Long-term biofouling formation mediated by extracellular proteins in Nannochloropsis gaditana microalga cultures at different medium N/P ratios.

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 whose cells secrete macromolecules, such as proteins, with a high capacity for forming a conditioning film before 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).

CFD-based prediction of initial microalgal adhesion to solid surfaces using force balances.

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.

Growth performance, body composition, and digestive functionality of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles fed diets including microalgae freeze-dried biomass.

Senegalese sole is one of the most promising fish species cultivated in the Southern European countries. This study was aimed at assessing the effects of microalgae biomass added to diets for Senegalese sole juveniles on fish growing and condition status. Three isoproteic (52%) and isolipidic (10%) were formulated containing 15% Tisochrysis lutea (TISO), Nannochloropsis gaditana (NAN), or Scenedesmus almeriensis (SCE) biomass, respectively. An experimental microalgae-free diet (CT) and a commercial diet (COM) were used as controls. Fish were fed at 3% of their body weight for 85 days. Final body weight of fish fed microalgae-supplemented diets did not differ from group fed CT diet. Fish-fed CT, TISO, NAN, and SCE showed higher growth performance and nutrient utilization figures than specimen-fed COM diet. The highest carcass lipid content was found in COM group (141 g kg-1), and no differences were observed in body protein content. Ash was significantly higher in TISO, NAN, and SCE groups compared to fish-fed CT. Muscle EPA and DHA contents were not modified owing to the different dietary treatments. The n3/n6 and EPA/DHA ratios in muscle were similar in all the experimental groups. The quantification of digestive proteolytic activities did not differ among experimental groups, although differences in the protease pattern in digestive extracts by zymography were revealed in those fish fed on COM diet. Both α-amylase activity in the intestinal lumen and leucine aminopeptidase in the intestinal tissue were significantly lower in COM fish. Specimens fed on SCE diet showed a higher leucine aminopeptidase activity associated to the intestinal tissue compared to NAN-fed fish (0.40 and 0.25 U g tissue-1, respectively). The ultrastructural study revealed that the dietary inclusion of algal biomass, especially T. lutea and N. gaditana, had a positive impact on the absorptive capacity of the intestinal mucosa. The highest values for the parameters microvilli length and microvilli absorption surface were observed in fish fed on NAN diet (1.99 µm and 45.93 µm2, respectively). Even though further studies aimed at optimizing commercial formulas for Senegalese sole are required prior to any large-scale practical utilization, the results obtained clearly suggest the potential of microalgae as dietary ingredients for this fish species.

A quantitative study of eicosapentaenoic acid (EPA) production by Nannochloropsis gaditana for aquaculture as a function of dilution rate, temperature and average irradiance.

Different pilot-scale outdoor photobioreactors using medium recycling were operated in a greenhouse under different environmental conditions and the growth rates (0.1 to 0.5 day(-1)) obtained evaluated in order to compare them with traditional systems used in aquaculture. The annualized volumetric growth rate for Nannochloropsis gaditana was 0.26 g l(-1) day(-1) (peak 0.4 g l(-1) day(-1)) at 0.4 day(-1) in a 5-cm wide flat-panel bioreactor (FP-PBR). The biomass productivity achieved in this reactor was 10-fold higher than in traditional reactors, reaching values of 28 % and 45 % dry weight (d.w.) of lipids and proteins, respectively, with a 4.3 % (d.w.) content of eicosapentaenoic acid (EPA). A model for predicting EPA productivity from N. gaditana cultures that takes into account the existence of photolimitation and photoinhibition of growth under outdoor conditions is presented. The effect of temperature and average irradiance on EPA content is also studied. The maximum EPA productivity attained is 30 mg l(-1) day(-1).

Economically viable bioprocess for inclusion of microalga Nannochloropsis gaditana in aquaculture feeds: Evaluation of antioxidant addition in preventing lipid oxidation during storage.

The rising prices of fishery derivatives limits their use in aquafeeds. Therefore, other alternatives are used to replace those ingredients. Among them, microalgae are of great interest both as an ingredient and as a potential stabilising agent against lipid oxidation. This study evaluates on the use of Nannochloropsis gaditana to prevent lipid oxidation in a set of 12 aquafeeds over 540 days of storage. Aquafeeds were formulated with/without 15 % N. gaditana combined with two antioxidants -butylhydroxytoluene (25-150 mg·kg-1) or vitamin E (500-3000 mg·kg-1). The effect of i) storage period, ii) presence of microalgae and iii) antioxidant addition on lipid oxidation was assessed. Results showed higher fatty acid degradation in diets lacking microalgae. The microalgae supplemented diets is enough for preserving feeds presenting the highest antioxidant effect at the end, without significant differences with the microalgae-supplemented feeds and those including antioxidants after 540 days of storage.

