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There is an increasing demand for bio-based fertilizers and phytostimulants. Microalgae biomass contains a number of compounds that have positive effects on plant growth (for instance, phenolic compounds). Other valuable substances are simultaneously produced in the biomass, enabling a biorefinery approach to be applied. Downstream processing optimization for sustainable economic biostimulant production must involve the use of microalgal wet paste instead of dry biomass. The present study investigated the effect of different parameters, such as the solvent, temperature, and time, on the extraction of biostimulant molecules from Scenedesmus almeriensis microalgal biomass. The extraction process optimization was determined by calculating the germination index in the watercress seed bioassays. Since phenols and other bioactives are extracted together, and purification should be avoided, biological methods are preferred for assessing biostimulation. Water was compared to organic solvents of lower polarity such as acetone, ethanol (96%), and an ethanol:hexane:water (76:18:6 v/v/v) mixture. Different solvent extraction ratios (0.5–8 mL solvent g−1 of dry biomass), temperatures (25–50 ºC), and extraction times (0.5–6 h) were tested, after which the best combination was selected for each solvent. The optimal conditions were obtained with organic green solvents (acetone or ethanol), which resulted in a Germination Index above 120% (at least 20% above the distilled water control). Consequently, from a biorefinery perspective, this process was considered to be the most suitable for microalgal biomass exploitation. Graphical Abstract

The nutrient content of a commercial seawater culture medium for growing the microalga Isochrysis galbana was optimized using a stochastic strategy based on genetic algorithms. For this, 210 experiments spread over seven generations were carried out. This strategy reduced the number of assays by more than 90% compared to a factorial design involving the optimization of twelve nutrients simultaneously. The optimized medium outperformed the reference medium in all aspects. The genetic algorithm strategy achieved a polyunsaturated fatty acids (PUFAs) productivity of 7.8 mg L−1 day−1 in a continuous culture of I. galbana, corresponding to an increase of 15% compared to the commercial formulation. Carotenoids, on the other hand, increased by 50% d.w. In addition, PUFA yields were significantly improved, which allowed us to reduce the requirement of several nutrients, for instance, N (25%), Mo (20%), Mn (60%), Co (60%), and Cu (60%).

There is growing interest in finding microalgae species that efficiently convert dissolved nutrients contained in aquaculture effluents into highly valuable biomass. The different nitrogen forms that are present in aquaculture effluents are particularly concerning. This study demonstrated that the dinoflagellate Amphidinium carterae can acclimate to both combined and sole nitrogen sources such as nitrate, ammonium, and urea over a wide concentration range. As far as is known, it is the first time that a species of the genus Amphidinium has been successfully cultured with urea as the sole source of nitrogen. In the presence of 882 μM of nitrate, A. carterae tolerated urea concentrations up to 5000 μM. With respect to ammonium-N tolerance, it has been observed that it is lethal at concentrations higher than 441 μM. A robust laboratory experimental design was critical for accurately assessing this acclimation. Alternative N sources did not affect the production of high-value specific polyketide secondary metabolites from A. carterae, such as amphidinols, with an average concentration of 0.435 ± 0.038% biomass d.w. An analysis of the symbiotic microbial assemblages developed in a long-term A. carterae culture in an open raceway pond, and the fact that it is able to metabolize all three nitrogen sources simultaneously, supports the idea that this microalga has the potential to be successfully cultured with aquaculture effluents.

There is broad scientific evidence on the health benefits of omega-3 polyunsaturated fatty acids (n-3 PUFAs), especially docosahexaenoic acid (DHA, 22:6n3) and eicosapentaenoic acid (EPA, 20:5n3). On the other hand, polar lipids (phospholipids, PLs, and glycolipids, GLs) are excellent emulsifying agents, making them useful both as food products and as excipients for drugs and cosmetics. The bioavailability of n-3 PUFAs in the form of PLs is greater than that of ethyl esters and triacylglycerols. This work has developed an easy method for the simultaneous extraction and fractionation of saponifiable lipids (SLs) from the microalga Nannochloropsis sp., using low-toxicity solvents. Firstly, a lipidic fraction very rich in neutral saponifiable lipids (NSLs) was obtained using hexane. Next, an EPA and polar lipids (PLs and GLs)-enriched fraction was obtained using ethanol (96%). Under optimal conditions, in the first extraction with hexane, an SL extract with 86.3% NSLs was obtained; this extract contained 88.9% of the biomass NSLs. Subsequently, in the second extraction step with ethanol (96%), an SL extract with 87.1% polar lipids and up to 35.2% EPA was obtained. This SL extract contained 87.1% of the polar lipids and 74.7% of the EPA from the Nannochloropsis sp. biomass.

