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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.

This work analyses the adhesion of flagellated microalgae to seven surfaces that have different water adhesion tension characteristics. Chlamydomonas reinhardtii and Isochrysis galbana, were cultivated in batch and fed-batch mode at four nitrogen/phosphorus (N/P) ratios (from 1.29 to 70) and subjected to four irradiance levels (50, 100, 200 and 400 µE·s·m) at 23 °C. Cell adhesion was greater in C. reinhardtii and a higher biomass concentration was obtained for this strain, reaching 2 g·L compared to 1 g·L for I. galbana. The adhesion of cells and exopolymeric substances was measured upon the batch and the first fed-batch reaching the stationary growth phase, observing a direct correlation between them and inversely to biomass generation in the cultures. The protein adhesion data for the different materials are comparable to those for cell adhesion coinciding with minimums of Baier's theory and Vogler. It is observed displacements in the curves as a function of the irradiance level., This work was supported by the Spanish Ministry of Science and Innovation grant RTI2018-101891-B-100, and by the European Regional Development Fund Program

An improved method that allows the robust characterization of surfaces is necessary to accurately predict the biofouling formation on construction materials of photobioreactors (PBR). Exopolymeric substances (EPS), such as proteins and polysaccharides, have been demonstrated to present a similar behavior to cells in terms of surface adhesion. In this work, these EPS were used to optimize parameters, such as EPS concentration or adsorption time, to evaluate accurately the adsorption capacity of surfaces and, with it, predict the biofouling formation in contact with microalgae cultures. Once the method was optimized, the characterization of seven commercial polymeric surfaces was submitted to different abrasive particles sizes, which modified the roughness of the samples, as well as protein and polysaccharide lawns, which were prepared and carried out in order to evaluate the characteristics of these substances. The characterization consisted of the determination of surface free energy, water adhesion tension, and critical tension determined from the measurement of the contact angle, roughness, surface zeta potential, and the EPS adhesion capacity of each material. This will be useful to understand the behavior of the surface in the function of its characteristics and the interaction with the solutions of EPS, concluding that the hydrophobic and smooth surfaces present good anti-biofouling characteristics., This work was supported by the Spanish Ministry of Science and Innovation grant RTI2018-101891-B-100, and by the European Regional Development Fund Program.

This study assessed the feasibility of an NMR metabolomics approach coupled to multivariate data analysis to monitor the naturally present or stresses-elicited metabolites from a long-term (>170 days) culture of the dinoflagellate marine microalgae Amphidinium carterae grown in a fiberglass paddlewheel-driven raceway photobioreactor. Metabolic contents, in particular, in two members of the amphidinol family, amphidinol A and its 7-sulfate derivative amphidinol B (referred as APDs), and other compounds of interest (fatty acids, carotenoids, oxylipins, etc.) were evaluated by altering concentration levels of the f/2 medium nutrients and daily mean irradiance. Operating with a 24 h sinusoidal light cycle allowed a 3-fold increase in APD production, which was also detected by an increase in hemolytic activity of the methanolic extract of A. carterae biomass. The presence of APDs was consistent with the antitumoral activity measured in the methanolic extracts of the biomass. Increased daily irradiance was accompanied by a general decrease in pigments and an increase in SFAs (saturated fatty acids), MUFAs (monounsaturated fatty acids), and DHA (docosahexaenoic acid), while increased nutrient availability lead to an increase in sugar, amino acid, and PUFA ω-3 contents and pigments and a decrease in SFAs and MUFAs. NMR-based metabolomics is shown to be a fast and suitable method to accompany the production of APD and bioactive compounds without the need of tedious isolation methods and bioassays. The two APD compounds were chemically identified by spectroscopic NMR and spectrometric ESI-IT MS (electrospray ionization ion trap mass spectrometry) and ESI-TOF MS (ESI time-of-flight mass spectrometry) methods.

Sustainable dinoflagellate microalgae-based bioprocess designed to produce secondary metabolites (SMs) with interesting bioactivities are attracting increasing attention. However, dinoflagellates also produce other valuable bioproducts (e.g polyunsaturated fatty acids, carotenoids, etc.) that could be recovered and should therefore be taken into account in the bioprocess. In this study, biomass of the marine dinoflagellate microalga Amphidinium carterae was used to assess and optimise three different methods in order to obtain three families of high-value biochemical compounds present in the biomass. The existing processes encompassed a multi-step extraction process for carotenoids, fatty acids and APDs individually and are optimized for the integral valorization of raw A. carterae biomass, with SMs being the primary target compounds. Total process recovery yields were 97% for carotenoids, 80% for total fatty acids and 100% for an extract rich in APDs (not purified).

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.

