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In this study the influence of day length on circadian rhythms of N. oleoabundans was investigated. N. oleoabundans was grown in a photobioreactor continuously operated (turbidostat), under various day/night (D/N) cycles; 20D4N, 16D8N and 12D12N. The following variables showed to be regulated by the circadian clock: maximum growth rate, start of starch synthesis and DNA replication to 4 and 8 copies. Timing of these processes was not influenced by day length. The length of the photoperiod caused changes in biomass composition, especially due to variations in starch content. In longer days, more starch was accumulated. Starch was used for cell division probably also when cell division occurred in the light. Therefore, also the timing of cell division should be considered in the production of microalgae biomass. This timing mechanism can be explored to produce biomass with a desired concentration of protein, lipids, carbohydrates or pigments.

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.

Algal growth rate was modeled from average irradiance and temperature. Indirect irradiation was estimated from actual horizontal irradiation. Photobioreactor productivity rate was modeled from irradiation and temperature. Photobioreactor productivity rate was measured from CO2-absorption.

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.

The effect of biomass concentration on areal productivity and photosynthetic efficiency of Nannochloropsis sp. CCAP211/78 was studied in three outdoor pilot-scale photobioreactors: an open raceway pond (OPR), a horizontal tubular (HT) photobioreactor and a vertically stacked horizontal tubular (VT) photobioreactor. The reactors were operated continuously as turbidostat at different biomass concentrations. For all systems highest areal productivities were obtained on days with a high light intensity, while the highest photosynthetic efficiencies were obtained on days with a low light intensity. Ground areal biomass concentration exceeding 51 g m − 2 had a negative effect on the areal productivity and photosynthetic efficiency. No significant effect of biomass concentration on the productivity was found for the HT at ground areal biomass concentration lower than 51 g m − 2 . Also for the VT, no significant effect of biomass concentration was found with the exception of the highest biomass concentration of 2.0 g L − 1 (68 g m − 2 ) resulting in decreased productivity. For the open raceway pond the highest biomass concentration (0.5 g L − 1 or 94 g m − 2 ) resulted in significantly lower areal productivity, compared to the lower biomass concentration (0.25 g L 47 g m − 2 ). Highest areal productivities were obtained for OPR and VT, most likely due to more efficient light interception. In this study we observed that night biomass loss was coupled to net growth. At lower biomass concentrations and concomitant higher growth rates the specific biomass loss rate was higher. Microalgal specific light absorption coefficient was correlated to biomass concentration; higher biomass concentrations resulted in higher specific absorption coefficients, resulting in a steeper light gradient in the microalgal cultures.

This chapter provides a comprehensive overview of the recent developments in microalgae cultivation and downstream processing with a focus on microalgae biorefinery. Microalgae are promising feedstocks for the production of a variety of bulk compounds in the near future. Current process designs typically consist of microalgae cultivation, harvesting, cell disruption and finally a product extraction/fractionation step. For every unit operation, the latest insights are discussed. From this overview, the current state-of-development and future directions towards large scale bulk-chemical production from microalgae are discussed.

In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25-145 gDW kg(-1)) over a range of agitator speeds (6-12 m s(-1)). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9-10 m s(-1) regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kgDW(-1).

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.

During the past few years, interest in microalgae has grown, mainly because of their potential for biofuel production. Botryococcus braunii, a green microalga that can accumulate more than half of its dry weight as hydrocarbons, is one of the most important examples. This microorganism grows in colonies and there has been no reliable viability protocol reported for this species as yet. Knowing the number of dead cells in cultures is essential for the development of efficient bioprocesses such as non-destructive extraction procedures (“milking”) to obtain lipid soluble substances from microalgal biomass. Our study presents a simple colorimetric method to determine the proportion of living to dead cells in cultures, based on the uptake of methylene blue in solution by dead B. braunii cells. The main parameters influencing this process were investigated and used to develop a protocol. This technique was validated using flow cytometry and Neochloris oleoabundans, and appears not to be limited to use with B. braunii.

Microalgae are considered to be one of the most promising next generation bio-based/food feedstocks with a unique lipid composition, high protein content, and an almost unlimited amount of other bio-active molecules. High-value components such as the soluble proteins, (poly) unsaturated fatty acids, pigments, and carbohydrates can be used as an important ingredient for several markets, such as the food/feed/chemical/cosmetics and health industries. Although cultivation costs have decreased significantly in the last few decades, large microalgae production processes become economically viable if all complex compounds are optimally valorized in their functional state. To isolate these functional compounds from the biomass, cost-effective, mild, and energy-efficient biorefinery techniques need to be developed and applied. In this review we describe current microalgae biorefinery strategies and the derived products, followed by new technological developments and an outlook toward future products and the biorefinery philosophy.