A step forward in sustainable pesticide production from Amphidinium carterae biomass via photobioreactor cultivation with urea as a nitrogen source.

This study addresses the problem of replacing nitrate and ammonium with urea as a greener nitrogen source in the mass cultivation of the microalga Amphidinium carterae for the development of amphidinol-based phytosanitary products. To solve this problem, a nuclear magnetic resonance assisted investigation evaluated the effect of nitrogen sources on growth and metabolic profiles in photobioreactors. Urea-fed cultures exhibited growth kinetics comparable to nitrate-fed cultures (µmax = 0.30 day-1, Pbmax = 43 mgL-1day-1). Urea-fed cultures had protein, lipid, and carbohydrate contents of 39.5%, 14.5%, and 42.4%, respectively, while nitrate-fed cultures had 27.9 %, 17.5% and 48.1%, respectively. Metabolomics revealed nitrogen source-dependent metabotypes and a correlation between amphidinols and polyunsaturated fatty acids. The amphidinol-to-nitrogen yield coefficient in urea-fed cultures (135 mg/g) was approximately 2.5 times higher than in nitrate-fed cultures. The potent antiphytopathogenic activity exhibited by extracts from urea-fed cultures underscores the potential of urea as a sustainable nitrogen source in microalgae-based biorefineries.

Life-cycle assessment of a microalgae-based fungicide under a biorefinery approach.

The aim of this work was to perform a life-cycle analysis of the production process of a fungicide based on amphidinols. Two scenarios were evaluated: (1) biorefinery process -biofungicide, fatty acids and carotenoids were considered as co-products-, and (2) biofungicide as only product. Inventory data were taken and scaled-up from previous work on pilot-scale reactors, as well as lab-scale downstream equipment. A yearly production of 22,000 L of fungicide, was selected as the production objective. Despite, photosynthetic biomass is a sink of anthropogenic CO2, harvesting and downstream processing have large carbon footprints that exceed the biomass fixed carbon. Producing the biofungicide resulted in 34.61 and 271.33 ton of CO2e (15 years) for the Scenarios 1 and 2, respectively. Different commercial agricultural fungicides were compared with the microalgal fungicide. A lower impact of the microalgal product for most of the indicators, including carbon footprint, was shown.

Alternatives to classic solvents for the isolation of bioactive compounds from Chrysochromulina rotalis.

This paper demonstrates a sequential partitioning method for isolating bioactive compounds from Chrysochromulina rotalis using a polarity gradient, replacing classic and hazardous solvents with greener alternatives. Seventeen solvents were evaluated based on their Hansen solubility parameters and for having a similar polarity to the solvents they would replace, four of which were selected as substitutes in the classic fractionation process. Considering the fatty acid and carotenoid recovery yields obtained for each of the solvents, it has been proposed to replace hexane (HEX), toluene (TOL), dichloromethane (DCM) and n-butanol (BUT) with cyclohexane, chlorobenzene, isobutyl acetate and isoamyl alcohol, respectively. In addition, cytotoxic activity was observed when the TOL and DCM solvent extracts were tested against tumour cell lines, demonstrating the antiproliferative potential of compounds containing, for example, fucoxanthin, fatty acids, peptides, isoflavonoids or terpenes, among others.

Assessment of the marine microalga Chrysochromulina rotalis as bioactive feedstock cultured in an easy-to-deploy light-emitting-diode-based tubular photobioreactor.

Marine microalgae have potential to be low-cost raw materials. This depends on the exploitation of different biomass fractions for high-value products, including unique compounds. Chrysochromulina rotalis, an under-explored haptophyte with promising properties, was the focus of this study. For the first time, C. rotalis was successfully cultivated in an 80 L tubular photobioreactor, illuminated by an easy-to-use light-emitting-diode-based system. C. rotalis grew without certain trace elements and showed adaptability to different phosphorus sources, allowing a significant reduction in the N:P ratio without compromising biomass yield and productivity. The design features of the photobioreactor provided a protective environment that ensured consistent biomass production from this shear-sensitive microalgae. Carotenoid analysis showed fucoxanthin and its derivatives as major components, with essential fatty acids making up a significant proportion of the total. The study emphasizes the tubular photobioreactor's role in sustainable biomass production for biorefineries, with C. rotalis as a valuable bioactive feedstock.