The biofouling formation of the marine microalga Nannochloropsis gaditana on nontoxic surfaces was quantified on rigid materials, both coated (with fouling release coatings and nanoparticle coatings) and noncoated, to cover a wide range of surface properties from strongly hydrophobic to markedly hydrophilic under conditions similar to those prevailing in outdoor massive cultures of marine microalgae. The effect of seawater on surfaces that presented the best antibiofouling properties was also evaluated. The adhesion intensity on the different surfaces was compared with the predictions of the biocompatibility theories developed by Baier and Vogler using water adhesion tension (τ0 ) as the quantitative parameter of surface wettability. For the most hydrophobic surfaces, τ0 ≤ 0, the microalgae adhesion density increased linearly with τ0 , following the Baier's theory trend. However, for the rest of the surfaces, τ0 ≥ 0, a tendency toward minimum adhesion was observed for amphiphilic surfaces with a τ0 = 36 mJ/m2 , a value close to that which minimizes cell adhesion according to Vogler's theory. The understanding and combination of the two biocompatibility theories could help to design universal antibiofouling surfaces that minimize the van der Waals forces and prevent foulant adsorption by using a thin layer of hydration.

The prevention of biofouling in the inner walls of closed photobioreactors (PBRs) becomes a critical step in improving the performance of photosynthetic microalgae bioprocesses. Selection of antifouling materials implies approaches at laboratory scale. This work reports the use of a flow cell of the modified Robbins device (MRD) type coupled to a PBR, operated in both batch and continuous modes with the model marine microalgae Nannochloropsis gaditana to study the biofouling formation on diverse materials. The fluid-dynamics within the MRD was studied via CFD-aided simulations. At separation distances lower than the cells' diffusion layer thickness, a diffusion-controlled transport of the cells to the material surface was postulated. Results suggested that the flow density of cells in the MRD (Jz), governed by cell concentration gradients, is a significant factor in the adhesion intensity (B) when the PBR is operated in batch mode; not in the continuous mode where the differences observed in B between materials were mainly attributed to the type of material. Polyvinylchloride (PVC) was clearly the best anti-biofouling material compared to polycarbonate, polystyrene, borosilicate glass and stainless steel. The B maximum occurred at the end of the stationary phase in batch culture mode. Continuous culture operation seemed to be preferable since once steady state is achieved, the B value remained low and constant, indicating equilibrium between the number of adhered cells per surface unit and the cell concentration in the culture broth – this was because the adhered cells did not grow on the surface due to phosphate limitation. The PBR-coupled MRD has demonstrated to be well-suited for the screening of antifouling materials under fluid-dynamic conditions relevant in PBRs.

Animal production inevitably causes the emission of greenhouse gases and the generation of large amounts of slurry, both representing a serious environmental problem. Photosynthetic microorganisms such as microalgae and cyanobacteria have been proposed as alternative strategies to bioremediate agricultural waste while consuming carbon dioxide and producing valuable biomass. The current study assessed the potential of the microalga Scenedesmus sp. to remove nutrients from piggery wastewater (PWW) and the influence of the microalga on the microbial consortia. Maximum N-NH4+ consumption was 55.3 &plusmn, 3.7 mg&middot, L&minus, 1&middot, day&minus, 1 while P-PO43&minus, removal rates were in the range 0.1&ndash, 1.9 mg&middot, 1. N-NH4+ removal was partially caused by the action of nitrifying bacteria, which led to the production of N-NO3&minus, N-NO3&minus, production values where lower when microalgae were more active. This work demonstrated that the photosynthetic activity of microalgae allows us to increase nutrient removal rates from PWW and to reduce the coliform bacterial load of the effluent, minimising both their environmental impact and health risks. Microalgae assimilated part of the N-NH4+ present in the media to produce biomass and did not to convert it into N-NO3&minus, as in traditional processes.

Adhesion of microalgal cells to photobioreactor walls reduces productivity resulting in significant economic losses. The physico-chemical surface properties and the fluid dynamics present in the photobioreactor during cultivation are relevant. However, to date, no multiphysical model has been able to predict biofouling formation in these systems. In this work, to model the microalgal adhesion, a Computational Fluid Dynamic simulation was performed using a Eulerian-Lagrangian particle-tracking model. The adhesion criterion was based on the balance of forces and moments included in the XDLVO model. A cell suspension of the marine microalga Nannochloropsis gaditana was fed into a commercial flow cell composed of poly-methyl-methacrylate coupons for validation. Overall, the simulated adhesion criterion qualitatively predicted the initial distribution of adhered cells on the coupons. In conclusion, the combined Computational Fluid Dynamics-Discrete Phase Model (CFD-DPM) approach can be used to overcome the challenge of predicting microalgal cell adhesion in photobioreactors.

Primary urban wastewater was treated in outdoor raceways using microalgae-bacteria consortia dominated by Scenedesmus almeriensis. The current study aimed at assessing the effect of operational conditions, namely, culture depth and dilution rate, on the following: (i) biomass productivity, (ii) the nutrient removal capacity and (iii) the composition of the microalgae-bacteria consortium and the presence of unwanted microorganisms. Optimum dilution rates to process large quantities of wastewater during summer and achieve high biomass productivities were 0.3–0.5 day−1. Under the optimum operational conditions, nitrogen and phosphorus removal rates were higher than 90% while removal of chemical oxygen demand was 70%. Operating at different culture depths had a striking effect on the composition of the microalgae-bacteria consortium. The relative abundance of nitrifiers increased with culture depth and was minimised at 0.05 m: larger culture depths led to enhanced nitrifying activity and therefore to nitrate production and accumulation in the system. Results demonstrate the potential of microalgae-based wastewater treatment processes and the importance of selecting suitable operational conditions to maximise both, biomass production and nutrient removal by minimising the occurrence of nitrifying bacteria.