The purpose of this study was to develop an economical agricultural fertilizer-based medium for the mass production of Isochrysis galbana Parke, a marine microalga producing docosahexaenoic acid (DHA) and considered an excellent feed for use in aquaculture. Different fertilizers were used to replace the main nutrients, nitrogen and phosphorus, and the micronutrients of the well-known commercial medium, ALGAL. The obtained results showed that I. galbana preferred nitrate, instead of ammonium, as the nitrogen source and phosphate as the phosphorus source. Nonetheless, a nitrate concentration above 4 mM inhibited cell growth. The alternative medium proposed achieved a biomass productivity 22% higher than that for the ALGAL medium, reducing the medium’s contribution to the overall biomass cost by over 95%, with protein, lipid, carbohydrate, and DHA contents at 44, 36, 25, and 1.47% of dry weight, respectively, thus providing a cheaper biomass with excellent nutritional value for use in aquaculture.

Current research indicates that n-3 polyunsaturated fatty acids (PUFAs) bind to polar lipids (phospholipids and glycolipids) seem to exert a greater bioavailability compared to their neutral forms. The aim of this work was to obtain eicosapentaenoic acid (EPA) rich polar lipids from the saponifiable lipids (SLs) extracted from the microalga Nannochloropsis sp. (33.4 ± 0.1% of EPA; 60 ± 0.6% polar lipids) by fractionation using silica-gel columns and importantly, non-polar and polar (ethanol) non-toxic solvents. Nowadays, few studies have been conducted towards the extraction and purification of polar lipids. Firstly, the solvent type for obtaining the neutral saponifiable lipid (NSL) fraction (ethyl acetate, EA, butyl acetate, BA) and the SL/silica-gel, SL/BA, and SL/ethanol ratios were optimized in a small silica-gel cartridge (0.69 g silica gel). The optimized conditions were an SL/silica-gel ratio of 22.6 mg/g, an SL/BA ratio of 1.56 mg/mL and an SL/ethanol ratio of 0.312 mg/mL. Next, the fractionation scale was increased to a column containing 10 g of silica-gel. At this scale, a BA SL fraction was obtained with 96.2 ± 0.5% of NSLs, and an ethanol SL fraction containing 97.7 ± 0.3% of polar lipids and 44.9 ± 0.2% of EPA. In the ethanol fraction, 86.6 ± 0.2% of the polar lipids and 71.5 ± 0.4% of the EPA from the SL microalgal extract were recovered. Consequently, EPA-rich polar lipids were obtained at high yields and purities, which could be used as a source of n-3 PUFAs with greater bioavailability than those based on neutral lipids.

Microalgae are potential sources of high-value lipids and colorants for use in foods, cosmetics, and other applications. Biomass and metabolite productivities of photoautotrophic algae cultures are low because of limited availability of light. Therefore, mixotrophic cultures were investigated in parallel with photoautotrophic controls. In mixotrophy, some of the energy and carbon are supplied in the form of dissolved organic substrates in addition to inorganic carbon and light being available. The aim was to compare productivities of biomass, fatty acids, and carotenoid pigments in outdoor and indoor mixotrophic and photoautotrophic batch and continuous cultures. The edible and safe marine microalga Nannochloropsis gaditana was used in these studies. The alga could be grown mixotrophically using glucose and glycerol, but not acetate. Optimal concentrations of the organic carbon sources were 5 g L−1 for glucose and 1 g L−1 for glycerol. Mixotrophy substantially increased the biomass concentration and productivity relative to photoautotrophy. The maximum biomass productivity in mixotrophic batch cultures using glucose or glycerol was identical at 170 mg L−1 day−1, being 30% greater than control cultures. In continuous outdoor culture with glucose (5 g L−1) mixotrophy at 12 °C, the total carotenoids in the biomass were 83% higher compared to photoautotrophic control biomass, and the eicosapentaenoic acid (EPA) productivity was 2.2-fold higher relative to controls. The maximum EPA productivity was 11 mg L−1 day−1. Glucose mixotrophy increased the total lipids content in the biomass by 34% relative to photoautotrophic operation.