Background Day/night cycles regulate the circadian clock of organisms to program daily activities. Many species of microalgae have a synchronized cell division when grown under a day/night cycle, and synchronization might influence biomass yield and composition. Therefore, the aim of this study was to study the influence of day/night cycle on biomass yield and composition of the green microalgae Neochloris oleoabundans. Hence, we compared continuous turbidostat cultures grown under continuous light with cultures grown under simulated day/night cycles. Results Under day/night cycles, cultures were synchronized as cell division was scheduled in the night, whereas under continuous light cell division occurred randomly synchronized cultures were able to use the light 10–15% more efficiently than non-synchronized cultures. Our results indicate that the efficiency of light use varies over the cell cycle and that synchronized cell division provides a fitness benefit to microalgae. Biomass composition under day/night cycles was similar to continuous light, with the exception of starch content. The starch content was higher in cultures under continuous light, most likely because the cells never had to respire starch to cover for maintenance during dark periods. Day/night cycles were provided in a ‘block’ (continuous light intensity during the light period) and in a ‘sine’ (using a sine function to simulate light intensities from sunrise to sunset). There were no differences in biomass yield or composition between these two ways of providing light (in a ‘block’ or in a ‘sine’). Conclusions The biomass yield and composition of N. oleoabundans were influenced by day/night cycles. These results are important to better understand the relations between research done under continuous light conditions and with day/night cycle conditions. Our findings also imply that more research should be done under day/night cycles.

Microalgae are considered to be one of the most promising next generation bio-based/food feedstocks with a unique lipid composition, high protein content, and an almost unlimited amount of other bio-active molecules. High-value components such as the soluble proteins, (poly) unsaturated fatty acids, pigments, and carbohydrates can be used as an important ingredient for several markets, such as the food/feed/chemical/cosmetics and health industries. Although cultivation costs have decreased significantly in the last few decades, large microalgae production processes become economically viable if all complex compounds are optimally valorized in their functional state. To isolate these functional compounds from the biomass, cost-effective, mild, and energy-efficient biorefinery techniques need to be developed and applied. In this review we describe current microalgae biorefinery strategies and the derived products, followed by new technological developments and an outlook toward future products and the biorefinery philosophy.

Our goal in the present study was to evaluate the potential for lipid production of two cell populations of the marine microalgae Chlorococcum littorale under different climate conditions. We selected, in a previous study and via fluorescence activated cell sorting (FACS), a new cell population of Chlorococcum littorale , namely S5. S5 showed a stable doubled triacylglycerol (TAG) productivity in comparison with the original population. A previously developed model was expanded to include day:night cycles and validated to predict biomass and outdoor TAG productivities at different locations. Four different locations were chosen to simulate the response of C. littorale to different day lengths and light intensities (the Netherlands, Norway, Brazil and Spain). Indoor experiments (simulated summer) were carried out with Original and S5, showing that S5 had a doubled TAG productivity under N-starvation. Finally, simulations of biomass and TAG productivities of Original and S5 at different locations were performed. At locations with lower light intensities, Norway and the Netherlands, biomass productivities were higher than at locations with higher light intensities, Brazil/Spain. Such results might be associated with light-saturation effects. TAG productivities, however, showed no effect of local light intensity. Locations at higher latitudes, Norway/Netherlands, cannot sustain phototrophic year-round production, hence, the yearly average TAG productivities were doubled in Brazil/Spain (from 1.4–1.6 to 3.0–3.2 g m − 2 d − 1 ). Likewise, C. littorale S5 was simulated with doubled TAG productivities when compared with Original, at all locations (2.5–2.7 (low light) to 4.7–5.2 g m − 2 d − 1 (high light)). The present results confirm the industrial potential of Chlorococcum littorale , both Original and S5, as a source of TAG. Furthermore, our results can be used for comparison and to estimate future production scenarios.

The disintegration of three industry relevant algae (Chlorella vulgaris, Neochloris oleoabundans and Tetraselmis suecica) was studied in a lab scale bead mill at different bead sizes (0.3–1 mm). Cell disintegration, proteins and carbohydrates released into the water phase followed a first order kinetics. The process is selective towards proteins over carbohydrates during early stages of milling. In general, smaller beads led to higher kinetic rates, with a minimum specific energy consumption of ⩽0.47 kWh kgDW−1 for 0.3 mm beads. After analysis of the stress parameters (stress number and stress intensity), it appears that optimal disintegration and energy usage for all strains occurs in the 0.3–0.4 mm range. During the course of bead milling, the native structure of the marker protein Rubisco was retained, confirming the mildness of the disruption process.