Photo-respirometric tecniques are applied for evaluating photosynthetic activity in phototrophic organisms. These methods allow to evaluate photosynthetic response under different conditions. In this work, the influence of nutrient availability (nitrate, ammonium, and phosphate) on the photosynthesis and respiration of Scenedesmus almeriensis was studied using short photo-respirometric measurements. Both photosynthesis and respiration increasing until saturation value and consecutively diminishing, presenting inhibition by high concentrations. Regarding the influence of phosphorus concentration in microalgae cells, a similar hyperbolic trend was observed but no inhibition was observed at high concentration. Based on these experimental data, the respiration, and the photosynthesis rate of S. almeriensis were modelled using Haldane equation for nitrate and ammonium data, and Monod equation for phosphate data. In addition, experiments were performed to determine the yield coefficients for both nitrogen and phosphorus in S. almeriensis cultures. The data showed that the nitrogen and phosphorous coefficient yields are not constant, being modified as a function of nutrients concentration, presenting the luxury uptake phenomena. Finally, the proposed models were incorporated into a simulation tool to evaluate the photosynthetic activity and the nutrient yield coefficients of S. almeriensis when different culture media and wastewaters are used as a nitrogen and phosphorous source for its growth.Key points• Microalgal photosynthesis/respiration vary as a function of nutrients availability.• Photosynthesis inhibition appears at high N-NO3-and N-NH4+concentrations.• Nutrient yield coefficients are influenced by luxury uptake phenomenon. Graphical abstract

Biofouling represents an important limitation in photobioreactor cultures. The biofouling propensity of different materials (polystyrene, borosilicate glass, polymethyl methacrylate and polyethylene terephthalate glycol-modified) and coatings (two spray-applied and nanoparticle-based superhydrophobic coatings and a hydrogel-based fouling release coating) was evaluated by means of a short-term protein test, using bovine serum albumin (BSA) as a model protein, and by the long-term culture of the marine microalga Nannochloropsis gaditana under practical conditions. The results from both methods were similar, confirming that the BSA test predicts microalgal biofouling on surfaces exposed to microalgae cultures; these secrete macromolecules, such as proteins, that have a high capacity for forming a conditioning film prior to cell adhesion. The hydrogel-based coating showed significantly reduced BSA and N. gaditana adhesion, whereas the other surfaces failed to control biofouling. Microalgal biofouling was associated with an increased concentration of sticky extracellular proteins at low N/P ratios (below 15).

Extraction of carotenoids and fatty acids from microalgae is a technological bottleneck in processing. An improved extraction process was developed to scale the production of these bioproducts from Nannochloropsis gaditana. Different cell disruption methods were evaluated in terms of carotenoids release. Ethanol was substituted by isopropyl alcohol in a three-component solution of water:isopropyl alcohol:hexane (WIH), in which the extracts were separated by solution partitioning. This resulted in higher carotenoid and fatty acid recovery yields if compared to the standard method. The extraction method was replicated on a pilot scale obtaining similar carotenoid recovery yields, higher than those of the standard method. Although fatty acid recovery was lower than that of the small-scale tests, yields above 85% were obtained. This demonstrated that the method was scalable for the extraction of high- value products from microalgae up to 10 L reactor volume. The use of isopropyl alcohol, which is cheaper than ethanol, and the separation of the solution phases by partitioning (avoiding drying) could contribute to reduce operation costs of downstream processing.

Microalgae are becoming an important source of high-value products such as biostimulants, biopesticides, and other low-value products such as biofertilizers or aquafeed (fatty acids and carotenoids), among others. However, extracting the biomolecules contained in the microalgae cells is difficult due to the structure and composition of the microalgal cell wall. To overcome this difficulty, mechanical cell disruption methods, such as high-pressure homogenization (HPH), can be applied to facilitate the extraction of the compounds of interest. This work focuses on optimizing the extraction of biostimulants from microalgae biomass and obtaining amino acid concentrates that can be used in several applications. The research aims to study the effect of HPH pretreatment on the production of these different bioproducts using Scenedesmus sp. biomass grown in pig sludge as the raw material. For each product, the HPH pretreatment of the wet biomass must be optimized to select the optimal conditions and the number of passes. Soft treatment of the wet biomass at 200 bar is sufficient to observe a 10% increase in the germination index (GI) of watercress seeds, while greater treatment intensities reduce the GI with respect to the untreated biomass, possibly because this increases the concentration of the molecules responsible for the bioactivity, thus causing a toxic effect on the seeds. In the case of the amino acid concentrates, better results were obtained at 600 bar, achieving a hydrolysis level of 72.9%. Lastly, the concentrations of the phenolic compounds and the salicylic acid, related with enhanced biostimulant activity, were determined using spectrophotometric methods and High-Performance Liquid Chromatography- diode array (HPLC), respectively.

