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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.

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.

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).

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.

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 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.

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.

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

The dinoflagellate Karlodinium veneficum was grown in bubble column photobioreactors and a genetic algorithm-based stochastic search strategy used to find optimal values for the culture parameters gas flow rate, culture height, and nozzle sparger diameter. Cell production, concentration of reactive oxygen species (ROS), membrane fluidity and photosynthetic efficiency were studied throughout the culture period. Gas-flow rates below 0.26Lmin(-1), culture heights over 1.25m and a nozzle diameter of 1.5mm were found to provide the optimal conditions for cell growth, with an increase of 60% in cell production with respect to the control culture. Non-optimal conditions produced a sufficiently high shear stress to negatively affect cell growth and even produce cell death. Cell physiology was also severely affected in stressed cultures. The production of ROS increased by up to 200%, whereas cell membrane fluidity decreased by 60% relative to control cultures. Photosynthetic efficiency decreased concomitantly with membrane fluidity.

The aim of this work was to obtain biodiesel (methyl esters) from the saponifiable lipids (SLs) fraction of the microalga Nannochloropsis gaditana, whose biomass dry weight contains 12.1 wt% of these lipids. SLs were extracted from the microalga as free fatty acids (FFAs) for subsequent transformation to methyl esters (biodiesel) by enzymatic esterification. Extraction as FFAs rather than as SLs allows them to be obtained with higher purity. Microalgal FFAs were obtained by direct saponification of lipids in the biomass and subsequent extraction-purification with hexane. Esterification of FFAs with methanol was catalysed by lipase Novozym 435 from Candida antarctica. Stability studies of this lipase in the operational conditions showed that the esterification degree (ED) attained with the same batch of lipase remained constant over six reaction cycles (36 h total reaction time). The optimal conditions attained for 4 g of FFAs were 25°C, 200 rpm, methanol/FFA molar ratio of 1.5:1, Novozym 435/FFA ratio of 0.025:1 w/w and 4 h reaction time. In these conditions the ED attained was 92.6%, producing a biodiesel with 83 wt% purity from microalgal FFAs. Several experimental scales were tested (from 4 to 40 g FFAs), and in all cases similar EDs were obtained.

This work studied outdoor pilot scale production of Nannochloropsis gaditana in tubular photobioreactors. The growth and biomass composition of the strain were studied under different culture strategies: continuous-mode (varying nutrient supply and dilution rate) and two-stage cultures aiming lipid enhancement. Besides, parameters such as irradiance, specific nitrate input and dilution rate were used to obtain models predicting growth, lipid and fatty acids production rates. The range of optimum dilution rate was 0.31–0.35 1/day with maximum biomass, lipid and fatty acids productivities of 590, 110 and 66.8 mg/l day, respectively. Nitrate limitation led to an increase in lipid and fatty acids contents (from 20.5% to 38.0% and from 16.9% to 23.5%, respectively). Two-stage culture strategy provided similar fatty acids productivities (56.4 mg/l day) but the neutral lipids content was doubled.

The baculovirus-insect cell expression system is widely used in producing recombinant proteins. This review is focused on the use of this expression system in developing bioprocesses for producing proteins of interest. The issues addressed include: the baculovirus biology and genetic manipulation to improve protein expression and quality; the suppression of proteolysis associated with the viral enzymes; the engineering of the insect cell lines for improved capability in glycosylation and folding of the expressed proteins; the impact of baculovirus on the host cell and its implications for protein production; the effects of the growth medium on metabolism of the host cell; the bioreactors and the associated operational aspects; and downstream processing of the product. All these factors strongly affect the production of recombinant proteins. The current state of knowledge is reviewed.

Continuous cultures of Nannochloropsis gaditana, Tetraselmis chuii, Tetraselmis suecica and Phaeodactylum tricornutum were carried out at different dilution rates and culture media in order to check their influence on biomass productivity. N. gaditana attained maximum biomass productivity of 0.49 g/l day at a dilution rate of 0.42 1/day. The influence of nitrate concentration on biomass productivity was tested by continuous cultures of N. gaditana. At 8.0 mM nitrate and dilution rates ranging between 0.30 and 0.40 1/day, maximum biomass productivities were achieved. To enhance lipid accumulation, a two-stage culture strategy consisting in a first stage of nitrate-replete conditions followed by a nitrate-depleted phase was performed. The accumulated productivity was 51 mgFATTY ACIDS/l day. Results showed an important change in the fatty acids profile and an increase in the neutral lipids content, representing 73.1% of total lipids. Additionally, the combination of nitrogen depletion and light stress was proved to contribute to lipid enhancement.

