all tested light conditions. Biomass yield on light decreased with increasing light conditions from 0.43 ± 0.21 g mol−1 to 0.18 ± 0.04 g mol−1 at 0–10 molph m−2 d−1 to 30–40 molph m−2 d−1. Biomass productivities increased with increasing light from 0.09 ± 0.04 g l−1 d−1 to 0.19 ± 0.06 g l−1 d−1, for 0–10 and 30–40 molph m−2 d−1. We obtained a 2–5 fold increase in biomass productivity compared to previous reports on Rhodomonas sp. cultivation using artificial light at large scale. Our results show that Rhodomonas sp. can be grown at pilot scale using sunlight conditions and further improvements can be reached in the future.

In this research, the gene expression of genes involved in lipid metabolism of the eustigmatophyte alga Nannochloropsis gaditana was measured by transcriptomic data. This microalga can be used as a source of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). Insight in TAG and EPA production and regulation are needed to improve their productivity. Nitrogen starvation induces TAG accumulation in N. gaditana. Previous research showed that during nitrogen starvation, EPA was translocated from the polar lipids to TAG and de novo synthesized in N. gaditana. Therefore, the expression levels of genes involved in fatty acid translocation and de novo TAG synthesis were measured. Furthermore, the genes involved in de novo EPA synthesis such as elongases and desaturases were studied. The expression levels were measured during the first hours of nitrogen starvation and the subsequent period of 14 days. One phospholipid:diacylglycerol acyltransferase (PDAT) gene involved in translocation of fatty acids from membrane lipids to TAG was upregulated. In addition, several lipases were upregulated, suggesting that these enzymes might be responsible for the translocation of EPA to TAG. Most desaturases and elongases involved in de novo EPA synthesis were downregulated during nitrogen starvation, except for Δ9 desaturase which was upregulated. This upregulation correlates with the increase in oleic acid. Due to the presence of many hypothetical genes, improvement in annotation is needed to increase our understanding of these pathways and their regulation.

Leptolyngbya sp. QUCCCM 56 was investigated as a possible alternative to A. platensis, for the production of phycocyanin-rich biomass under desert conditions. Under elevated temperatures and light intensities, of up to 40 °C and 1800 μmol·m−2·s−1, the strain's biomass productivity was up to 45% higher as compared to reported productivities for A. platensis, with comparable phycocyanin content. Increasing temperatures were found to improve the biomass productivity and phycocyanin content, which, at 40 °C, were 1.09 ± 0.03 gX·L−1·d−1 and 72.12 ± 3.52 mgPC·gX−1, respectively. The optimum biomass productivity was found at a light intensity of 300 μmol·m−2·s−1, with higher light intensities causing a decrease of 15%. Furthermore, of the various phycocyanin extraction methods tested, bead-beating in phosphate buffer had the highest combined phycocyanin yield (169.9 ± 3.6 mgPC·gX) and purity (7.37 ± 0.16) for Leptolyngbya sp. For A. platensis, this extraction method also resulted in the highest extract purities (3.78 ± 0.04). The extract purities obtained for Leptolyngbya sp. are considerably higher than other reported phycocyanin purities, and further investigation is recommended to study the scale-up of both Leptolyngbya sp. and bead-beating for commercial scale high-grade phycocyanin production under desert conditions. The authors would like to thank Ms. Ghamza Al Ghazal, Mr. Mohammad Dadrahim Mollazehi, and the QDVC team for their support. This work was supported by QDVC and Qatar University [Project: QUEX-CAS-QDVC-14/15-7].