In this article, the influence of culture conditions (irradiance, temperature, pH, and dissolved oxygen) on the photosynthesis and the respiration rates of microalgae–bacteria consortia in wastewater treatment was analyzed. Specifically, some short photo-respirometric experiments, simulating outdoor raceway reactors, were performed to evaluate the response of microalgae, heterotrophic bacteria, and nitrifying bacteria to variations in environmental parameters. Results demonstrate that irradiance is the most dominant variable to determine microalgae photosynthesis rates. However, reduction in microalgae activity was not observed at higher irradiance, ruling out the existence of photoinhibition phenomena. Related to heterotrophic and nitrifying bacteria, their activities were strongly affected by the influence of temperature and pH. Moreover, the effect of dissolved oxygen concentrations on microalgae, and bacteria activities was studied, displaying a reduced photosynthetic rate at dissolved oxygen concentrations above 20 mg/L. Data have been used to develop an integrated model for each population (microalgae, heterotrophic bacteria, and nitrifying bacteria) based on considering the simultaneous influence of irradiance, temperature, pH, and dissolved oxygen. The models fit the experimental results in the range of culture conditions tested, and they were validated using data obtained by the simultaneous modifications of the variables. These individual models serve as a basis for developing a global biologic microalgae–bacteria model for wastewater treatment to improve the optimal design and management of microalgae-based processes, especially outdoors, where the cultures are subject to variable daily culture conditions.

In this chapter, the most relevant factors determining the selection, design, and performance of microalgae production systems are analyzed. The starting point is to define the final purpose of the facility, because this determines the overall design of the process. Next, the requirements of the selected microalgae strains are discussed, because these determine the requirements to be satisfied by the photobioreactor such as light, temperature, pH/CO2 and dissolved oxygen concentration. The precise design of the reactor must be completed on the basis of these requirements. It includes the geometry of the reactor, the fluid-dynamic and power consumption/mixing, the mass transfer, and the heat transfer capacity of the reactor. Also on the basis of this design, adequate control systems must be developed to adjust the capacity of the reactor to provide adequate culture conditions to the requirements of the cells, such as CO2 supply and dissolved oxygen removal modify along the daylight period also modifying along the annual cycle due to additional temperature variations at different seasons. Fundamentals of design and operation of photobioreactors for large-scale production of microalgae are summarized. Additionally, the most relevant reactor designs, such as raceway, thin-layer, and tubular reactors are reviewed, the major aspects of the design of these reactors being analyzed to determine the reliability of large scale industrial reactors.

Production of phytoplankton (microalgae and cyanobacteria) in commercial raceway ponds and other systems is adversely impacted by phytoplankton pathogens, including bacteria, fungi and viruses. In addition, cultures are susceptible to productivity loss, or crash, through grazing by contaminating zooplankton such as protozoa, rotifers and copepods. Productivity loss and product contamination are also caused by otherwise innocuous invading phytoplankton that consume resources in competition with the species being cultured. This review is focused on phytoplankton competitors, pathogens and grazers of significance in commercial culture of microalgae and cyanobacteria. Detection and identification of these biological contaminants are discussed. Operational protocols for minimizing contamination, and methods of managing it, are discussed.

In this study, the outdoor production of marine microalgae in raceway photobioreactors was investigated, modifying the centrate percentage in the culture medium (20, 30, 40 and 50%) and using two different dilution rates (0.2 day−1 and 0.3 day−1). The data obtained showed that microalgae are capable of producing biomass in addition to recovering the nutrients contained in the centrate. The best results for biomass productivity and light efficiency were obtained when the centrate was set at 20% with a dilution rate of 0.3 day−1. The biomass productivity was 32.42 g m−2·day−1 while the photosynthetic efficiency was 0.74 gbiomass·E−1 (3.66%). Regarding the nutrients, nitrogen (the majority being in the form of ammonium [NH4+]) and phosphorus were only fixed into biomass when optimal conditions were set; if this was not the case, they were lost to stripping or precipitation. The maximal nutrient removal capacities under the optimal conditions were 28.72 mgN·l−1·day−1 and 3.99 mgP·l−1·day−1. Population changes were determined by the dilution rate set whilst the centrate percentage had little effect. Four strains were present in the culture, Nannochloropsis g. being the main one. Biochemical changes did not vary greatly between the conditions set for the culture, with a composition rich in proteins and carbohydrates being observed. One can conclude that to produce marine microalgal biomass for a range of potential commodities such as feed, biofertilizers and biofuels, it is possible to use centrate from anaerobic digestion as the sole nutrient source, as a way of reducing costs.

The production of fatty acid methyl esters (FAMEs, biodiesel) was optimized from wet Nannochloropsis gaditana microalgal biomass (81.8 wt% water and 28.1 wt% saponifiable lipids, SLs, from the dry biomass). FAME production was carried out by direct acid-catalyzed methylation of the SLs from the microalgal biomass and FAME extraction with hexane. A three-variable, three-level Box-Behnken design (BBD) was applied to optimize the sulphuric acid (catalyst) concentration, the methanol/SL ratio and the hexane/SL ratio. The best FAME yield was obtained with a methanol/SL ratio of 254 mL/g, a sulphuric acid concentration of 3.7% (v/v) and a hexane/SL ratio of 107.7 mL/g at 100 °C and for 105 min. Under these conditions, 100% of the SLs were transformed into FAMEs. Finally, the FAME purity was increased from 78.7 to 86.8 wt% using an adsorption treatment with bentonite.