Since the infection strategy in the baculovirus-insect cell system mostly affects production of the vector itself or the target product, and given that individual infection parameters interact with each other, the optimal combination must be established for each such specific system. In this work an artificial neural network was used to model infection strategy, including the cell concentration at infection, the multiplicity of infection, the medium recycle, and agitation intensity, and to evaluate the relative importance of each factor in the baculovirus production obtained. The results demonstrate that this model can be used to select an optimal infection strategy. For the baculovirus-insect cell system used in this study, this includes low multiplicity of infection and agitation intensity, along with high cell concentration at infection and medium recycle. Our model is superior to regression methods and predicts baculovirus production more precisely, thus meaning that it could be useful for the development of feasible processes, thereby improving process performance and economy.

Background: Microalgae are attracting much attention as a promising feedstock for renewable energy production, while simultaneously providing environmental benefits. So far, comparison studies for microalgae selection for this purpose were mainly based on data obtained from batch cultures, where the lipid content and the growth rate were the main selection parameters. The present study evaluates the performance of native microalgae strains in semi-continuous mode, considering the suitability of the algal-derived fatty acid composition and the saponifiable lipid productivity as selection criteria for microalgal fuel production. Evaluation of the photosynthetic performance and the robustness of the selected strain under outdoor conditions was conducted to assess its capability to grow and tolerate harsh environmental growth conditions. Results: In this study, five native microalgae strains from Tunisia (one freshwater and four marine strains) were isolated and evaluated as potential raw material to produce biofuel. Firstly, molecular identification of the strains was performed. Then, experiments in semi-continuous mode at different dilution rates were carried out. The local microalgae strains were characterized in terms of biomass and lipid productivity, in addition to protein content, and fatty acid profile, content and productivity. The marine strain Chlorella sp. showed, at 0.20 1/day dilution rate, lipid and biomass productivities of 35.10 mg/L day and 0.2 g/L day, respectively. Moreover, data from chlorophyll fluorescence measurements demonstrated the robustness of this strain as it tolerated extreme outdoor conditions including high (38 ° C) and low (10 ° C) temperature, and high irradiance (1600 µmol/m2 s). Conclusions: Selection of native microalgae allows identifying potential strains suitable for use in the production of biofuels. The selected strain Chlorella sp. demonstrated adequate performance to be scaled up to outdoor conditions. Although experiments were performed at laboratory conditions, the methodology used in this paper allows a robust evaluation of microalgae strains for potential market applications. This study was supported by the Marine Microalgae Biotechnology Group at the University of Almer'a (BIO 173) and the Campus de Excelencia Internacional Agroalimentario (ceiA3) within the joint framework of supervised theses between the University of Almeria, Spain and the University of Sfax, Tunisia. Scopus

A solid understanding of the effect of hydrodynamic forces encountered by microalgae in bioprocesses would benefit existing bioprocesses, eventually allowing an increase in their productivity. For this purpose, a sensitive method able to quantify cell lysis is crucial. Most of the available protocols and methods intended for this purpose were developed for animal or insect cells. In the case of microalgae, the commercial kits tested were unable to determine the cell lysis extension. The method proposed here was developed to relate the release of a cytoplasmic component (enzyme lactate dehydrogenase (LDH)) with cell lysis by measuring the NADH (reduced form of nicotinamide cofactor adenine dinucleotide) produced by LDH. Although different commercial kits based on similar processes are available, they are more complicated to use and not applicable to microalgae nor when longer-term tests are to be performed.

The aim of this paper is to solve problems that may arise in microalgae cultivation facilities at a hatchery in such a way that its activities are not disrupted. Thus, the effect of storage conditions (time, initial biomass concentration, presence or absence of light, addition of preservatives and conservation atmospheres) on cell viability, evolution of sample concentration, contamination, fatty acid and pigment content for long-term preservation of Nannochloropsis gaditana biomass has been studied. The polyunsaturated fatty acid content was maintained and the eicosapentaenoic acid (EPA, 20:5n3) content was kept stable at 3.6 % (d.w.). The total pigments content was also preserved at 0.85 % (d.w.), what allowed maintaining the antioxidant activity of samples. The results show that samples preserved both as concentrated inocula ready to run a photobioreactor, at an initial biomass (N. gaditana) concentration of 5 g L−1, and as pastes to be used as live food if any incident happens in a hatchery, at 150 g L−1, can be stored for up to 4 months at 4 °C under nitrogen or air atmosphere of conservation maintaining the cell viability and the sample concentration stable and at low incident irradiance (35 μmol photons m−2 s−1). This allows the photosynthetic activity of cells to be preserved in the case of samples at 5 g L−1. The bacterial load was kept at low level by the regular addition of preservative agents.

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).