Treatment of secondary urban wastewater with a low ammonium-tolerant marine microalga using zeolite-based adsorption.

The low tolerance of marine microalgae to ammonium and hyposalinity limits their use in urban wastewater (UWW) treatments. In this study, using the marine microalga Amphidinium carterae, it is demonstrated for the first time that this obstacle can be overcome by introducing a zeolite-based adsorption step to obtain a tolerable UWW stream. The maximum ammonium adsorption capacities measured in the natural zeolite used are among the highest reported. The microalga grows satisfactorily in mixtures of zeolite-treated UWW and seawater at a wide range of proportions, both with and without adjusting the salinity, as long as the ammonium concentration is below the threshold tolerated by the microalgae (6.3 mg L-1). A proof of concept performed in 10-L bubble column photobioreactors with different culture strategies, including medium recycling, showed an enhanced biomass yield relative to a control with no UWW. No noticeable effect was observed on the production of specialty metabolites.

Carboxymethyl cellulose and Pluronic F68 protect the dinoflagellate Protoceratium reticulatum against shear-associated damage.

The red-tide dinoflagellate Protoceratium reticulatum is shown to be protected against turbulence-associated damage by the use of the additives Pluronic F68 (PF68) and carboxymethyl cellulose (CMC) in the culture medium. Relative to agitated controls, these additives had a dose-dependent protective effect at concentrations of up to 0.4 and 0.5 g L(-1) for CMC and F68, respectively. In static cultures, these additives inhibited growth directly or indirectly at a concentration of >0.5 g L(-1). Compared to CMC, PF68 was a better protectant overall. Cell-specific production of yessotoxins was enhanced under elevated shear stress regimens so long as the turbulence intensity was insufficient to damage the cells outright. Shear-induced production of reactive oxygen species and direct effects of turbulence on the cell cycle contributed to the observed shear effects.

An integrated approach for the efficient separation of specialty compounds from biomass of the marine microalgae Amphidinium carterae.

An amphidinol-prioritized fractioning approach was for the first time developed to isolate multiple specialty metabolites such as amphidinols, carotenoids and fatty acids using the biomass of the marine microalgae Amphidinium carterae. The biomass was produced in a raceway photobioreactor and the exhausted culture media were reused, thus fulfilling sustainability criteria employing a circular economy concept. The integrated bioactive compounds-targeted approach presented here consisted of four steps with which recovery percentages of carotenoids, fatty acids and amphidinols of 97%, 82% and 99 %, respectively, were achieved. The proposed process was proved to be a better extraction system for this microalga than another based on a sequential gradient partition with water and four water-immiscible organic solvents (hexane, carbon tetrachloride, dichloromethane and n-butanol). The proposed process could be scaled-up as a commercial solid-phase extraction technology well-established for industrial bioprocesses.

Improved extraction of bioactive compounds from biomass of the marine dinoflagellate microalga Amphidinium carterae.

The extraction of three families of compounds (carotenoids, fatty acids and amphidinols) from the biomass of two strains of Amphidinium carterae (ACRN03 and Dn241EHU) was improved by tuning cell disruption and solvent extraction operations. The extraction of carotenoids was evaluated using alkaline saponification (0%-60% KOH d.w.) at different temperatures (25-80 °C). High levels of carotenoids were obtained at 60 °C using freeze-dried biomass, not subjected to cell disruption methods. The ACRN03 strain required 20% KOH whereas the Dn241EHU strain did not require saponification since carotenoid degradation was observed. The extraction efficiencies were determined with a wide range of pure solvents and mixtures thereof. Two empirical non-linear equations were used to correlate extraction percentages for each family of compounds with the Hildebrand solubility parameter (δT) and the polarity index of the solvents (PI). Thresholds of δT and PI of around 20 MPa1/2 and 6, respectively, were determined for the extraction of amphidinols, consistent with antiproliferative activity measurements.

Assessment of multi-step processes for an integral use of the biomass of the marine microalga Amphidinium carterae.

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).

Data on the Amphidinium carterae Dn241EHU isolation and morphological and molecular characterization.

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].