In this paper, worldwide research trends in the microalgae field are analyzed based on a bibliometric study. We have reviewed the number of publications and their distribution, as well as the most relevant journals and keywords, to determine the evolution and latest tendencies in this field. The results confirm that this is a fast-growing area in terms of the number of publications. The most relevant journals on this subject are Bioresource Technology and Algal Research. Although the majority of papers come out of the USA, the institutions with larger number of publications are actually located in China, France and Spain. The most frequently cited strains are Chlorella and Chlamydomonas. The main keywords that appear in over 1000 articles are generally related to microalgae cultivation applications such as ‘biomass, biofuel, and lipids’ while others are related to the methodology; for instance, ‘bioreactor’. Of all the keywords, ‘biomass’ stands out, as it appears in almost 20% of publications. Bibliographic analysis confirms that Microalgae Biotechnology is a very active field, where scientific productivity has exponentially increased over recent years in tandem with industrial production. Therefore, expectations are high in this field for the near future.

We present the data corresponding to the isolation and morphological and molecular characterization of a strain of Amphidinium carterae, isolated in Mallorca Island waters and now deposited in the microalgae culture collection of the Plant Biology and Ecology Department of the University of the Basque Country under the reference Dn241Ehu. The morphological characterization was made using two different techniques of microscopy and the molecular characterization by using the 28S rDNA sequences of D1 and D2 domains. This strain has been used for a culture study in an indoor LED-lighted pilot-scale raceway to determine its production of carotenoids and fatty acids, "Long-term culture of the marine dinoflagellate microalga Amphidinium carterae in an indoor LED-lighted raceway photobioreactor: Production of carotenoids and fatty acids." (Molina-Miras et al., 2018) [1]. (C) 2018 The Authors. Published by Elsevier Inc. This research was funded by the Spanish Ministry of Economy and Competitiveness (CTQ2014-55888-C3-02), the European Regional Development Fund Program, and the projects PPG17/67 from the UPV/EHU and IT1040-16 from the Basque Government.

Microalgae are an interesting source of natural pigments that have valuable applications. However, further research is necessary to develop processes that allow us to achieve high levels of carotenoid recovery while avoiding degradation. This work presents a comprehensive study on the recovery of carotenoids from several microalgae genera, optimizing carotenoid extraction using alkaline saponification at various temperatures and KOH concentrations. Results show that I. galbana requires a temperature of 60 °C and10% KOH, N. gaditana and K. veneficum require 60 °C and no saponification, P. reticulatum requires 40 °C and 10% KOH, T. suecica and H. pluvialis require 25 °C and 40% KOH while C. sp. and S. almeriensis require 80 °C and 40% KOH. The influence of the solvent on carotenoid recovery was also studied. In general terms, an ethanol:hexane:water (77:17:6 v/v/v) mixture results in good yields.

A pilot-scale bioprocess was developed for the production of karlotoxin-enriched extracts of the marine algal dinoflagellate Karlodinium veneficum. A bubble column and a flat-panel photobioreactors (80–281 L) were used for comparative assessment of growth. Flow hydrodynamics and energy dissipation rates (EDR) in the bioreactors were characterized through robust computational fluid dynamic simulations. All cultures were conducted monoseptically outdoors. Bubble column (maximum cell productivity in semicontinuous operation of 58 × 103 cell mL−1 day−1) proved to be a better culture system for this alga. In both reactors, the local EDR near the headspace, and in the sparger zone, were more than one order of magnitude higher than the average value in the whole reactor (=4 × 10−3 W kg−1). Extraction of the culture and further purification resulted in the desired KTXs extracts. Apparently, the alga produced three congeners KTXs: KmTx-10 and its sulfated derivative (sulfo-KmTx-10) and KmTx-12. All congeners possessed hemolytic activity.