Microalgae are a promising source for proteins, lipids, and carbohydrates for the cosmetic, nutraceutical, chemical, food/feed, and biofuel industry. In comparison with soy and palm oil, microalgae can be produced in a more sustainable way. To make microalgae production economically feasible, all biomass ingredients need to be efficiently utilized, similar to petroleum refineries in which oil is fractionated in fuels and a variety of products with higher value. However severe conditions can affect the properties of some components in the biomass. To overcome this, focus needs to be put on biorefinery techniques which are mild and effective. Microalgal biorefinery is a linear process consisting of harvesting, cell disintegration, sequential extraction, and further fractionation. Among these steps, the cell disintegration often represents a bottleneck for the extraction of hydrophilic or hydrophobic components, due to the presence of a tough cell wall in many strains. State of the art knowledge on both novel and classical techniques for product extraction within cell disintegration is presented. Comparison is made on the basis of two main criteria: yield of disintegration and energy consumption. The current work gives also a comprehensive outlook on business cases for microalgae biorefinery.

The microalgae Tetradesmus obliquus is able to maintain a high photosynthetic efficiency under nitrogen limitation and is considered a promising green microalgae for sustainable production of diverse compounds, including biofuels. Here, we report the first draft whole-genome shotgun sequencing of T. obliquus . The final assembly comprises 108,715,903 bp with over 1,368 scaffolds.

Pulsed Electric Field (PEF) is currently discussed as promising technology for mild and scalable cell disintegration of microalgae. In this study Chlorella vulgaris and Neochloris oleoabundans have been subjected to batch and continuous PEF treatments under a wide range of operating conditions (1–40 pulses, 0.05–5 ms pulses, 7.5–30 kV cm − 1 , 0.05–150 kWh kg DW − 1 ). In many cases after treatment, both algal species show release of ions, which indicates that PEF treatment resulted in permeabilization of the algal cell. However, the electroporation effect was not sufficient to substantially release intracellular proteins. Even at severe energy input (10 to 100 times higher than bead milling) only up to 13% of proteins released from the cells in comparison to 45–50% after bead milling.

Microalgae gained much interest from industry as promising sustainable feedstock for the production of food, feed, bulk chemicals, and biofuels. Pilot scale research on microalgae is needed to bridge the gap between laboratory scale research and commercial applications. The AlgaePARC (Algae Production And Research Center) pilot facility was constructed to bridge this gap. The objective of this pilot center is to compare and improve photobioreactors and operational strategies under outdoor conditions. The pilot plant facility consists of four production systems (raceway pond, horizontal tubular reactor, vertically stacked tubular reactor and flat panels) and allows comparison of performance of these systems under identical climatological conditions. This paper describes the development of this pilot facility, decisions made during the building process and discusses the production systems including technical specifications, measurements and supporting facilities.

Background Day/night cycles regulate the circadian clock of organisms to program daily activities. Many species of microalgae have a synchronized cell division when grown under a day/night cycle, and synchronization might influence biomass yield and composition. Therefore, the aim of this study was to study the influence of day/night cycle on biomass yield and composition of the green microalgae Neochloris oleoabundans. Hence, we compared continuous turbidostat cultures grown under continuous light with cultures grown under simulated day/night cycles. Results Under day/night cycles, cultures were synchronized as cell division was scheduled in the night, whereas under continuous light cell division occurred randomly synchronized cultures were able to use the light 10–15% more efficiently than non-synchronized cultures. Our results indicate that the efficiency of light use varies over the cell cycle and that synchronized cell division provides a fitness benefit to microalgae. Biomass composition under day/night cycles was similar to continuous light, with the exception of starch content. The starch content was higher in cultures under continuous light, most likely because the cells never had to respire starch to cover for maintenance during dark periods. Day/night cycles were provided in a ‘block’ (continuous light intensity during the light period) and in a ‘sine’ (using a sine function to simulate light intensities from sunrise to sunset). There were no differences in biomass yield or composition between these two ways of providing light (in a ‘block’ or in a ‘sine’). Conclusions The biomass yield and composition of N. oleoabundans were influenced by day/night cycles. These results are important to better understand the relations between research done under continuous light conditions and with day/night cycle conditions. Our findings also imply that more research should be done under day/night cycles.