In order to obtain microalgal saponifiable lipids (SLs) fractions containing different polar lipid (glycolipids and phospholipids) contents, SLs were extracted from wet Nannochloropsis gaditana microalgal biomass using seven extraction systems, and the polar lipid contents of some fractions were reduced by low temperature acetone crystallization. We observed that the polar lipid content in the extracted lipids depended on the polarity of the first solvent used in the extraction system. Lipid fractions with polar lipid contents between 75.1% and 15.3% were obtained. Some of these fractions were transformed into fatty acid methyl esters (FAMEs, biodiesel) by methanolysis, catalyzed by the lipases Novozym 435 and Rhizopus oryzae in tert-butanol medium. We observed that the reaction velocity was higher the lower the polar lipid content, and that the final FAME conversions achieved after using the same lipase batch to catalyze consecutive reactions decreased in relation to an increase in the polar lipid content.

Microalgae have been proposed as an option for wastewater treatment since the 1960s, but still, this technology has not been expanded to an industrial scale. In this paper, the major factors limiting the performance of these systems are analysed. The composition of the wastewater is highly relevant, and especially the presence of pollutants such as heavy metals and emerging compounds. Biological and engineering aspects are also critical and have to be improved to at least approximate the performance of conventional systems, not just in terms of capacity and efficiency but also in terms of robustness. Finally, the harvesting of the biomass and its processing into valuable products pose a challenge; yet at the same time, an opportunity exists to increase economic profitability. Land requirement is a major bottleneck that can be ameliorated by improving the system’s photosynthetic efficiency. Land requirement has a significant impact on the economic balance, but the profits from the biomass produced can enhance these systems’ reliability, especially in small cities.

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).

Dinoflagellate microalgae are an important source of marine biotoxins. Bioactives from dinoflagellates are attracting increasing attention because of their impact on the safety of seafood and potential uses in biomedical, toxicological and pharmacological research. Here we review the potential applications of dinoflagellate toxins and the methods for producing them. Only sparing quantities of dinoflagellate toxins are generally available and this hinders bioactivity characterization and evaluation in possible applications. Approaches to production of increased quantities of dinoflagellate bioactives are discussed. Although many dinoflagellates are fragile and grow slowly, controlled culture in bioreactors appears to be generally suitable for producing many of the metabolites of interest.

The cost of lipases and the relatively slower reaction rate remain as the major obstacles for enzymatic production of biodiesel as opposed to the conventional chemical processes. This paper reviews the starting oils usually employed in biodiesel production, the processes for transforming them to biodiesel placing particular emphasis on enzymatic transesterification. The pros and cons of the lipase-based process, the key operational variables and the technological alternatives for attenuating lipase deactivation are also discussed. Finally, suggestions are made for future studies, paying particular attention to the use of whole cell immobilization in the production process, as this methodology may reduce both the cost of the biocatalyst and dependence on lipase manufacturers.

In this paper, the biomass and lutein productivity of the lutein-rich new strain Scenedesmus almeriensis is modelled versus irradiance and temperature. The results demonstrate that S. almeriensis is a mesophile microorganism with an optimal growth temperature of 35 degrees C, and capable of withstanding up to 48 degrees C, which caused culture death. This strain is also tolerant to high irradiances, showing no signs of photoinhibition even at the maximum irradiance essayed of 1625 microE m(-2) s(-1) accumulating up to 0.55% dry weight (d.wt.) of lutein. The optimal conditions that maximise the biomass productivity also favour the lutein productivity, lutein being a primary metabolite. Maximal biomass and lutein productivities of 0.87 g l(-1) day(-1) and 4.77 mg l(-1) day(-1), respectively, were measured. The analysis of light availability inside the cultures, quantified as average irradiance, demonstrates that the cultures were mainly photo-limited, although photosaturation also took place at high external irradiances. The effect of temperature was also investigated finding that the specific maximal growth rate is modified by the temperature according to the Arrhenius equation. The influence of both light availability and temperature was included in an overall growth model, which showed, as a result, capable of fitting the whole set of experimental data. An overall lutein accumulation rate model was also proposed and used in a regression analysis. Simulations performed using the proposed models show that under outdoor conditions a biomass productivity of 0.95 g l(-1) day(-1) can be expected, with a lutein productivity up to 5.31 mg l(-1) day(-1). These models may be useful to assist the design and operation optimisation of outdoor cultures of this strain.

This paper reports on a methodology for increasing proliferation and monoclonal antibody (mAb) production in hybridoma cultures. The 55-6 murine B cell hybridoma line (CD40 and CD19-deficient expression) was treated with increasing concentrations of lipopolysaccharide (LPS). Expression of CD69, CD40, and CD19 surface antigens on 55-6 cells did not show significant changes from untreated cells. The specific growth rate decreased at higher concentrations of LPS, but the monoclonal antibody production rate was highest at the highest LPS concentration assayed. These data are in agreement with the lowest growth rate found at this concentration of LPS. Furthermore, cells were cultured with anti-mouse surface immunoglobulin G antibody (anti-mIgG) plus LPS to find out whether LPS-derived signals and anti-mIgG stimuli are synergistic. CD69, CD40, and CD19 expression was greater than for either untreated cells (control culture) or cells stimulated with LPS alone. Moreover, LPS stimulation in combination with anti-mIgG enhanced both the growth rate and IgG2a production over the control culture and cells stimulated with LPS alone. Maximum antibody concentration increased almost 500% compared to the control and about 100% with respect to culture stimulated with LPS alone. The maximum specific IgG2a production rate was about 300% higher than in the control culture and about 30% higher than in culture stimulated only with LPS.