In this paper, the biomass and lutein productivity of the lutein-rich new strain Scenedesmus almeriensis is modelled versus irradiance and temperature. The results demonstrate that S. almeriensis is a mesophile microorganism with an optimal growth temperature of 35 degrees C, and capable of withstanding up to 48 degrees C, which caused culture death. This strain is also tolerant to high irradiances, showing no signs of photoinhibition even at the maximum irradiance essayed of 1625 microE m(-2) s(-1) accumulating up to 0.55% dry weight (d.wt.) of lutein. The optimal conditions that maximise the biomass productivity also favour the lutein productivity, lutein being a primary metabolite. Maximal biomass and lutein productivities of 0.87 g l(-1) day(-1) and 4.77 mg l(-1) day(-1), respectively, were measured. The analysis of light availability inside the cultures, quantified as average irradiance, demonstrates that the cultures were mainly photo-limited, although photosaturation also took place at high external irradiances. The effect of temperature was also investigated finding that the specific maximal growth rate is modified by the temperature according to the Arrhenius equation. The influence of both light availability and temperature was included in an overall growth model, which showed, as a result, capable of fitting the whole set of experimental data. An overall lutein accumulation rate model was also proposed and used in a regression analysis. Simulations performed using the proposed models show that under outdoor conditions a biomass productivity of 0.95 g l(-1) day(-1) can be expected, with a lutein productivity up to 5.31 mg l(-1) day(-1). These models may be useful to assist the design and operation optimisation of outdoor cultures of this strain.

An integrated process for the indoor production of 13C labelled PUFA from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor from which the exhaust gas from the reactor is returned to the culture by a low pressure compressor. To avoid accumulation of dissolved oxygen in the culture medium, the exhaust gas is bubbled through a sodium sulphite solution before returning it to the reactor. Carbon is removed from the medium before inoculating the alga, then labelled 13CO2 is injected for pH control and carbon supply. The reactor has been operated in semicontinuous mode at a dilution rate of 0.01 h−1, a biomass productivity of 0.1 g L−1 d−1 being obtained. Under this conditions both pH and dissolved oxygen were correctly controlled and the adequacy of the system for autotrophic production of labelled biomass was demonstrated. Analysis by GC-MS revealed that the fatty acids content of the biomass obtained was 10% d.wt., the content of eicosapentaenoic acid was 2.5% d.wt. All the fatty acids were labelled, more that 90% of the carbon present in these fatty acids was 13C. Element analysis of biomass and supernatant showed that 59.5% of injected carbon was assimilated into the biomass whereas 33% remained in the supernatant, and 7.5% remained undetected. Due to the high cost of 13CO2 different strategies for the optimisation of labelled carbon use are proposed.

Outdoor chemostat cultures of the marine microalgaIsochrysis galbana at constant dilution rate (0.034 h−1 ) have been carried out under different weather conditions. Steady-state biomass concentrations were 1.61±0.03 kg m−3 in May and 0.95±0.04 kg m−3 in July, resulting in biomass output rates of 0.54 kg m−3 d−1 and 0.32 kg m−3 d−1 in May and July, respectively. Two patterns of daily variation with the solar cycle were observed in the fatty acid content. Saturated and mono-unsaturated fatty acids (16:0 and 16:1n7) show significant variation with the solar cycle, associated with short-term changes in environmental factors. Palmitic and palmitoleic acids are generated during daylight and consumed during the dark period. However, polyunsaturated fatty acids do not show a significant response to the solar cycle and their changes are associated with long-term variation in environmental factors. The maximum EPA productivity was obtained in May, 14.1 g m−3 d−1, which is close to that found in the literature for indoor continuous cultures. Nonetheless, the outdoor EPA content (up to 2.61 % d.wt) was lower than the indoor EPA content from a previous study (5% d.wt).

Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid productivities from chemostat cultures of an isolate of Isochrysis galbana have been studied. The productivities reached in the interval of dilution rates between 0.0295 h−1 and 0.0355 h−1 were 1.5mg·1−1·h−1 for lipids, 300 μg·1−1·h−1 for EPA and 130μg1·1−1·h−1 for DHA. Furthermore, light attenuation by mutual shading, and agitation speed influences on growth and fatty acid composition were analysed. A model relating steady-state dilution rates to internal average light intensity has been proposed, the parameter values of which obtained by non-linear regression were: maximum specific growth rate (μmax)=0.0426 h−1; the affinity of cells to light (Ik) = 10.92 W·m−2; the exponent (n) = 5.13; regression coefficient (r2)=0.9999.