Microalgae are a promising source for proteins, lipids and carbohydrates for the food/feed and biofuel industry. In comparison with soya and palm oil, microalgae can be grown in a more efficient and sustainable way. To make microalgae production economically feasible it is necessary to optimally use all produced compounds. To accomplish this focus needs to be put on biorefinery techniques which are mild and effective. Of the techniques described, Pulsed Electric Field (PEF) seems to be the most developed technique compared to other cell disruption applications. For separation technology ionic liquids seems most promising as they are able to both separate hydrophobic and hydrophilic compounds. But additional studies need to be evolved in the coming years to investigate their relevance as novel cell disruption and separation methods. We propose a complete downstream processing flow diagram that is promising in terms of low energy use and state of the art knowledge.

article i nfo The effect of cell cycle stage on biomass composition of the green microalgae Neochloris oleoabundanswas inves- tigated. N. oleoabundans was grown under constant light conditions in a flat panel photobioreactor operated as a turbidostat. Even though light conditions were constant, a synchronized cell division was observed with the cells dividing by multiple fission during the natural night. Presumably, the circadian clock was responsible for 'gating' cell division to this specific time frame. Oscillations in starch, protein and pigment content were observed during the cell cycle. These oscillations could be solely contributed to the cell cycle stage of the synchronized culture, since all experimental conditions were kept constant. A maximum in starch, protein and fatty acid content was obtained just before cell division. Biomass yield was also greatly influenced by the cell cycle and declined to a minimum during cell division. These findings highlight that knowledge of the cell cycle is of importance in microalgae process optimization.

CO2 fixation by phototrophic microalgae has been addressed as a possible global carbon emissions reducer, whilst simultaneously producing useful products. Especially in Qatar, the prospect of using microalgae for CO2 abatement is promising: high solar irradiance, large areas of non-arable land, and large amounts of CO2 emissions make it seemingly the ideal place for algae cultivation. In order to promote high biomass productivities, and subsequent CO2 uptake rates, effective CO2 supply to the cultivation system is of high importance. However, the low solubility of CO2 in water, as well as the limiting tolerance of microalgae to increased CO2 concentrations, results in low efficiency of CO2 capture by microalgal production systems. In order to overcome these hurdles, this research focused on selecting local desert microalgae strains with high tolerance to increased CO2 levels, and developing growth media in order to increase the solubility of CO2. Forty-five locally isolated marine microalgae strains were screened for growth under increased CO2 concentrations, ranging from 0.04% to 30% (v/v). A number of different trends in CO2 tolerance could be identified from the results; a number of strains showed a clear inhibition of growth with CO2 concentrations of 5% and higher, whilst others showed increasing growth rates for increasing CO2 concentrations up to 30%. The trend in growth rate suggests that even higher CO2 concentration could be applied without growth-limiting effects, and could even stimulate higher growth-rates. In order to further increase the productivity of high CO2-tolerant strains, as well as to investigate the effects of pH on the CO2 tolerance of low-tolerant strains, various strains were cultivated in alkaline media and high CO2 concentrations. Besides leading to an increased solubility of CO2 in the culture media, increasing the pH is thought to balance the acidification effect of CO2 – possibly leading to higher CO2 tolerances. Overall, applying these strains and media adaptations for large-scale applications is expected to increase the CO2 transfer efficiency to the culture, resulting in decreased operational costs and higher overall productivities.

Background Despite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity. Results 1. We successfully identified sub-populations of cells with increased TAG productivity using Fluorescence assisted cell sorting (FACS). 2. We sequentially sorted cells after repeated cycles of N-starvation, resulting in five sorted populations (S1–S5). 3. The comparison between sorted and original populations showed that S5 had the highest TAG productivity [0.34 against 0.18 g l−1 day−1 (original), continuous light]. 4. Original and S5 were compared in lab-scale reactors under simulated summer conditions confirming the increased TAG productivity of S5 (0.4 against 0.2 g l−1 day−1). Biomass composition analyses showed that S5 produced more biomass under N-starvation because of an increase only in TAG content and, flow cytometry showed that our selection removed cells with lower efficiency in producing TAGs. Conclusions All combined, our results present a successful strategy to improve the TAG productivity of Chlorococcum littorale, without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production. Graphical abstract

The synergistic effect of temperature (25-65 °C) and total specific energy input (0.55-1.11 kWh kgDW(-1)) by pulsed electric field (PEF) on the release of intracellular components from the microalgae Chlorella vulgaris was studied. The combination of PEF with temperatures from 25 to 55 °C resulted in a conductivity increase of 75% as a result of cell membrane permeabilization. In this range of temperatures, 25-39% carbohydrates and 3-5% proteins release occurred and only for carbohydrate release a synergistic effect was observed at 55 °C. Above 55 °C spontaneous cell lysis occurred without PEF. Combined PEF-temperature treatment does not sufficiently disintegrate the algal cells to release both carbohydrates and proteins at yields comparable to the benchmark bead milling (40-45% protein, 48-58% carbohydrates).