Ethanol was used for the extraction and purification of lipids from the biomass of the microalga Phaeodactylum tricornutum. This microalga is an oil-rich substrate with a high proportion of eicosapentaenoic acid (EPA). The process consisted of two steps. First, ethanol (96% vol/vol) was used to extract the lipids from the lyophilized biomass. Second, a biphasic system was formed by adding water and hexane to the extracted crude oil. In this way, most of the lipids were transferred to the hexanic phase while most impurities remained in the hydroalcoholic phase. The first step was carried out by two consecutive extractions at room temperature, each with 5 mL ethanol per gram of biomass, for 10 and 1.25 h, respectively. Under these conditions, over 90% of the saponifiable lipids in the biomass were extracted. In the second step, the percentage of water in the hydroalcoholic phase, the hexane/hydroalcoholic phase ratio and the number of extraction steps were optimized. A water content of 40% vol/vol in the hydroalcoholic phase provided the highest lipid recovery. A recovery yield of 80% was obtained by four consecutive extractions with a hexane/hydroalcoholic phase ratio of 0.2 (vol/vol). Equilibrium distribution data of the lipids between the hydroethanolic and the hexanic phases were also obtained in order to predict the lipid recovery yield of the extraction. This process is an alternative to the traditional methods of lipid extraction, which uses less toxic solvents and reduces the total amount of solvents used.

In previous experiments with the 55-6 hybridoma cell line, we showed that cell stimulation with anti-mouse surface immunoglobulin G antibody (anti-mIgG) increased both CD40 expression and specific monoclonal antibody (mAb) production rate. Cell preincubation with lipopolysaccharide (LPS) prior to anti-mIgG stimulation enhanced these results. Moreover, the expression of both CD40 and surface immunoglobulin G (sIgG) were higher for cells in the G1 phase of the cell cycle. Therefore, to determine the relationship between cell cycle position, CD40 expression, and mAb productivity, in this work cells were synchronized in the G1 phase by thymidine block. In addition, synchronized cells were subjected to different treatments with anti-mIgG. Although synchronized cells showed a slight increase in both CD40 expression and maximum specific growth rate (mu max) compared with unsynchronized cells, specific productivity did not show significant changes. However, the stimulation of synchronized cells with anti-mIgG increased over 65% the expression of CD40 and over 50% the specific productivity in comparison with that obtained on unsynchronized cells after anti-mIgG stimulation. These data improved additionally over 15 and 60%, respectively, by adding 2 mM thymidine to the culture medium. These results suggest that the effect of the positive association between G1 phase, CD40 expression, and specific productivity is subordinated to the effect of anti-mIgG stimulation, which is enhanced by increasing the percentage of cells on the G1 phase of the cell cycle. Contrary to expectations, LPS preincubation of synchronized cells prior to anti-mIgG stimulation did not increase the specific productivity in comparison with non-preincubated cells, and the expression of CD40 was minor compared to that on non-preincubated cells.

In this work, we studied the potential use of secondary-treated wastewater as nutrient source in the production of freshwater microalgae strains. Experiments were performed indoors in a semicontinuous mode, at 0.3 day(-1), simulating outdoor conditions. We demonstrated that all the tested strains can be produced by using only secondary-treated wastewater as the nutrient source. The utilization of secondary-treated wastewater imposes nutrient-limiting conditions, with maximal biomass productivity dropping to 0.5 g l(-1) day(-1) and modifies the biochemical composition of the biomass by increasing the amount of lipids and carbohydrates while reducing the biomass protein content. We measured fatty acid content and productivity of up to 25 %d.wt. and 110 mg l(-1) day(-1), respectively. We demonstrated that all the tested strains were capable of completely removing the nitrogen and phosphorus contained in the secondary-treated wastewater, and while the use of this effluent reduced the cells' photosynthetic efficiency, the nitrogen and phosphorus coefficient yield increased. Muriellopsis sp. and S. subpicatus were selected as the most promising strains for outdoor production using secondary-treated wastewater as the culture medium; this was not only because of their high productivity but also their photosynthetic efficiency, of up to 2.5 %, along with nutrient coefficient yields of up to 96 gbiomass gN (-1) and 166 gbiomass gP (-1). Coupling microalgae production processes to tertiary treatment in wastewater treatment plants make it possible to recover nutrients contained in the water and to produce valuable biomass, especially where nutrient removal is required prior to wastewater discharge.