The biomass composition, namely the fatty composition, of microalgae used in aqua feed is of great importance for the nutritional value of the biomass. Day:night cycles of light and temperature could influence the growth and biomass composition of microalgae. To study the effect of these cycles on Rhodomonas sp. the algae were grown under 16:8 day:night cycles in lab-scale photobioreactors running as turbidostat. Different temperature and light intensities were applied during the light phase. Synchronized cell cycles were observed for Rhodomonas sp. under day:night conditions with oscillating cell size, cell number, biomass concentration and fatty acid content and composition. Cells increased in size during the light phase, storing energy, with cell division scheduled in the dark phase. The introduction of a 16:8 day:night cycle did not affect the biomass yield on light, when operating at optimal conditions of light (150 μmol m−2 s−1) and temperature (21 °C). However, under high light (600 μmol m−2 s−1) or temperature (25 °C), an increased biomass yield on light of up to 22% was found under day:night cycles in comparison to continuous conditions under equal light and temperature levels. Implementation of a day:night cycle increased the maximum daytime temperature for Rhodomonas sp. from 25 °C to 30 °C. The fatty acid content and composition was influence by the implementation of day:night cycles. Daily fluctuations in total fatty acid content from 76 ± 2 mg gDW−1 at the end of the light phase to 94 ± 2 mg gDW−1 in the first hours of the light phase are found. The eicosapentaenoic acid and docosahexaenoic acid content fluctuated by 30% (12.1–16.1 mgEPA+DHA gDW−1) on a daily basis. These daily fluctuations can be exploited in aqua feed applications by selecting a specific time of the day to harvest algae.

The microalgae species Rhodomonas sp. is commonly used in aquaculture for its high nutritional value due to the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content. Understanding the effect of cultivation parameters on biomass production rate and composition is presently limited, however essential in further commercialization of this strain. Under nutrient replete conditions, light intensity and temperature are the main factors determining biomass growth and composition. Therefore, the combined effect of light and temperature on the biomass production rate and biomass composition of Rhodomonas sp. was studied using a statistical Design of Experiment approach. Rhodomonas sp. was cultivated under continuous (turbidostat) conditions in lab-scale reactor systems (1.8 l) under different temperature (15–20–25–30 °C) and light conditions (60–195–330–465–600 μmol m−2 s−1). Stable biomass production was observed under all conditions except experiments performed at 30 °C, which led to cell death. Under optimized growth conditions, high growth rates (>1.0d−1) and high biomass production rates, up to 1.5 g l−1 d−1, were obtained in this study. The biomass production rate reported here is >10-fold higher than values reported in literature on Rhodomonas sp. The optimal temperature for maximal growth was found at T = 22–24 °C under all light conditions. The maximum biomass yield on light (Yx,ph – 0.87 g mol−1) was found at light levels between 110 and 220 μmol m−2 s−1. The fatty acid profile was only significantly influenced by temperature, with higher EPA and DHA contents at lower temperatures (15 °C). A total fatty acid (TFA) content of 8–10% of the total dry-weight was found for all tested conditions. The EPA content fluctuated between 9 and 16% of TFA and DHA content between 6 and 9% of TFA, only affected by temperature. A maximum EPA + DHA production rate of 114 mg l‐−1 d−1 was obtained at 20 °C and high light (600 μmol m−2 s−1) conditions. Paid Open Access

Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019), PhD grant SFRH/BD/108894/2015 of FCT/MCTES. Online monitoring of algal biotechnological processes still requires development to support economic sustainability. In this work, fluorescence spectroscopy coupled with chemometric modelling is studied to monitor simultaneously several compounds of interest, such as chlorophyll and fatty acids, but also the biomass as a whole (cell concentration). Fluorescence excitation-emission matrices (EEM) were acquired in experiments where different environmental growing parameters were tested, namely light regime, temperature and nitrogen (replete or deplete medium). The prediction models developed have a high R2 for the validation data set for all five parameters monitored, specifically cell concentration (0.66), chlorophyll (0.78), and fatty acid as total (0.78), saturated (0.81) and unsaturated (0.74). Regression coefficient maps of the models show the importance of the pigment region for all outputs studied, and the protein-like fluorescence region for the cell concentration. These results demonstrate for the first time the potential of fluorescence spectroscopy for in vivo and real-time monitoring of these key performance parameters during Nannochloropsis oceanica cultivation. publishersversion published

