A. Sánchez Mirón

Carboxymethyl cellulose protects algal cells against hydrodynamic stress

The harmful effect of direct air sparging on Phaeodactylum tricornutum microalgal cultures was investigated in bubble columns and airlift photobioreactors with various superficial air velocities and two types of spargers which generated different sizes of bubbles. Small bubbles bursting at the surface of the culture were apparently the main cause of cell damage in batch cultures in laboratory-scale bubble columns. Other mechanisms of cell damage also were a contributing factor to the observed cell loss in outdoor pilot-scale bubble columns. Supplementation of the microalgal culture medium with carboxymethyl cellulose at concentrations of 0.02% and greater is shown to protect the algal cells against aeration-induced hydrodynamic stress.

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.

Causes of shear sensitivity of the toxic dinoflagellate Protoceratium reticulatum.

Dinoflagellates have proven extremely difficult to culture because they are inhibited by low‐level shear forces. Specific growth rate of the toxic dinoflagellate Protoceratium reticulatum was greatly decreased compared with static control culture by intermittent exposure to a turbulent hydrodynamic environment with a bulk average shear rate that was as low as 0.3 s−1. Hydrodynamic forces appeared to induce the production of reactive oxygen species (ROS) within the cells and this caused peroxidation of cellular lipids and ultimately cell damage. Exposure to damaging levels of shear rate correlated with the elevated level of lipoperoxides in the cells, but ROS levels measured directly by flow cytometry did not correlate with shear induced cell damage. This was apparently because the measured level of ROS could not distinguish between the ROS that are normally generated by photosynthesis and the additional ROS produced as a consequence of hydrodynamic shear forces. Continuously subjecting the cells to a bulk average shear rate value of about 0.3 s−1 for 24‐h caused an elevation in the levels of chlorophyll a, peridinin and dinoxanthin, as the cells apparently attempted to counter the damaging effects of shear fields by producing pigments that are potential antioxidants. In static culture, limitation of carbon dioxide produced a small but measureable increase in ROS. The addition of ascorbic acid (0.1 mM) to the culture medium resulted in a significant protective effect on lipid peroxidation, allowing cells to grow under damaging levels of shear rates. This confirmed the use of antioxidant additives as an efficient strategy to counter the damaging effects of turbulence in photobioreactors where shear sensitive dinoflagellates are cultivated.

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 Octopusaqueous 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.

Assessment of the photosynthetically active incident radiation in outdoor photobioreactors using oxalic acid/uranyl sulfate chemical actinometer

A simple actinometric method was evaluated formeasuring the photosynthetically active incidentphoton flux on outdoor photobioreactors. The method isbased on uranyl sulfate catalyzed photodecompositionof oxalic acid in presence of light. The uranyl–oxalate chemical actinometer absorbs radiation ofwavelengths below 535 nm. In the present work, thephotobioreactor wall material did not transmit lightenergy of wavelengths below 350 nm and the effectiveabsorptivity method was used to evaluate the photonflux between 350–535 nm. The standard solar spectrumof the American Society for Testing and Materials(ASTM) was employed for estimating the ratio betweenthe photosynthetically active radiation (400–700 nm)and the solar radiation in the 350–535 nm range. Thisratio (2.21) was taken to be equal to the quotientbetween the photosynthetically active radiation (PAR)and the incident photon flux on the photobioreactorssurface (for the solar radiation between 350–535 nm).PAR measurements with 4π spherical and 2πquantum sensors were used to validate the method.

A bioreaction-diffusion model for growth of marine sponge explants in bioreactors

Marine sponges are sources of high-value bioactives. Engineering aspects of in vitro culture of sponges from cuttings (explants) are poorly understood. This work develops a diffusion-controlled growth model for sponge explants. The model assumes that the explant growth is controlled by diffusive transport of at least some nutrients from the surrounding medium into the explant that generally has a poorly developed aquiferous system for internal irrigation during early stages of growth. Growth is assumed to obey Monod-type kinetics. The model is shown to satisfactorily explain the measured growth behavior of the marine sponge Crambe crambe in two different growth media. In addition, the model is generally consistent with published data for growth of explants of the sponges Disidea avara and Hemimycale columella. The model predicted that nutrient concentration profiles for nutrients, such as dissolved oxygen within the explant, are consistent with data published by independent researchers. In view of the proposed model’s ability to explain available data for growth of several species of sponge explants, diffusive transport does play a controlling role in explant growth at least until a fully developed aquiferous system has become established. According to the model and experimental observations, the instantaneous growth rate depends on the size of the explant and all those factors that influence the diffusion of critical nutrients within the explant. Growth follows a hyperbolic profile that is consistent with the Monod kinetics.