A bubble column and two airlift photobioreactors (a draft-tube sparged vessel and a split-cylinder device) of the same general design (0.19 m column diameter, 2 m tall, 0.06 m3 working volume) were evaluated for outdoor continuous culture of the microalga Phaeodactylum tricornutum at a dilution rate of 0.03 h−1. At a daily averaged irradiance (photosynthetically active) value of 900 μE m−2 s−1, all bioreactors attained a quasi steady-state biomass concentration of ∼1 kg m−3 and a biomass productivity of ∼0.3 kg m−3 per day when the aeration velocity was 0.01 m s−1. The microalgal cells were susceptible to aeration-associated hydrodynamic stress if the superficial aeration velocity exceeded 0.01 m s−1. Supplementing the culture medium with 0.02% or more carboxymethyl cellulose (CMC), allowed stable culture under conditions that had previously damaged the cells. The average elemental composition of the biomass was: 49.2% C, 6.3% H, 0.8% N, and 1.3% S. The chlorophylls, carotenoids, and pigments content of the biomass changed with irradiance within a given day. Low irradiance favored accumulation of the light capture pigments. Increasing daily irradiance led to accumulation of carbohydrates. Some of the carbohydrate accumulated during the day was consumed at night and partly converted to proteins. Eicosapentaenoic acid (EPA, 20:5n3) constituted between 27 and 30% of the total fatty acids present, or 2.6–3.1% of the dry biomass. The other main fatty acids present were palmetic acid (16:0), palmoleic acid (16:1n7), and myristic acid (14:0). On average, these three fatty acids constituted 16.9% (16:0), 14.0% (16:1n7) and 9.4% (14:0) of the total fatty acids present.

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. This review focuses on methods of producing sponge biomass to overcome supply limitations. Production techniques discussed include aquaculture in the sea, the controlled environments of aquariums, and culture of sponge cells and primmorphs. Cultivation in the sea and aquariums are currently the only practicable and relatively inexpensive methods of producing significant quantities of sponge biomass. In the future, metabolite production from cultured sponge cells and primmorphs may become feasible. Obtaining a consistent biomass yield in aquariums requires attention to many factors that are discussed in this work.

An integrated process for the indoor production of 13C labeled polyunsaturated fatty acids (PUFAs) from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor operating with recirculation of the exhaust gas using a low-pressure compressor. Oxygen accumulation in the system is avoided by bubbling the exhaust gas from the reactor in a sodium sulfite solution before returning to it. To achieve a high 13C enrichment in the biomass obtained, the culture medium is initially stripped of carbon, and labeled 13CO(2) is automatically injected on-demand during operation for pH control and carbon supply. The reactor was operated in both batch and semicontinuous modes. In semicontinuous mode, the reactor was operated at a dilution rate of 0.01 h(-1), resulting in a biomass productivity of 0.1 g l(-1) per day. The elemental analysis of the inlet and outlet flows of the reactor showed that 64.9% of carbon was turned into microalgal biomass, 34.9% remained in the supernatant mainly as inorganic compounds. Only 3.8% of injected carbon was effectively fixed as the target labeled product (EPA). Regarding the isotopic composition of fatty acids, results showed that fatty acids were not labeled in the same proportion, the higher the number of carbons the lower the percentage of 13C. Isotopic composition of EPA ranged from 36.5 to 53.5%, as a function of the methodology used (GC-MS, EA-IRMS or gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS)). The low carbon uptake efficiency combined with the high cost of 13CO(2) make necessary to redefine the designed culture system to increase the efficiency of the conversion of 13CO(2) into the target product. Therefore, the possibility of removing 12C from the fresh medium, and recovering and recirculating the inorganic carbon in the supernatant and the organic carbon from the EPA depleted biomass was studied. The inorganic carbon of the fresh medium was removed by acidification and stripping with N(2). The inorganic carbon of the supernatant was recovered also by acidification and subsequent stripping with N(2). The operating conditions of this step were optimized for gas flow rate and type of contactor. A carbon recovery step for the depleted biomass was designed based on the catalytic oxidation to CO(2) using CuO (10 wt.%) as catalyst with an oxygen enriched atmosphere (80% O(2) partial pressure). In this way, the carbon losses reduced an 80.2% and the efficiency of the conversion of carbon in EPA was increased to 19.5%, which is close to the theoretical maximum. Further increase in 13CO(2) use efficiency is only possible by additionally recovering other labeled by-products present in the biomass: proteins, carbohydrates, lipids, and pigments.

