Long-lasting biofouling formation on transparent fouling-release coatings for the construction of efficient closed photobioreactors.

In order to build an efficient closed-photobioreactor (PBR) in which biofouling formation is avoided, a non-toxic coating with high transparency is required, which can be applied to the interior surface of the PBR walls. Nowadays, amphiphilic copolymers are being used to inhibit microorganism adhesion, so poly(dimethylsiloxane)-based coatings mixed with poly(ethylene glycol)-based copolymers could be a good option. The 7 poly(dimethylsiloxane)-based coatings tested in this work contained 4% w/w of poly(ethylene glycol)-based copolymers. All were a good alternative to glass because they presented lower cell adhesion. However, the DBE-311 copolymer proved the best option due to its very low cell adhesion and high transmittance. Furthermore, XDLVO theory indicates that these coatings should have no cell adhesion at time 0 since they create a very high-energy barrier that microalgae cells cannot overcome. Nevertheless, this theory also shows that their surface properties change over time, making cell adhesion possible on all coatings after 8 months of immersion. The theory is useful in explaining the interaction forces between the surface and microalgae cells at any moment in time, but it should be complemented with models to predict the conditioning film formation and the contribution of the PBR's fluid dynamics over time.

Bioactives Overproduction through Operational Strategies in the Ichthyotoxic Microalga Heterosigma akashiwo Culture.

The red tide-forming microalga Heterosigma akashiwo has been associated with massive events of fish deaths, both wild and cultured. Culture conditions are responsible for the synthesis or accumulation of some metabolites with different interesting bioactivities. H. akashiwo LC269919 strain was grown in a 10 L bubble column photobioreactor artificially illuminated with multi-coloured LED lights. Growth and production of exopolysaccharides, polyunsaturated fatty acids (PUFAs), and carotenoids were evaluated under different culture modes (batch, fed-batch, semicontinuous, and continuous) at two irradiance levels (300 and 700 µE·s-1·m-2). Continuous mode at the dilution rate of 0.2·day-1 and 700 µE·s-1·m-2 provided the highest production of biomass, PUFAs (132.6 and 2.3 mg·L-1·day-1), and maximum fucoxanthin productivity (0.16 mg·L-1·day-1). The fed-batch mode accumulated exopolysaccharides in a concentration (1.02 g·L-1) 10-fold over the batch mode. An extraction process based on a sequential gradient partition with water and four water-immiscible organic solvents allowed the isolation of bioactive fucoxanthin from methanolic extracts of H. akashiwo. Metabolites present in H. akashiwo, fucoxanthin and polar lipids (i.e., eicosapentaenoic acid (EPA)), or probably such as phytosterol (ß-Sitosterol) from other microalgae, were responsible for the antitumor activity obtained.

Adsorption Analysis of Exopolymeric Substances as a Tool for the Materials Selection of Photobioreactors Manufacture.

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.

The Isolation of Specialty Compounds from Amphidinium carterae Biomass by Two-Step Solid-Phase and Liquid-Liquid Extraction.

The two main methods for partitioning crude methanolic extract from Amphidinium carterae biomass were compared. The objective was to obtain three enriched fractions containing amphidinols (APDs), carotenoids, and fatty acids. Since the most valuable bioproducts are APDs, their recovery was the principal goal. The first method consisted of a solid-phase extraction (SPE) in reverse phase that, for the first time, was optimized to fractionate organic methanolic extracts from Amphidinium carterae biomass using reverse-phase C18 as the adsorbent. The second method consisted of a two-step liquid-liquid extraction coupled with SPE and, alternatively, with solvent partitioning. The SPE method allowed the recovery of the biologically-active fraction (containing the APDs) by eluting with methanol (MeOH): water (H2O) (80:20 v/v). Alternatively, an APD purification strategy using solvent partitioning proved to be a better approach for providing APDs in a clear-cut way. When using n-butanol, APDs were obtained at a 70% concentration (w/w), whereas for the SPE method, the most concentrated fraction was only 18% (w/w). For the other fractions (carotenoids and fatty acids), a two-step liquid-liquid extraction (LLE) method coupled with the solvent partitioning method presented the best results.

Effect of Nitrogen, Phosphorous, and Light Colimitation on Amphidinol Production and Growth in the Marine Dinoflagellate Microalga Amphidinium carterae.