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0475-4) contains supplementary material, which is available to authorized users.

Our society needs new sustainable biobased feedstocks to meet population growth and reduce dependence on fossil fuels. Microalgae are considered one of the most promising feedstocks for sustainable production of food, feed, chemicals, materials and fuels. Our mission is to develop a commercial and sustainable production chain for commodity products from microalgae. Estimations of biomass production costs for a 100 ha plant facility have been done. Projections of different scenarios allowed us to compare effect of variables, such as location of the facility, type of cultivation system and operational parameters. As result, this tool shows the most suitable location and system for algae production, as well as the main cost factors. The biomass produced follows to the biorefinery process to be fractionated into the main components: proteins, lipids, carbohydrates and pigments. Several chains have been specifically designed for different market scenarios. The overall turnover coming from the exploitation of the different biomass fractions depends on the end product. A market analysis has been conducted looking to different scenarios according to the biomass value pyramid: biofuels, chemicals, food, feed, specialties in food, cosmetics and a combination of these. Projections show that production of high-value products from microalgae could be profitable nowadays and commodities will become profitable within 10 years.

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.

In this feature, leading researchers in the field of microbial biotechnology speculate on the technical and conceptual developments that will drive innovative research and open new vistas over the next few years

The carbon footprint caused by unsustainable development and its environmental and economic impact has become a major concern in the past few decades. Photosynthetic microbes such as microalgae and cyanobacteria are capable of accumulating value-added compounds from carbon dioxide, and have been regarded as environmentally friendly alternatives to reduce theusage of fossil fuels, thereby contributing to reducing the carbon footprint. This light-driven generation of green chemicals and biofuels has triggered the research for metabolic engineering of these photosynthetic microbes. CRISPR-Cas systems are successfully implemented across a wide range of prokaryotic and eukaryotic species for efficient genome editing. However, the inception of this genome editing tool in microalgal and cyanobacterial species took off rather slowly due to various complications. In this review, we elaborate on the established CRISPR-Cas based genome editing in various microalgal and cyanobacterial species. The complications associated with CRISPR-Cas based genome editing in these species are addressed along with possible strategies to overcome these issues. It is anticipated that in the near future this will result in improving and expanding the microalgal and cyanobacterial genome engineering toolbox.

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). Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0283-2) contains supplementary material, which is available to authorized users.

An economically feasible microalgal lipid industry heavily relies on the selection of suitable strains. Because microalgae lipid content increases under a range of adverse conditions (e.g. nutrient deprivation, high light intensity), photosynthetic activity is usually strongly reduced. As a consequence, lipid productivity rapidly declines overtime, after reaching a maximum within the first days of cultivation. The microalgae Chlorella vulgaris, Chlorococcum littorale, Nannochloropsis oculata, Nannochloropsis sp., Neochloris oleoabundans, Stichococcus bacillaris and Tetraselmis suecica were compared on fatty acid content and productivity, and also on photosynthetic activity under nitrogen (N) starvation. Cultures in N-replete conditions were used as reference. Photosystem II (PSII) maximum efficiency was followed during the experiment, as proxy for the change in photosynthetic activity of the cells. Strains with a high capacity for both lipid accumulation as well as high photosynthetic activity under N starvation exhibited a high lipid productivity over time. Among the tested strains, Nannochloropsis sp. showed highest fatty acid content (45 % w/w) and productivity (238 mg L−1 day−1) as well as PSII maximum efficiency, demonstrating to be the most suitable strain, of those tested, for lipid production. This study highlights that for microalgae, maintaining a high photosynthetic efficiency during stress is the key to maintain high fatty acid productivities overtime and should be considered when selecting strains for microalgal lipid production.