Relatively large (0.19 m column diameter, 2 m tall, 0.06 m3 working volume) outdoor bubble column and airlift bioreactors (a split-cylinder and a draft-tube airlift device) were compared for monoseptic fed-batch culture of the microalga Phaeodactylum tricornutum. The three photobioreactors produced similar biomass versus time profiles and final biomass concentration (∼4 kg m−3). The maximum specific growth rate observed within a daily illuminated period in the exponential growth phase, had a value of 0.08 h−1 on the third day of culture. Because of night-time losses of biomass, the specific growth rate averaged over the 4-days of exponential phase was 0.021 h−1 for the three reactors. The biomass in the vertical column reactors did not experience photoinhibition under conditions (photosynthetically active daily averaged irradiance value of 1150±52 μE m−2 s−1) that are known to cause photoinhibition in conventional thin-tube horizontal loop reactors. Because of good gas-liquid mass transfer, the dissolved oxygen concentration in the reactors at peak photosynthesis remained

Saponifiable lipids (SLs) were extracted with hexane from wet biomass (86 wt% water) of the microalga Nannochloropsis gaditana in order to transform them into fatty acid methyl esters (FAMEs, biodiesel). The influence of homogenization pressure on SL extraction yield at low temperature (20-22 °C) was studied. Homogenization at 1700 bar tripled the SL extraction yield. Two biomass batches with similar total lipid content but different lipidic compositions were used. Batch 1 contained fewer SLs (12.0 wt%) and neutral saponifiable lipids (NSLs, 7.9 wt%) than batch 2 (21.6 and 17.2 wt%, respectively). For this reason, and due to the selectivity of hexane toward NSLs, high SL yield (69.1 wt%) and purity (71.0 wt%) were obtained from batch 2. Moreover, this extract contains a small percentage of polyunsaturated fatty acids (16.9 wt%), thereby improving the biodiesel quality. Finally, up to 97.0% of extracted SLs were transformed to FAMEs by acid catalyzed transesterification.

The free fatty acids eicosapentaenoic acid (C20:5ω3, EPA) and docosahexaenoic acid (C22:6ω3), obtained from the microalgae Phaeodactylum tricornutum, and the EPA methyl ester were compared with regard to their extent of peroxidation using different storage conditions. Several series were stored according to selected variables: hexane addition versus no addition, 4 °C versus 25 °C, and antioxidant addition (octyl gallate) versus no antioxidant addition, always in the dark. Previously, the EPA and methyl EPA structures were confirmed by NMR spectra to discard the formation of conjugated dienes after the downstream process. The results showed that the stability was higher for methyl EPA than for the free fatty acid, and that peroxidation can be retarded by low temperature storage and mainly by hexane addition. The peroxidation process was evaluated by the peroxide value (spectrophotometric method by iodine absorption), although the conjugated diene absorbance and the loss in percentage of the fatty acid have been tested as good indicators of the peroxidation process. A simple kinetic model that explains the peroxidation process during the initiation and propagation steps is given.

Bubble columns and airlift photobioreactors can be useful for culturing phototrophic organisms requiring light as a nutrient. Light availability in bubble columns and airlift devices is influenced by aeration rate, gas holdup, and the liquid velocity (mixing and turbulence). The photosynthetically generated oxygen also needs to be removed, as excessive dissolved oxygen suppresses photosynthesis. Oxygen removal capacity is governed by the magnitude of the overall gas–liquid mass-transfer coefficient, kLaL. This work characterizes the relevant hydrodynamic and mass-transfer parameters in three air-agitated reactors: bubble column, split-cylinder airlift device and concentric draft-tube sparged airlift vessel. The reactors are then evaluated for culture of the microalga Phaeodactylum tricornutum. All reactors were about 0.06 m3 in working volume, and the working aspect ratio was about 10. Data were obtained in tap water for a base-line comparison and in Mediterranean seawater, as a potential medium for algal culture. A theoretical relationship was developed and proved between kLaL and the aeration rate. In addition, a method based on mechanistic relationships was proved for predicting the liquid circulation velocity and kLaL in airlift reactors. Existing correlations applied satisfactorily to gas holdup and kLaL data obtained in the bubble column. Aqueous solution of sodium chloride (0.15 M) closely resembled seawater in terms of its hydrodynamics and oxygen transfer behavior. Under the conditions tested, all three reactors attained a biomass concentration of about 4 kg·m−3 after ∼260 h. The mean maximum specific growth rate was 0.022 h−1 in all cases at a power input of 109 W·m−3.

Engineering analyses combined with experimental observations in horizontal tubular photobioreactors and vertical bubble columns are used to demonstrate the potential of pneumatically mixed vertical devices for large-scale outdoor culture of photosynthetic microorganisms. Whereas the horizontal tubular systems have been extensively investigated, their scalability is limited. Horizontal tubular photobioreactors and vertical bubble column type units differ substantially in many ways, particularly with respect to the surface–to–volume ratio, the amount of gas in dispersion, the gas–liquid mass transfer characteristics, the nature of the fluid movement and the internal irradiance levels. As illustrated for eicosapentaenoic acid production from the microalga Phaeodactylum tricornutum, a realistic commercial process cannot rely on horizontal tubular photobioreactor technology. In bubble columns, presence of gas bubbles generally enhances internal irradiance when the Sun is low on the horizon. Near solar noon, the bubbles diminish the internal column irradiance relative to the ungassed state. The optimal dimensions of vertical column photobioreactors are about 0.2 m diameter and 4 m column height. Parallel east–west oriented rows of such columns located at 36.8°N latitude need an optimal inter-row spacing of about 3.5 m. In vertical columns the biomass productivity varies substantially during the year: the peak productivity during summer may be several times greater than in the winter. This seasonal variation occurs also in horizontal tubular units, but is much less pronounced. Under identical conditions, the volumetric biomass productivity in a bubble column is ∼60% of that in a 0.06 m diameter horizontal tubular loop, but there is substantial scope for raising this value.