Long-term culture of the marine dinoflagellate microalga Amphidinium carterae in an indoor LED-lighted raceway photobioreactor: Production of carotenoids and fatty acids.

The feasibility of the long-term (>170 days) culture of a dinoflagellate microalga in a raceway photobioreactor is demonstrated for the first time. Amphidinium carterae was chosen for this study as it is producer of interesting high-value compounds. Repeated semicontinuous culture provided to be a robust operational mode. Different concentration levels of the f/2 medium nutrients (i.e. f/2×1-3) were assayed. The composition f/2×3 (N:P = 5), combined with a sinusoidal irradiance pattern (L/D = 24:0) with a 570 µE m-2 s-1 daily mean irradiance, maximized the biomass productivity (2.5 g m-2 day-1) and production rate of the valuable carotenoid peridinin (19.4 ±â€¯1.35 mg m-2 L-1 with nearly 1% of the biomass d.w.). Several carotenoids and polyunsaturated fatty acids were also present in significant percentages in the harvested biomass (EPA, 1.69 ±â€¯0.31% d.w.; DHA, 3.47 ±â€¯0.24% d.w.), which had an average P-molar formulate of C40.7O21.2H73.9N3.9S0.3P1.

Maximizing carotenoid extraction from microalgae used as food additives and determined by liquid chromatography (HPLC).

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 and <10% 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.

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640.

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.

Genetic algorithm for the medium optimization of the microalga Nannochloropsis gaditana cultured to aquaculture.

A genetic algorithm has been used to optimize the composition of the culture medium for growing the microalga Nannochloropsis gaditana, based on the nutrients composition of the commercial medium ALGAL. This strategy was carried out through the implementation of 270 experiments spread over nine generations, which allowed achieving an eicosapentaenoic acid (EPA) productivity of 17.8 m gL(-1) d(-1) in a continuous culture of N. gaditana, with an increase of 23% compared to the commercial medium. The EPA yield on nitrogen and phosphorous, 0.042 and 1.146 g(EPA) g(s)(-1), respectively, were 40% and 5-fold higher, respectively, than the values obtained with the nitrogen-optimized ALGAL medium. This improvement was obtained with the medium G-8, which also allowed reducing the requirement of several nutrients such as P, Mo, Mn in 74%, 69% and 66%, respectively, as well as the thiamine content a 46%.

Pilot-plant-scale outdoor mixotrophic cultures of Phaeodactylum tricornutum using glycerol in vertical bubble column and airlift photobioreactors: studies in fed-batch mode.

Pilot-scale (0.19 m column diameter, 2 m tall, and 60 L working volume) outdoor vertical bubble column (BC) and airlift photobioreactors (a split-cylinder (SC) and a draft-tube airlift device (DT)) were compared for fed-batch mixotrophic culture of the microalga Phaeodactylum tricornutum UTEX 640. The cultures were started photoautotrophically until the onset of a quasi-steady-state biomass concentration of 3.4 g L(-)(1). After this, the cultures were supplemented with organic nutrient (glycerol 0.1 M) and a reduced nitrogen source, resulting in an immediate growth rate boost, which was repeated with successive additions of nutrients in all three photobioreactors. During this period the biomass productivity was enhanced compared to photoautotrophic cultures in the three reactors, although differences were found among them. These could be attributed to the different hydrodynamic behavior influencing the transport phenomena inside the cultures. A 25.4 g L(-)(1) maximum biomass concentration was attained in the SC. Further additions of nutrients did not promote any more growth. The consumption of glycerol was quantitative in the first additions but slowed at high biomass concentration, suggesting that a minimum amount of light is needed to sustain growth. No significant effect of the supplied organic nutrient on carotenoids and chlorophylls content was observed, although it had a profound effect on the fatty acid composition. Eicosapentaenoic acid (EPA) content was increased up to 3% (DW) in mixotrophic growth, giving a productivity of 56 mg L(-)(1) d(-)(1), a significant increase compared to the photoautotrophic control, which yielded a maximum EPA content of 1.9% (DW) and a productivity of 18 mg L(-)(1) d(-)(1). The maximum biomass and EPA volumetric yields obtained in this work are comparable with those reported for commercial photoautotrophic monoculture of large quantities of P. tricornutum in closed continuous-run tubular loop bioreactors with tubes that are typically less than 0.08 m in diameter. When the comparison is established in terms of productivities based on the land area occupied, the vertical airlift and bubble-column bioreactors are extraordinarily more productive.