Mathematical models were developed to predict biomass and hydrocarbon productivities and colony size (ouputs) of Botryococcus braunii showa cultures based on light intensity, temperature and dilution rate (inputs). These models predicted the following maximum values: biomass productivity, 1.3 g L−1 d−1; hydrocarbon productivity, 1.5 mg L−1 d−1; colony size, 320 µm under different culture conditions respectively. These values were confirmed experimentally. Additionally, the combination of inputs that simultaneously maximize all the possible outputs combinations were determined. The prediction for biomass-hydrocarbon-colony size were 1 g L−1 d−1, 12.05 mg L−1 d−1 and 156.8 µm respectively; biomass productivity-hydrocarbon productivities: 1 g L−1 d−1 and 13.94 mg L−1 d−1 respectively; biomass productivity-colony size: 1 g L−1 d−1 and 172.8 µm respectively; hydrocarbon productivity-colony size: 9 mg L−1 d−1 and 240 µm respectively. All these predictions were validated experimentally. These models might be very useful to implement a Botryococcus braunii showa large scale production. publishedVersion

International audience; Microalgae are promising natural resources for biofuels, chemical, food and feed products. Besides their economic potential, the environmental sustainability must be examined. Cultivation has a significant environmental impact that depends on reactor selection and operating conditions. To identify the main environmental bottlenecks for scale-up to industrial facilities this study provides a comparative life cycle assessment (LCA) of open raceway ponds and tubular photobioreactors at pilot scale. The results are based on experimental data from real pilot plants operated in summer, fall and winter at AlgaePARC (Wageningen, The Netherlands). The energy consumption for temperature regulation presented the highest environmental burden. The production of nutrients affected some categories. Despite limited differences compared to the vertical system, the horizontal PBR was found the most efficient in terms of productivity and environmental impact. The ORP was, given the Dutch climatic conditions, only feasible under summer operation. The results highlight the relevance of LCA as a tool for decision-making in process design. Weather conditions and availability of sources for temperature regulation were identified as essential factors for the selection of geographic locations and for microalgal cultivation systems based on environmental criteria. Simulation of large-scale reactors with optimized temperature regulation systems lead to environmental improvements and energy demand reductions ranging from 17% up to 90% for systems operated in favorable summer conditions.

Botryococcus braunii can produce both long-chain hydrocarbons as well as carbohydrates in large quantities, and is therefore a promising industrial organism for the production of biopolymer building blocks. Many studies describe the use of different strains of Botryococcus braunii but differences in handling and cultivation conditions make the comparison between strains difficult. In this study, 16 B. braunii strains obtained from six culture collections were compared for their biomass productivity and hydrocarbon and carbohydrate content. Biomass productivity was highest for AC768 strain with 1.8 g L−1 day−1, while hydrocarbon production ranged from none to up to 42% per gram biomass dry weight, with Showa showing the highest hydrocarbon content followed by AC761. The total carbohydrate content varied from 20% to 76% per gram of the biomass dry weight, with CCALA777 as the highest producer. Glucose and galactose are the main monosaccharides in most strains and fucose content reached 463 mg L−1 in CCALA778.

Background Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR–Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout. Results In this study, we report for the first time an alternative approach for CRISPR–Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines. Conclusion We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Micro-algae are a new and promising source of nutrients. The main products obtainable are dried algae with high nutrients content and high-value compounds such as fatty acids, pigments and anti-oxidants. This paper analyses the market and the economic opportunities of micro-algae-based food and feed sectors in the EU through an integrated methodology composed by literature search, interviews to experts and Delphi survey. Results show that the quantities produced and the market size of nutrients obtained from micro-algae are still significantly smaller in comparison to the ones derived from cereals and other commodity crops, but that the sector has seen an impressive and unique growth. Despite the challenges due to the climatic conditions together with the insufficient domestic demand and the complexity of the EU Novel Food regulation, the survey revealed that the EU can improve its market position in the next decade, thanks to its scientific and technological capacity and its dominant position in the global agri-food markets. New micro-algae-based products can be developed for foreign markets, and the improved global production share of European firms (presently of about 5%) may be the result of strategic acquisitions of foreign companies.