A well established analysis of energy dissipation in the riser, downcomer and the bottom sections of split-cylinder airlift bioreactors (aspect ratio=7.6 and 14.5; equal riser-to-downcomer cross-sectional area ratios of 1.0) was used to characterize shear rates in those systems for application to animal cell culture. Shear rates were evaluated for suspensions of typical microcarriers (loading =0–30 kg m −3 ; particle diameter=(150–300)×10 −6 m; density 1030–1050 kg m −3 ) encountered in anchorage-dependent cell culture and for microcarrier-free liquids. For the reactors tested, the highest shear rates were encountered in the bottom zone; the riser had lower shear rate values, while the downcomer was the most quiescent. The shear rates in various zones ranged over 0–12 000 s −1 for a riser superficial gas velocity range of 0–6.7×10 −3 m s −1 which is typical for cell culture. In all zones, the shear rates increased with increasing aeration rate. Shear rates declined with increasing loading of microcarriers, but were not substantially affected by the carrier diameter or density. Relative to the microcarrier free system, even small amounts of carriers (6 kg m −3 ) lowered the maximum prevailing shear rate to about 4000 s −1 . The shear rates were extremely sensitive to the length scale of the fluid eddies when the eddy length-to-carrier diameter ratio was less than or equal to unity. The results showed quantitatively how the shear rate in various zones of airlift reactors may be manipulated by modifications to operational and geometric parameters. The methodology presented allowed for characterization of shear rates in the bulk flow, unlike existing studies that provide information only on wall shear rates which are not particularly relevant to shear sensitive bioprocesses.

The aim of this paper was to study the effect of spent medium recycle on Spodoptera exigua Se301 cell line proliferation, metabolism, and baculovirus production when grown in batch suspension cultures in Ex-Cell 420 serum-free medium. The results showed that the recycle of 20% of spent medium from a culture in mid-exponential growth phase improved growth relative to a control culture grown in fresh medium. Although both glucose and glutamine were still present at the end of the growth phase, glutamate was always completely exhausted. The pattern of the specific glucose and lactate consumption and production rates, as well as the specific glutamine and glutamate consumption rates, suggests a metabolic shift at spent medium recycle values of over 60%, with a decrease in the efficiency of glucose utilization and an increase in glutamate consumption to fuel energy metabolism. Baculovirus infection provoked a change in the metabolic pattern of Se301 cells, although a beneficial effect of spent medium recycle was also observed. Both growth rate and maximum viable cell density decreased relative to uninfected cultures. The efficiency of glucose utilization was dramatically reduced in those cultures containing the lowest percentages of spent medium, whereas glutamine and glutamate consumption was modulated, thereby suggesting that infected cells were devoted to virus replication, retaining their ability to incorporate the nutrients required to support viral replication. Recycle of 20% of spent medium increased baculovirus production by around 90%, thus showing the link between cell growth and baculovirus production.

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).

The cell growth and monoclonal antibody production of the 55-6 hybridoma cell co-cultured with the murine thymoma cell line EL-4 at different initial 55-6:EL-4 ratios were investigated. Both populations were seeded in co-culture without previous stimulation and therefore with low constitutive CD40 and CD40 ligand (CD154) expression levels, and in the absence of exogenous co-stimuli. Viable cell density and growth rate data seem to suggest a competition for nutrients, which is detrimental for both cells in terms of biomass production and also of growth rate for 55-6. Final concentrations of antibody and specific antibody production rates were affected by the initial 55-6:EL-4 ratio. The 4:1 ratio yielded the highest IgG2a concentration, whereas the highest specific antibody production rate was obtained at the 2:1 ratio. Changes mainly in CD154 and also in CD40 expression in co-cultures could suggest cross-talk between both populations. In conclusion, different types of interactions are probably present in this co-culture system: competition for nutrients, cognate interaction and/or autocrine or paracrine interactions that influence the proliferation of both cells and the hybridoma antibody secretion. We are hereby presenting a pre-scale-up process that could speed up the optimization of large-scale monoclonal antibodies production in bioreactors by emulating the in vivo cell-cell interaction between B and T cells without previous stimulation or the addition of co-stimulatory molecules. © 2013 Springer Science+Business Media Dordrecht, The authors acknowledge and appreciate the financial support received from Junta de Andalucía-Spain (P07-CVI-03193) and Ministerio de Ciencia e Innovación-Spain (BIO2008-06505)