Background: Microalgae are a potential source of sustainable commodities of fuels, chemicals and food and feed additives. The current high production costs, as a result of the low areal productivities, limit the application of microalgae in industry. A first step is determining how the different production system designs relate to each other under identical climate conditions. The productivity and photosynthetic efficiency of Nannochloropsis sp. CCAP 211/78 cultivated in four different outdoor continuously operated pilot-scale photobioreactors under the same climatological conditions were compared. The optimal dilution rate was determined for each photobioreactor by operation of the different photobioreactors at different dilution rates. Results: In vertical photobioreactors, higher areal productivities and photosynthetic efficiencies, 19-24 g m-2 day-1 and 2.4-4.2 %, respectively, were found in comparison to the horizontal systems; 12-15 g m-2 day-1 and 1.5-1.8 %. The higher ground areal productivity in the vertical systems could be explained by light dilution in combination with a higher light capture. In the raceway pond low productivities were obtained, due to the long optical path in this system. Areal productivities in all systems increased with increasing photon flux densities up to a photon flux density of 30 mol m-2 day-1. Photosynthetic efficiencies remained constant in all systems with increasing photon flux densities. The highest photosynthetic efficiencies obtained were; 4.2 % for the vertical tubular photobioreactor, 3.8 % for the flat panel reactor, 1.8 % for the horizontal tubular reactor, and 1.5 % for the open raceway pond. Conclusions: Vertical photobioreactors resulted in higher areal productivities than horizontal photobioreactors because of the lower incident photon flux densities on the reactor surface. The flat panel photobioreactor resulted, among the vertical photobioreactors studied, in the highest average photosynthetic efficiency, areal and volumetric productivities due to the short optical path. Photobioreactor light interception should be further optimized to maximize ground areal productivity and photosynthetic efficiency.

Acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed at a constant acceleration rate, leading to different average light intensities inside the photobioreactor, can supply more information and reduce experimental time compared with chemostat cultivations. The A-stat was used to optimize the biomass and product yield of continuous cultures of the microalgae D. tertiolecta in a flat-panel reactor. In this study, four different accelerations were studied, a pseudo steady state was maintained at acceleration rates of 0.00016 and 0.00029 h-2 and results were similar to those of the chemostat. An increase in the acceleration rate led to an increase in the deviation between results obtained in the A-stat and in the chemostats. We concluded that it is advantageous to use the A-stat instead of chemostats to determine culture characteristics and optimize a specific photobioreactor. The effect of average light intensity inside the photobioreactor on the production of vitamins C and E, lutein, and β-carotene was studied using the A-stat. The highest concentrations of these products were 3.48 ± 0.46, 0.33 ± 0.06, 5.65 ± 0.24, and 2.36 ± 0.38 mg g-1 respectively. These results were obtained at different average light intensities, showing the importance of optimizing each product on light intensity. © 2004 Wiley Periodicals, Inc.

The present work established an efficient staining method for fluorescence activated cell sorting (FACS) with Chlorococcum littorale maintaining cellular viability. The method was designed to detect high-lipid cells and to guarantee cellular viability. BODIPY505/515 (BP) was more suitable to FACS when compared to Nile red. The optimum concentrations were 0.4 μg ml−1 of BP, 0.1% DMSO or 0.35% ethanol. Both ethanol and DMSO were equally efficient and assured cellular viability after the staining and sorting. Here a method is presented to rapidly screen and sort lipid rich cells of C. littorale with FACS, which can be used to produce new inoculum with increased cellular lipid content.

Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp. produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l(-1) h(-1), under a light intensity of 680 micromol photons m(-2) s(-1), and a maximum light efficiency of 6.2% under a light intensity of 43 micromol photons m(-2) s(-1). Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease in the hydrogen evolved from 214 to 27 ml H2 per vessel.

The prospect of sustainable production of food ingredients from photoautotrophic microalgae was reviewed. Clearly, there is scope for microalgal oils to replace functions of major vegetable oils, and in addition to deliver health benefits to food products. Furthermore, with a limited production surface, a substantial portion of the European Union market could be supplied with edible oils and proteins from microalgae. Yet, before microalgal ingredients can become genuinely sustainable and cost effective alternatives for current food commodities, major breakthroughs in production technology and in biorefinery approaches are required. Moreover, before market introduction, evidence on safety of novel microalgal ingredients, is needed. In general, we conclude that microalgae have a great potential as a sustainable feedstock for food commodities.

Microalgae are a promising feedstock for sustaineble production of biofuela due to their unique capacity to reach high lipid productivities. Although the promises are there, production costs and energy requirements are high and the technology is still ammature for the production of bulk products. It is an enormous challange to transfer technology so far used for production of high-value niche products to a technology to be used at industrial scale for commodities. A new mind-set is needed and knowledge needs to be gained in different scientific disciplines, intergrated, applied, and demontrated in an industrial context. Biofuels from microalgae can only become realistic if the cost of production of algae is reduced, if the produntivity of lipids in microalgae is maximized, and if besides lipids, other compounds are validated for applications for food, feed, chemistry, and materials using a microalgal biorefinery.