Marine dinoflagellate microalgae belonging to the genus Amphidinium are a key source of an interesting group of polyketide metabolites with potent bioactivities, wide-ranging functional diversity and stereochemical complexity, but low natural availabilities. The feasibility of a microalgae dinoflagellate-based sustainable bioprocess for producing amphidinols (APDNs) by photoautotrophic culture of Amphidinium carterae in a pilot-scale LED-illuminated bubble column photobioreactor (PBR) was therefore investigated. A fed-batch culture mode with pulse feeding strategy provided a growth pattern strongly limited by the availability of phosphate content in the culture medium that stimulated the production of cellular APDNs. Since A. carterae was found to be much more shear-sensitive than other shear-tolerant non-dinoflagellate microalgae, the culture height and air flow rate were established to ensure the absence of damaging levels of hydrodynamic stress. The biomass capacity yielded by the PBR at the end of the culture (0.540 g d.w. L−1 equivalent to 1.70 × 106 cell mL−1) corresponded to that estimated stoichiometrically from the experimentally determined biomass P-molar formula (C329 O126 H732 N69 S3 P1) and the total phosphorus and nitrogen balances. The downstream processing section was initially conceived to recover APDNs excreted by cells into the supernatant. A dry APDN-enriched extract concentration of 49 mg per liter of supernatant was obtained. This purification process led to partitioning of the extract into several fractions and sub-fractions thereof. Only two sub-fractions were studied, yielding thereof highly pure (>95% pure) luteophanol D and lingshuiol A, and a new, roughly purified (>80% pure) APDN, namely amphidinol 20. The percentages of luteophanol D, lingshuiol A and amphidinol 20 by dry weight of the APDN-enriched extract obtained were 1%, 0.39% and 0.31%, respectively, thus representing a concentration in the culture supernatant of 490, 191 and 152 μg L−1, respectively.

A study has been conducted to assess cell damage during centrifugation and to optimize this operation for the insect cell line Se301. Experiments were carried out in a discontinuous centrifuge using Falcon tubes of different sizes as containers. The cells were easily recovered from the cell suspension and the time required for complete sedimentation was found to be independent of tube size or design. Cell damage was observed once the cells were sedimented, therefore this process depends on both the residence time of the cells in the pellet and on the acceleration applied. The sensitivity of the cells to this operation was higher than for other naked microalgae or for very sensitive red blood cells. Indeed, excessive treatment produced cell damage that reduced the productivity of subsequent cultures. CFD simulations were carried out in 2D and a good agreement was found between the CFD and experimental values in terms of the time required for full sedimentation. In addition, this technique allowed the calculation of shear stress, a key variable in the study of cell sensitivity to flow.

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.

The economic and/or energetic feasibility of processes based on using microalgae biomass requires an efficient cultivation system. In photobioreactors (PBRs), the adhesion of microalgae to the transparent PBR surfaces leads to biofouling and reduces the solar radiation penetrating the PBR. Light reduction within the PBR decreases biomass productivity and, therefore, the photosynthetic efficiency of the cultivation system. Additionally, PBR biofouling leads to a series of further undesirable events including changes in cell pigmentation, culture degradation, and contamination by invasive microorganisms; all of which can result in the cultivation process having to be stopped. Designing PBR surfaces with proper materials, functional groups or surface coatings, to prevent microalgal adhesion is essential for solving the biofouling problem. Such a significant advance in microalgal biotechnology would enable extended operational periods at high productivity and reduce maintenance costs. In this paper, we review the few systematic studies performed so far and applied the existing thermodynamic and colloidal theories for microbial biofouling formation in order to understand microalgal adhesion on PBR surfaces and the microalgae-microalgae cell interactions. Their relationship to the physicochemical properties of the solid PBR surface, the microalgae cell surfaces, and the ionic strength of the culture medium is discussed. The suitability and the applicability of such theories are reviewed. To this end, an example of biofouling formation on a commercial glass surface is presented for the marine microalgae Nannochloropsis gaditana. It highlights the adhesion dynamics and the inaccuracies of the process and the need for further refinement of previous theories so as to apply them to flowing systems, such as is the case for PBRs used to culture microalgae.

This paper addresses a complete set of procedures to study and to better understand the emerging problem of the biofouling formation in photobioreactors (PBRs). Methodologies are described in detail for: (i) PBR and microalgae mat surface preparation, (ii) contact angles (CA) and zeta potential (ZP) measurements for both microalgal cells and PBR surfaces, and (iii) microscopic methods for studying the evolution of adhesion intensity. The impact that these methodologies may have on the photosynthetic apparatus of the cells, the biomass concentration and cell viability are also quantified. A lab-scale flat-plate PBR was used to perform a long-term batch culture of the marine microalgae Nannochloropsis gaditana , in which a devised rack of 25 PBR glass surfaces were submerged. To study the cell-to-cell and cell-to-PBR surface interactions, the existing surface thermodynamics and colloidal theories (XDLVO) were used. The major outcomes were: (1) that N. gaditana has a exposure time threshold to the electric field produced by the ZP meter; (2) a linear equation is provided for predicting the PBR surface potential as a function of the culture medium's ionic strength; (3) the biofouling growth curve on the PBR surface varied in line with the growth kinetic followed by the freely suspended culture cells; (4) the devised PBR slide rack system offers a versatile experimental platform for generating biofouling results, making it suitable for the in situ efficiency evaluation of antibiofouling coatings; (5) there was a significant variation in the surface free energy of the PBR surfaces and algal mats with respect to that present at the beginning of the culture, and, consequently, the application of thermodynamic theories failed to predict cell adhesion over long-term cultivation. However, the XDLVO model satisfactorily explained the dynamics of the adhesion studied. The reported results might be useful for research in the microalgal production and PBR engineering area.