Growth and fatty acid composition of Isochrysis galbana ALII-4 in batch and semicontinuous outdoor culture in a closed tubular photobioreactor are reported. Specific growth rate at the exponential phase was 0.38 d −1 , higher than that obtained in previous indoor experiments. At biomass concentration above 2 g l −1 net biomass productivity obtained in three successive semicontinuous cultures was 0.32 g l −1 d −1 . A daily cyclic variation in the fatty acid profile was observed. From morning to afternoon, 14:0, 16:0 and 16:1 n 7 content increased, although it decreased during darkness renewing the cycle again with daylight. On the other hand, no daily variation was observed in polyunsaturated fatty acid content. The EPA generation rate during linear growth, with an average content of 2.56%, was 8.2 mg l −1 d −1 . The present study was conducted uninterruptedly in February and March, 1994, in Almeria, Spain (latitude 36° 50′ N, longitude 2° 27′ W).

As chemical pesticides are being banned as control agents for agricultural pests, the use of the highly specific, safe to non-target organisms baculoviruses has been proposed. These viruses can be produced either in vivo or in vitro. In vitro production requires appropriated host insect cell lines with the ability for growing as freely-suspended cells. In this work, the Spodoptera exigua Se301 cell line was used to produce the commercially available S. exigua nucleopolyhedrovirus (SeMNPV) in suspension. Se301 cells showed to be very sensitive to the hydrodynamic shear rates developed in bioreactors. A process of progressive adaptation to freely-suspended cultures using protective additives against shear stress and disaggregant was proposed. The best combinations were polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) with the disaggregant dextran sulfate (DS). Both static and freely-suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Production of occluded baculovirus (OB) increased with the multiplicity of infection (MOI > 0.1).

The commonly used shear protective agent Pluronic F68 (PF68) was toxic to the marine dinoflagellate microalga Protoceratium reticulatum, but had a shear-protective effect on it at concentrations of ≤ 0.5 g L(-1) . Supplementation of P. reticulatum cultures with PF68 actually increased the fluidity of the cell membrane; therefore, the shear protective effect of PF68 could not be ascribed to reduced membrane fluidity, an explanation that has been commonly used in relation to its shear protective effect on animal cells. Data are reported on the membrane fluidity of P. reticulatum and its response to the presence of PF68 under sublethal and lethal turbulence regimens. The membrane fluidity was found to depend strongly on the level of lipoperoxides in the cells produced under lethal agitation.

The feasibility of a one-step method for the continuous production of astaxanthin by the microalga Haematococcus pluvialis has been verified outdoors. To this end, influence of dilution rate, nitrate concentration in the feed medium, and irradiance on the performance of continuous cultures of H. pluvialis was firstly analyzed indoors in bubble column reactors under daylight cycles, and then outdoors, using a tubular photobioreactor. At the laboratory scale, the behavior of the cultures agreed with that previously recorded in continuous illumination experiences, and attested that the major factors determining biomass and astaxanthin productivity were average irradiance and specific nitrate supply. The rate of astaxanthin accumulation was proportional to the average irradiance inside the culture, provided that a nitrate limiting situation had been established. The accumulation of astaxanthin under daylight cycles was maximal for a specific nitrate input of 0.5 mmol/g day. The recorded performance has been modeled on the basis of previously developed equations, and the validity of the model checked under outdoor conditions. Productivity values for biomass and astaxanthin of 0.7 g/L day and 8.0 mg/L day respectively, were obtained in a pilot scale tubular photobioreactor operating under continuous conditions outdoors. The magnitude of the experimental values, which matched those simulated from the obtained model, demonstrate that astaxanthin can be efficiently produced outdoors in continuous mode through a precise dosage of the specific nitrate input, taking also into consideration the average irradiance inside the culture

Continuous cultivation of Haematococcus pluvialis under moderate nitrogen limitation represents a straightforward strategy, alternative to the classical two-stage approach, for astaxanthin production by this microalga. Performance of the one-step system has now been validated for more than 40 combinations of dilution rate, nitrate concentration in the feed medium, and incident irradiance, steady state conditions being achieved and maintained in all instances. Specific nitrate input and average irradiance were decisive parameters in determining astaxanthin content of the biomass, as well as productivity of the system. The growth rate of the continuous photoautotrophic cultures was a hyperbolic function of average irradiance. As long as specific nitrate input was above the threshold value of 2.7 mmol/gday, cells performed green and astaxanthin was present at basal levels only. Below the threshold value, under moderate nitrogen limitation conditions, astaxanthin accumulated to reach cellular levels of up to 1.1% of the dry biomass. Increasing irradiance resulted in enhancement of astaxanthin accumulation when nitrogen input was limiting, but never under nitrogen sufficiency. Mean daily productivity values of 20.8 ± 2.8 mg astaxanthin/L day (1.9 ± 0.3 g dry biomass/L day) were consistently achieved for a specific nitrate input of about 0.8 mmol/gday and an average irradiance range of 77-110 μE/m2 s. Models relating growth rate and astaxanthin accumulation with both average irradiance and specific nitrate input fitted accurately experimental data. Simulations provided support to the contention of achieving efficient production of the carotenoid through convenient adjustment of the determining parameters, and yielded productivity estimates for the one-step system higher than 60 mg astaxanthin/L day. The demonstrated capabilities of this production system, as well as its product quality, made it a real alternative to the current two-stage system for the production of astaxanthin-rich biomass. © 2007 Wiley Periodicals, Inc.