Worldwide, microalgal biofuel production is being investigated. It is strongly debated which type of production technology is the most adequate. Microalgal biomass production costs were calculated for 3 different micro algal production systems operating at commercial scale today: open ponds, horizontal tubular photobioreactors and flat panel photobioreactors. For the 3 systems, resulting biomass production costs including dewatering, were 4.95, 4.15 and 5.96 € per kg, respectively. The important cost factors are irradiation conditions, mixing, photosynthetic efficiency of systems, medium- and carbon dioxide costs. Optimizing production with respect to these factors, a price of € 0.68 per kg resulted. At this cost level microalgae become a promising feedstock for biodiesel and bulk chemicals.Photobioreactors may become attractive for microalgal biofuel production.

The feasibility of microalgae production for biodiesel was discussed. Although algae are not yet produced at large scale for bulk applications, there are opportunities to develop this process in a sustainable way. It remains unlikely, however, that the process will be developed for biodiesel as the only end product from microalgae. In order to develop a more sustainable and economically feasible process, all biomass components (e.g. proteins, lipids, car- bohydrates) should be used and therefore biorefi ning of microalgae is very important for the selective separation and use of the functional biomass components. If biorefi ning of microalgae is applied, lipids should be fractionated into lipids for biodiesel, lipids as a feedstock for the chemical industry and w -3 fatty acids, proteins and carbohydrates for food, feed and bulk chemicals, and the oxygen produced should be recovered also. If, in addition, production of algae is done on residual nutrient feedstocks and CO2, and production of microalgae is done on a large scale against low production costs, production of bulk chemicals and fuels from microalgae will become economically feasible. In order to obtain that, a number of bottlenecks need to be removed and a multidisciplinary approach in which sys- tems biology, metabolic modeling, strain development, photobioreactor design and operation, scale-up, biorefi ning, integrated production chain, and the whole system design (including logistics) should be addressed. © 2010 Society of Chemical Industry and John Wiley & Sons, Ltd

Microalgae are considered one of the most promising feedstocks for biofuels. The productivity of these photosynthetic microorganisms in converting carbon dioxide into carbon-rich lipids, only a step or two away from biodiesel, greatly exceeds that of agricultural oleaginous crops, without competing for arable land. Worldwide, research and demonstration programs are being carried out to develop the technology needed to expand algal lipid production from a craft to a major industrial process. Although microalgae are not yet produced at large scale for bulk applications, recent advances—particularly in the methods of systems biology, genetic engineering, and biorefining—present opportunities to develop this process in a sustainable and economical way within the next 10 to 15 years.

In the present work we identified and quantified the effect of hydrodynamic stress on two different microalgae strains, Dunaliella tertiolecta and D. salina, cultivated in bench-scale bubble columns. The cell death rate constant increased with increasing gas-entrance velocity at the sparger. Dunaliella salina was slightly more sensitive than D. tertiolecta. The critical gas-entrance velocities were approximately 50 and 30 m s(-1) for D. tertiolecta and D. salina, respectively. The effects of gas-flow rate, culture height, and nozzle diameter on the death rate constant were also studied. From these results it was concluded that bubble rising and bubble bursting are not responsible for cell death. Regarding nozzle diameter, small nozzles were more detrimental to cells. The bubble formation at the sparger was found to be the main event leading to cell death.

The slow development of microalgal biotechnology stems from the failure in the design of large-scale photobioreactors where light energy is efficiently utilized. Due to the light gradient inside the reactor and depending on the mixing properties, algae are subjected to certain light/dark cycles where the light period is characterized by a light gradient. These light/dark cycles will determine productivity and biomass yield on light energy. Air-lift reactors can be used for microalgae cultivation and medium-frequency light/dark cycles will be found in these systems. Light/dark cycles are associated with two basic parameters: first, the light fraction, i.e., the ratio between the light period and the cycle time and second, the frequency of the light/dark cycle. In the present work, light/dark cycles found in air-lift reactors were simulated taking into account the light gradient during the light period. The effect of medium-frequency cycle time (10-100 s) and light fraction (0.1-1) on growth rate and biomass yield on light energy of the microalgae Dunaliella tertiolecta was studied. The biomass yield and growth rates were mainly affected by the light fraction, while cycle time had little influence. Response surface methodology was used and a statistical model describing the effect of light fraction and cycle time on growth rate and biomass yield on light energy was developed. The use of the model as a reactor design criterion is discussed.