This study assessed the feasibility of an NMR metabolomics approach coupled to multivariate data analysis to monitor the naturally present or stresses-elicited metabolites from a long-term (170 days) culture of the dinoflagellate marine microalgae

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

In this chapter, a techno-economic analysis of the production of microalgae for different applications is presented. First, a costs estimation methodology previously validated is shown. Next, a market analysis of different microalgae applications is performed, and from this it is concluded that agriculture- and aquaculture-related applications today are highly interesting. The cost analysis methodology is then used to determine the production cost of microalgae for these markets in different scenarios. A case study considering a 5-ha facility using open reactors, raceway, and thin-layer cascade is analyzed in depth. Results confirm that microalgae biomass can be produced for these applications at 4.5 and 2.3 €/kg, using raceway and thin-layer cascade, respectively. To reduce the production cost, it is necessary to use wastewaters as a nutrient source; this enables the biomass production cost to reduce to 3.6 and 1.4 €/kg for raceway and thin-layer cascade, respectively. Further reduction requires a substantial decrease in manpower for the operation of the process. By reducing up to 0.1 person/ha, the biomass production cost can be reduced to 2.6 and 0.8 €/kg for raceway and thin-layer cascade, respectively. On the other hand, when analyzing the process as a wastewater treatment process, the treatment cost can be reduced to minimum values of 1.1 and 1.2 €/m3 for raceway and thin-layer cascade, respectively. These costs are higher than those of conventional systems, of 0.2 €/m3. However, if including the service of wastewater treatment cost as an input for the production of biomass, the final biomass production cost can be reduced to minimum values of 2.1 and 0.6 €/kg using raceway and thin-layer cascade, respectively. Thus, techno-economic analysis is confirmed as a powerful tool to perform the optimization of any process, also allowing us to identify the target markets in which the biomass can be used, thus contributing to the expansion of microalgae-based processes.

The massive outdoor culture of microalgae rarely reaches biomass concentrations higher than 1 g/L. The dewatering step is known to be a significant barrier in the large-scale production of microalgae-based commodities and bioproducts. High cost and energy consumption, along with low effectiveness, are still key constraints for commonly used dewatering operations. The potential use of forward osmosis as a membrane-based technology for algae dewatering employing osmotic pressure instead of external hydraulic pressure has several advantages such as low energy use and remarkable algae cell recovery. Nevertheless, the main drawbacks of forward osmosis are membrane fouling, concentration polarization phenomena, and reverse solutes diffusion. A lot of research is being carried out to overcome these limitations by developing new membranes and optimizing the process design. This article aims to review the main studies performed to date on microalgae biomass dewatering with the focus on forward osmosis and critical challenges, particularly fouling phenomenon. The forward osmosis performance evaluation, with respect to water flux behavior and microalgae dewatering efficiency, has been discussed in detail as an integral part of this review. When one appraises the achievements of the research carried out up until now, it becomes clear that we are still far from developing forward osmosis for microalgae dewatering at the industrial scale, and novel solutions are needed to enhance its technological readiness level. Some of the remaining challenges include: improving module design and arrangements to mitigate concentration polarization, lab-scale evaluation of different available draw solutions in relation to the properties and composition of the feed solution (i.e. the pH, microalgae species, etc.) for good dewatering process design, the membrane cleaning method and the pilot-scale validation of forward osmosis for dewatering microalgae, all of which are necessary to evaluate the economic and methodological feasibility of the process in a real setting.

The aim of this work was to optimize the production of fatty acid methyl ester (FAME, biodiesel) from wet Nannchloropsis gaditana microalgal biomass by direct enzymatic transesterification. This was done in order to avoid the high cost associated with the prior steps of drying and oil extraction. Saponifiable lipids (SLs) from microalgal biomass were transformed to FAME using the lipase Novozyme 435 (N435) from Candida antarctica as the catalyst, and finally the FAME were extracted with hexane. t-Butanol was used as the reaction medium so as to decrease lipase deactivation and increase mass transfer velocity. A FAME conversion of 99.5% was achieved using wet microalgal biomass homogenized at 140 MPa to enhance cell disruption, a N435:oil mass ratio of 0.32, methanol added in 3 stages to achieve a total of 4.6 cm 3 g −1 of oil and 7.1 cm 3 g −1 oil of added t-butanol, with a reaction time of 56 h. The FAME conversion decreased to 57% after catalyzing three reactions with the same lipase batch. This work shows the influence of the polar lipids contained in the microalgal biomass both on the reaction velocity and on lipase activity.

This work is the last of a series of three papers. The previous ones were performed in tubular photobioreactors and raceways. In the present work, the outdoor continuous production of Nannochloropsis gaditana in flat-panel reactors is studied over a two-year period, analysing the influence of the dilution rate imposed in addition to the nitrate and phosphate supplied. Maximum biomass and lipid yields of 0.19 g/l day and 38.0 mg/l day were obtained when operating at a dilution rate of 0.35 1/day and concentrations of 10.0 mM NO 3 − and 0.8 mM PO 4 3 − in the culture medium. By decreasing the specific nitrate input (below 2 mmol N/g day), and exposing the culture to moderate light availability and temperature, the volumetric productivity of saturated and monounsaturated fatty acids increased to 10.0 mg/l day. The specific nitrate input, together with other important parameters such as the average irradiance, temperature and dilution rate were used to develop a model describing this species' behaviour. The model is a powerful tool for further improvement and scaling-up of this technology.