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.

Fed-batch and perfusion cultures were carried out in a traditional glass 2-L bioreactor with the toxic dinoflagellate Protoceratium reticulatum. The maximum cell concentration obtained was 2.3 x 10 5 cell·mL -1 , which is almost 1 order of magnitude higher than the maximum previously referenced for this species. L1 medium was shown to be clearly deficient in nitrate and phosphate for this strain, and addition of highly concentrated aliquots of these nutrients allowed higher cell concentrations to be obtained. This species consumed high amounts of nitrate and phosphate, 2.1 x 10 -3 and 2.3 x 10 -4 μmol·h -1 ·cell -1 , respectively. However, this consumption produced a very low number of cells compared to other classes of microalgae, indicating that this species is, like other dinoflagellates, a poor competitor in terms of utilization of inorganic nutrients. Higher production of toxins and pigments was strongly associated with cell number in the culture, with maximum values of 700 ng·mL -1 and 1321 μg·mL -1 , respectively. Most yessotoxins remained within the cells and not in the cell-free culture medium, and their production was not related to either the age of the culture or the cell growth phase.

The performance of Haematococcus pluvialis in continuous photoautotrophic culture has been analyzed, especially from the viewpoint of astaxanthin production. To this end, chemostat cultures of Haematococcus pluvialis were carried out at constant light irradiance, 1,220 μE/m2 · s, and dilution rate, 0.9/d, but varying the nitrate concentration in the feed medium reaching the reactor, from 1.7 to 20.7 mM. Both growth and biomass composition were affected by the nitrate supply. With saturating nitrate, the biomass productivity was high, 1.2 g/L · d, but astaxanthin accumulation did not take place, the C/N ratio of the biomass being 5.7. Under moderate nitrate limitation, biomass productivity was decreased, as also did biomass concentration at steady state, whereas accumulation of astaxanthin developed and the C/N ratio of the biomass increased markedly. Astaxanthin accumulation took place in cells growing and dividing actively, and its extent was enhanced in response to the limitation in nitrate availability, with a recorded maximum for astaxanthin cellular level of 0.8% of dry biomass and of 5.6 mg/L · d for astaxanthin productivity. The viability of a significant continued generation of astaxanthin-rich H. pluvialis cells becomes thus demonstrated, as also does the continuous culture option as an alternative to current procedures for the production of astaxanthin using this microalga. The intensive variable controlling the behavior of the system has been identified as the specific nitrate input, and a mathematical model developed that links growth rate with both irradiance and specific nitrate input. Moreover, a second model for astaxanthin accumulation, also as a function of irradiance and specific nitrate input, was derived. The latter model takes into account that accumulation of astaxanthin is only partially linked to growth, being besides inhibited by excess nitrate. Simulations performed fit experimental data and emphasize the contention that astaxanthin can be efficiently produced under continuous mode by adjustment of the specific nitrate input, predicting even higher values for astaxanthin productivity. The developed models represent a powerful tool for management of such an astaxanthin-generating continuous process, and could allow the development of improved systems for the production of astaxanthin-rich Haematococcus cells. © 2005 Wiley Periodicals, Inc.

Explants of the tropical sponge Pseudosuberites andrewsi were fed with the marine diatom Phaeodactylum tricornotum. The food was supplied either as intact algae or as a filtered crude extract. Growth (measured as an increase in underwater weight) was found in both experiments. The explants fed with intact algae increased to an average underwater weight of 255% of the initial weight in 45-60 days. The explants fed with crude extract increased to an average of 200% of the initial weight in 30 days. These results show that it is possible to grow a sponge using a single microorganism species as a food source. In addition, it was demonstrated that sponges are also capable of growing on non-particulate food. Therefore, this study is an important step forward towards the development of controlled, in vivo sponge cultures. (C) 2002 Elsevier Science B.V. All rights reserved.

Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid productivities from chemostat cultures of an isolate of Isochrysis galbana have been studied. The productivities reached in the interval of dilution rates between 0.0295 h−1 and 0.0355 h−1 were 1.5mg·1−1·h−1 for lipids, 300 μg·1−1·h−1 for EPA and 130μg1·1−1·h−1 for DHA. Furthermore, light attenuation by mutual shading, and agitation speed influences on growth and fatty acid composition were analysed. A model relating steady-state dilution rates to internal average light intensity has been proposed, the parameter values of which obtained by non-linear regression were: maximum specific growth rate (μmax)=0.0426 h−1; the affinity of cells to light (Ik) = 10.92 W·m−2; the exponent (n) = 5.13; regression coefficient (r2)=0.9999.