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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.

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 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.

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.

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.

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.

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.

In this paper, we have studied the outdoor production of the freshwater microalgae Scenedesmus sp. in two open reactors (32 m2): thin-layer (1.2 m3) and raceway (4.4 m3), using centrate from anaerobic digestion as the sole nutrient source. The aim was to recover valuable nutrients (nitrogen and phosphorus) from effluents in order to maximize biomass productivity. Experiments were performed in semicontinuous mode, modifying the centrate percentage within the culture medium. The optimal centrate percentage was 30% — above this value the culture's performance reduced, probably due to ammonium excess (above 122 mg l− 1). Using the raceway reactor, biomass productivity was 24 g m− 2 day− 1, whereas using the thin-layer reactor, it increased up to 42 g m− 2 day− 1. Nitrogen and phosphorus removal was demonstrated to be proportional to biomass productivity, with maximal values up to 38 mgN l− 1 day− 1 and 3.9 mgP l− 1 day− 1 being removed, respectively. Ammonium stripping was only relevant in the raceway reactor, due to its lower biomass productivity, with more than 40% of the inlet nitrogen being lost to the air. The thin-layer reactor also proved to be more photosynthetically efficient, with maximal values of 4.7% being measured. An economic analysis demonstrated that the thin-layer reactor allowed a reduction in the biomass production cost, in addition to utilizing waste as the nutrient source, with a minimum production cost of 0.9 €/kgbiomass being estimated. In conclusion, it is possible to use centrate from anaerobic digestion as the sole nutrient source for the large-scale production of Scenedesmus sp. biomass thus reducing the biomass production cost by avoiding the use of expensive and non-sustainable fertilizers; while also obtaining returns from the treatment of this type of residue. Such a combination helps to increase the possibility of producing commodities, or biofuels, from microalgae by coupling their production to treatment processes.

The effect of culture conditions on the productivity of biomass and high value products by microalgae has been studied in order to maximise these parameters and meet the nutritional requirements for aquaculture. To this end, a range of temperatures (15–30 °C), incident photon flux densities (PFD) (250–1600 μmol·s − 1 ·m − 2 ) and dilution rates (0.1–0.5 d − 1 ) have been tested in indoor Nannochloropsis gaditana cultures. Predictive models for biomass and high value product productivities are presented. Photoinhibition was observed in cultures at both high incident PFD and temperature. The maximum biomass, EPA, vaucheriaxanthin and β-carotene productivities (0.429 g·L − 1 ·d − 1 and 17.2, 1.05 and 1.31 mg·L − 1 ·d − 1 , respectively) were achieved at 25 °C, with an average irradiance of 170 μmol·s − 1 ·m − 2 and a dilution rate of 0.3 d − 1 .

In this paper freshwater microalgae production is studied using centrate from anaerobic digestion as the only nutrient source. The objective is not only to maximize biomass productivity but also to eliminate contaminants in order to release water which is clean enough for reuse in compliance with regulations. Experiments were performed indoors using two microalgae strains, Muriellopsis sp. and Pseudokirchneriella subcapitata, to establish the optimal centrate percentages within the culture medium. Results demonstrated that with 40–50% of centrate in the culture medium, productivity reached values of up to 1.13 and 1.02 g·l− 1·day− 1 for Muriellopsis sp. and P. subcapitata, respectively. In addition, the removal of nitrogen and phosphorous contained in the culture medium exceeded 90%, the COD at the outlet being lower than 100 mg·l− 1. Above 50% of centrate, toxicity exists and culture performance decreases. No nitrification was observed as the ammonium was completely consumed at centrate percentages lower than 20%. Muriellopsis sp. demonstrated itself to be the more robust strain tolerating higher ammonium concentrations as well as achieving a higher quantum yield, of up to 0.50 g·E− 1, and a higher nitrogen removal rate, of up to 47.5 mgN·l− 1·day− 1 and 3.8 mgP·l− 1·day− 1. Consequently, it is possible to use centrate from anaerobic digestion as the sole nutrient source for the production of freshwater microalgae, thus reducing the biomass production cost by avoiding the use of expensive and non-sustainable fertilizers; not to mention the returns obtained from treating this type of residue. This combination can help to increase the possibilities of producing biofuels from microalgae by means of coupling their production to other treatment processes.

In the present work, a study on the performance, culture parameters and modeling of the growth and lipid productivity of Nannochloropsis gaditana in outdoor pilot-scale raceway ponds is made. During the experiments, modifications to the dilution rate and nutrient supply were performed, with maximum productivities of 0.19 gBIOMASS/l day and 30.4 mgLIPIDS/l day obtained at 0.30–0.36 1/day and 10.5 mM NO3−. The impact of average irradiance within the culture (in the range of 280–350 μE/m2 s) and the specific nitrate input (SNI, below 0.4 mmol N/g day) was correlated with an enhancement of saturated and monounsaturated fatty acid contents, which attained maximum values of 29.5% and 33.0% of total FAs, respectively. The modeling of growth, lipid and fatty acid production rates was developed taking into account the irradiance, dilution rate and SNI, which might prove useful in the scaling-up of raceway ponds with this species.

A study has been conducted to assess clarification efficiency and cell damage during centrifugation, and to optimize this operation for the dinoflagellate microalga Amphidinium carterae. Although cells were easily recovered from the cell suspension, cell damage was observed in some experiments once the cells had sedimented. Cell damage depends on both the residence time of the cells in the pellet and on the g-force applied. 2D Computer fluid dynamics simulations were carried out to simulate and predict microalgal cell settling times, and a dimensionless number was used to obtain an operating window (combinations of g-force and centrifugation time) for optimal centrifugation of the microalga. The approach used in this study can be extrapolated to other cells and other centrifuges.