A feed-forward back-propagation neural network (FBN) was used as a tool for predicting the growth dynamics of the microalga Karlodinium veneficum in a culture medium with any specified concentrations of the key nutrients. A 3-layered FBN configuration of 27-25-1 nodes was used. This FBN satisfactorily represented the nonlinear interactions among all the nutrients of a culture medium containing up to 25 different components. The FBN model was trained using the growth dynamics data from more than 420 batch culture experiments involving different media compositions. The relative impact of individual nutrients, the initial cell concentration and the culture duration on growth profiles were determined through a systematic analysis of the partitioning of the FBN connection weights. Microelements and vitamins together had a higher relative impact on growth compared to the impact of the macronutrients. The trained FBN successfully predicted the cell concentration dynamics in cultures with previously untested initial conditions. The FBN proved to be an excellent tool for predicting the growth curves in the range of culture conditions that were relevant to this study.

In this paper, a new photo-respirometry method for determining the rates of the main metabolic processes of microalgae-bacteria consortia in microalgae-based wastewater treatment processes has been developed and tested. The proposed protocol consists on applying dark and light periods to a microalgae-bacteria consortium in the presence of different substrates and measuring the rate of oxygen production. This allows determining the activity of microalgae, heterotrophic bacteria and nitrifying bacteria separately. The method has been optimized in terms of the operation strategy, including the starvation period required, the biomass concentration and the irradiance during the measurements. Results show that a starvation period of one to three days is necessary depending on the nutrient concentration. The optimal experimental conditions determined were a biomass concentration of 0.5 g/L and an irradiance of 200 μmol photons/m2·s. Furthermore, microalgae-bacteria samples from seven photobioreactors (indoor and outdoor) with different nutrient sources have been evaluated applying the methodology proposed. Regardless of the wastewater type, the microalgae activity is the main metabolic process, with heterotrophic activity increasing along with the chemical oxygen demand (COD) in the wastewater. Nitrifying activity was only observed when high ammonium concentrations were present. The developed method is a powerful tool to adequately manage and operate wastewater treatment processes using microalgae/bacteria consortia, providing valuable information to model wastewater treatment systems with microalgae and determine kinetic parameters.

Amphidinium carterae is a marine dinoflagellate microalga that produces high-value bioactive polyketides such as amphidinols and amphidinolides, which have remarkable cytotoxic, antifungal and antitumoral activities. This species also produces docosahexaenoic acid and the carotenoid peridinin as high-value byproducts. The development of any sustainable microalgae-based bioprocess must comprise complete valorization of the biomass and reuse of the exhausted culture medium as much as possible to comply with the circular economy concept. In this work we have assessed the effect of recycling the cell-free supernatant on the growth kinetics and production of amphidinols by A. carterae photoautotrophically cultured in a pilot-scale raceway-type photobioreactor in semi-continuous mode. Acclimation to the recycled medium was first studied on a laboratory scale and was accomplished in the fourth sub-culture, except for pigment content, which decreased throughout the process. No measurably negative effect was detected in the cells. Maximum growth rates and cell productivities increased by 60% with use of 75% spent medium, as a result of mixotrophic consumption of the organic carbon present in that medium, remaining constant thereafter. In the photobioreactor, acclimation took 77 days and was also accomplished in the fourth sub-culture, with no negative effects on cell health. Biomass and cell productivities were higher with the recycled medium than with the fresh medium. Pigments decreased throughout the acclimation process, probably due to organic carbon-based mixotrophic growth. Amphidinols and docosahexaenoic acid biomass contents were not affected by medium recycling, which will significantly improve the economics of amphidinols production from A. carterae cultures on a circular economy basis.

Saponifiable lipids (SLs), rich in eicosapentaenoic acid (EPA), were extracted and purified from Nannochloropsis gaditana biomass. Firstly, the SLs were extracted using ethanol (96% v/v). The influence of the following operational variables was studied: water content of the wet biomass, solvent/biomass ratio, homogenization pressure and temperature. SL yields of 92–99 wt% and 80–90 wt% were achieved from dry and wet biomass, respectively, under various operational conditions from batches with different SL contents and lipid profiles. These SLs were extracted with only a 20–22 wt% purity, which was increased to 42% (100 wt% yield) by extracting the SLs with hexane. In this hexane extraction step, it was important to extract the SLs from a highly concentrated hydroethanolic solution. The SL purity was further increased to 95 wt% by acetone crystallization. Throughout this extraction-purification process, SLs were fractionated into neutral lipids, glycolipids and phospholipids - neutral lipids were recovered with higher yields than were glycolipids and phospholipids. The energy consumption per unit mass of extracted lipids was estimated, along with the solvent recovery, and compared for the dry and wet methods. When wet biomass was used, the presence of water significantly increased the energy required for ethanol. However, when cost and capacity of the main equipment (freeze-dryer, homogenizer and centrifuge) were considered, SL extraction from high pressure homogenized wet biomass resulted in a lower economic cost.