The marine dinoflagellate microalga Amphidinium carterae is a source of amphidinols, a fascinating group of polyketide metabolites potentially useful in drug design. However, Amphidinium carterae grows slowly and produces these toxins in tiny amounts, representing a hurdle for large-scale production. Understanding dinoflagellate growth kinetics under different photobioreactor conditions is imperative for promoting the successful implementation of a full-scale integrated bioproduct production system. This study evaluates the feasibility of growing Amphidinium carterae under different ranges of nitrogen concentration (NO3- = 882-2646 µM), phosphorus concentration (PO33- = 181-529 µM), and light intensity (Y0 = 286-573 µE m-2 s-1) to produce amphidinols. A mathematical colimitation kinetic model based on the "cell quota" concept is developed to predict both algal growth and nutrient drawdown, assuming that all three variables (nitrogen, phosphorous and light) can simultaneously colimit microalgal growth. The model was applied to the semicontinuous culture of the marine microalgae Amphidinium carterae in an indoor LED-lit raceway photobioreactor. The results show that both growth and amphidinol production strongly depend on nutrient concentrations and light intensity. Nonetheless, it was possible to increase Amphidinium carterae growth while simultaneously promoting the overproduction of amphidinols. The proposed model adequately describes Amphidinium carterae growth, nitrate and phosphate concentrations, and intracellular nitrogen and phosphorus storage, and has therefore the potential to be extended to other systems used in dinoflagellate cultivation and the production of bioproducts obtained therein.

Influence of abiotic conditions on the biofouling formation of flagellated microalgae culture.

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-1·m-2) at 23 °C. Cell adhesion was greater in C. reinhardtii and a higher biomass concentration was obtained for this strain, reaching 2 g·L-1 compared to 1 g·L-1 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.

Production of Amphidinols and Other Bioproducts of Interest by the Marine Microalga Amphidinium carterae Unraveled by Nuclear Magnetic Resonance Metabolomics Approach Coupled to Multivariate Data Analysis.

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.

Preparative Recovery of Carotenoids from Microalgal Biomass.

Carotenoids are widespread substances with important physiological roles, and some of them, such as lutein, astaxanthin, or vaucherioxanthin, are high-value products that can be used as high-quality food color and antioxidants, and some have an alleged role in the prevention of disorders such as AMD. Carotenoid extracts are currently obtained from plant sources, but microalgae have been demonstrated to be a competitive source likely to become an alternative. The extraction of carotenoids from microalgae possesses specific problems that arise from the different structure and composition of the source biomass. Here is presented a method for the recovery of carotenoid extracts from microalgal biomass in the kilogram scale.

Biofouling in photobioreactors for marine microalgae.

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.

Simultaneous effect of temperature and irradiance on growth and okadaic acid production from the marine dinoflagellate Prorocentrum belizeanum.

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I0). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·(m-2)·s(-1)), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µ(m)) was 0.204 day(-1) at 25 °C and 40 µE·(m-2)·s(-1). Photo-inhibition was observed at I0 > 40 µEm(-2)s(-1), leading to culture death at 120 µE·m(-2)·s(-1) and 28 °C. Cells at I0 ≥ 80 µE·m(-2)·s(-1) were photoinhibited irrespective of the temperature assayed. A mechanistic model for µ(m)-I0 curves and another empirical model for relating µ(m)-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I0-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors.

Stability of carotenoids in Scenedesmus almeriensis biomass and extracts under various storage conditions.

Scenedesmus almeriensis biomass is a source of carotenoids, particularly lutein, and is considered to be promising as an alternative source to marigold. One key question concerning alternative sources of lutein is the loss of carotenoids that takes place between harvesting and processing, which in the case of marigold is frequently up to 50%. The work described here involved a study into the stability of the main carotenoids (lutein, violaxanthin, and beta-carotene), as well as other components, under different storage conditions. The experiments were carried out with biomass in three forms: frozen, freeze-dried, and spray-dried. The stability of extracts of Scenedesmus biomass in acetone and olive oil was also studied. The results show that the most important factor in retaining carotenoids is a low temperature. At -18 degrees C the loss of carotenoids was negligible after the storage period, regardless of the biomass form used (frozen, freeze-dried, or spray-dried). On the other hand, the carotenoid content and fatty acid profile was increasingly affected with increasing temperature. However, the protein content is unaffected by storage conditions.