High-quality research in the field of marine biotechnology is one of the key-factors for successful innovation in exploiting the vast diversity of marine life. However, fascinating scientific research with promising results and claims on promising potential applications (e.g. for pharmaceuticals, nutritional supplements, (feed-)products for aquaculture and bioremediation solutions) is not the only factor to realise the commercial applications of marine biotechnology. What else is needed to exploit the promising potential of marine biotechnology and to create new industrial possibilities? In the study project 'Ocean Farming-Sustainable exploitation of marine organisms', we explore the possibilities of marine organisms to fulfill needs, such as safe and healthy food, industrial (raw) materials and renewable energy in a sustainable way. One of the three design groups is envisioning the future of strong land-based 'marine' market chains. Marine biotechnology is one of the foci of attention in this design group. This article provides a model of future-oriented thinking in which a variety of experts actively participate. (C) 2003 Elsevier B.V. All rights reserved.

The effect of high superficial gas velocities in continuous and batch cultures of the strains Dunaliella tertiolecta, Chlamydomonas reinhardtii wild-type and cell wall-lacking mutant was studied in bubble columns. No cell damage was found for D. tertiolecta and C. reinhardtii (wild-type) up to superficial gas velocities of 0.076 and 0.085 m s(-1), respectively, suggesting that high superficial gas velocities alone cannot be responsible for cell death and, consequently, bubble bursting cannot be the sole cause for cell injury. A death rate of 0.46 +/- 0.08 h(-1) was found for C. reinhardtii (cell wall-lacking mutant) at a superficial gas velocity of 0.076 m s(-1), and increased to 1.01 +/- 0.29 h(-1) on increasing superficial gas velocity to 0.085 m s(-1). Shear sensitivity is thus strain-dependent and to some extent the cell wall plays a role in the protection against hydrodynamic shear. When studying the effect of bubble formation at the sparger in batch cultures of D. tertiolecta by varying the number of nozzles, a death rate of 0.047 +/- 0.016 h(-1) was obtained at high gas entrance velocities. D. tertiolecta was cultivated in a pilot-plant reactor under different superficial gas velocities of up to 0.026 m s(-1), with relatively low gas entrance velocities and no cell damage was observed. There is some indication that the main parameter causing cell death and damage was the gas entrance velocity at the sparger.

Publisher Summary Hydrogen is recognized nowadays as the fuel of the future. For its large scale use, the production of large quantities of economic hydrogen is essential. However, biological hydrogen production, as an environmental-friendly resource of energy, must be developed in order to be an alternative technology, so that it can be used when other goals become more important than price. Hydrogen can be produced by photo-fermentation with photosynthetic bacteria, carried out with suitable nutrients, under anaerobic conditions, in the absence of nitrogen gas, with illumination and with stressful concentrations of nitrogen sources. The main enzymes involved are nitrogenase and hydrogenase. Valuable by-products can also be formed. These advantages put together with waste treatment give this system potential at a short term. The design of photobioreactors with efficient light transfer is still lacking. The optimal production of hydrogen by photosynthetic bacteria, to be economically attractive, has to take into account several parameters, such as the productivity, the light efficiency, and the yield on carbon source.

Astaxanthin concentration in the blood of rainbow trout was studied in a feeding trial with two different astaxanthin sources: green algae Haematococcus pluvialis and commercial beadlets of 8% astaxanthin content (CAROPHYLL® Pink), and two different dietary lipid levels. The green algae contained 1.4% of carotenoids on a dry matter basis: free astaxanthin (< 1%), astaxanthin monoester (24.3%); astaxanthin diester (70.2%) and lutein (4.8%). Algal biomass was mechanically ground to disrupt the cell wall before incorporation in the feed. Hydrolysis of astaxanthin esters from algae occurred during the pelletization even at a low process temperature (43°C). Rainbow trout with an initial mean body weight of 150 g were fed experimental diets supplemented at a rate of 100 mg pigment/kg diet combined with two different lipid levels (9 and 24%) during 5 days. Astaxanthin concentration in the serum ranged from 5.3 μg/ml (8.9 nmol/ml) to 9.0 μg/ml (15.1 nmol/ml). Astaxanthin concentration in the serum was higher for fish fed high lipid level diets, independently of the astaxanthin source. No differences in the astaxanthin serum concentration were found for fish fed diets supplemented with either natural or synthetic astaxanthin, respectively 9.0 ± 1.9 and 8.4 ± 2.4 μg astaxanthin/ml serum, when dietary lipid level was high (24%). On the other hand, there was a higher blood astaxanthin concentration in fish fed diets supplemented with algal biomass (7.0 ± 2.4 μg astaxanthin/ml serum) compared to synthetic astaxanthin (5.3 ± 2.0 μg astaxanthin/ml serum) when dietary lipid level was low (9%).