Respirometric assessment of bacterial kinetics in algae-bacteria and activated sludge processes.

Algae-bacteria (AB) consortia can be exploited for effective wastewater treatment, based on photosynthetic oxygenation to reduce energy requirements for aeration. While algal kinetics have been extensively evaluated, bacterial kinetics in AB systems are still based on parameters taken from the activated sludge models, lacking an experimental validation for AB consortia. A respirometric procedure was therefore proposed, to estimate bacterial kinetics in both activated sludge and AB, under different conditions of temperature, pH, dissolved oxygen, and substrate availability. Bacterial activities were differently influenced by operational/environmental conditions, suggesting that the adoption of typical activated sludge parameters could be inadequate for AB modelling. Indeed, respirometric results show that bacteria in AB consortia were adapted to a wider range of conditions, compared to activated sludge, confirming that a dedicated calibration of bacterial kinetics is essential for effectively modelling AB systems, and respirometry was proven to be a powerful and reliable tool to this purpose.

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

Adhesion of microalgal cells to photobioreactor walls reduces productivity resulting in significant economic losses. The physico-chemical surface properties and the fluid dynamics present in the photobioreactor during cultivation are relevant. However, to date, no multiphysical model has been able to predict biofouling formation in these systems. In this work, to model the microalgal adhesion, a Computational Fluid Dynamic simulation was performed using a Eulerian-Lagrangian particle-tracking model. The adhesion criterion was based on the balance of forces and moments included in the XDLVO model. A cell suspension of the marine microalga Nannochloropsis gaditana was fed into a commercial flow cell composed of poly-methyl-methacrylate coupons for validation. Overall, the simulated adhesion criterion qualitatively predicted the initial distribution of adhered cells on the coupons. In conclusion, the combined Computational Fluid Dynamics-Discrete Phase Model (CFD-DPM) approach can be used to overcome the challenge of predicting microalgal cell adhesion in photobioreactors.

Modelling of photosynthesis, respiration, and nutrient yield coefficients in Scenedemus almeriensis culture as a function of nitrogen and phosphorus.

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-NO 3 - and N-NH4+ concentrations. • Nutrient yield coefficients are influenced by luxury uptake phenomenon.

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

Biofouling represents an important limitation in photobioreactor cultures. The biofouling propensity of different materials (polystyrene, borosilicate glass, polymethyl methacrylate, and polyethylene terephthalate glycol-modified) and coatings (two spray-applied and nanoparticle-based superhydrophobic coatings and a hydrogel-based fouling release coating) was evaluated by means of a short-term protein test, using bovine serum albumin (BSA) as a model protein, and by the long-term culture of the marine microalga Nannochloropsis gaditana under practical conditions. The results from both methods were similar, confirming that the BSA test predicts microalgal biofouling on surfaces exposed to microalgae cultures whose cells secrete macromolecules, such as proteins, with a high capacity for forming a conditioning film before cell adhesion. The hydrogel-based coating showed significantly reduced BSA and N. gaditana adhesion, whereas the other surfaces failed to control biofouling. Microalgal biofouling was associated with an increased concentration of sticky extracellular proteins at low N/P ratios (below 15).

Evaluation of native microalgae from Tunisia using the pulse-amplitude-modulation measurement of chlorophyll fluorescence and a performance study in semi-continuous mode for biofuel production.

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.

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

Sustainable dinoflagellate microalgae-based bioprocess designed to produce secondary metabolites (SMs) with interesting bioactivities are attracting increasing attention. However, dinoflagellates also produce other valuable bioproducts (e.g polyunsaturated fatty acids, carotenoids, etc.) that could be recovered and should therefore be taken into account in the bioprocess. In this study, biomass of the marine dinoflagellate microalga Amphidinium carterae was used to assess and optimise three different methods in order to obtain three families of high-value biochemical compounds present in the biomass. The existing processes encompassed a multi-step extraction process for carotenoids, fatty acids and APDs individually and are optimized for the integral valorization of raw A. carterae biomass, with SMs being the primary target compounds. Total process recovery yields were 97% for carotenoids, 80% for total fatty acids and 100% for an extract rich in APDs (not purified).

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

We present the data corresponding to the isolation and morphological and molecular characterization of a strain of Amphidinium carterae, isolated in Mallorca Island waters and now deposited in the microalgae culture collection of the Plant Biology and Ecology Department of the University of the Basque Country under the reference Dn241Ehu. The morphological characterization was made using two different techniques of microscopy and the molecular characterization by using the 28S rDNA sequences of D1 and D2 domains. This strain has been used for a culture study in an indoor LED-lighted pilot-scale raceway to determine its production of carotenoids and fatty acids, "Long-term culture of the marine dinoflagellate microalga Amphidinium carterae in an indoor LED-lighted raceway photobioreactor: Production of carotenoids and fatty acids." (Molina-Miras et al., 2018) [1].

Utilization of centrate for the outdoor production of marine microalgae at pilot-scale in flat-panel photobioreactors.

The outdoor production of marine microalgae biomass in pilot scale flat panels photobioreactors, under not sterile conditions and using centrate as nutrients source, was studied. Experiments were performed modifying the centrate percentage, dilution rate and orientation of the photobioreactors. The strain Geitlerinema sp. was that one prevailing independently of the culture conditions. The higher productivity of 47.7 gbiomass m-2·day-1 dry weight and photosynthetic efficiency of 2.8%, was achieved when using 20% centrate and a dilution rate of 0.3 day-1, whatever the orientation of the reactor, maximal nutrient removal capacities of 82%, 85% and 100% for carbon, nitrogen and phosphorus being obtained. Under non-optimal conditions up to 80% of the nitrogen and 60% of the phosphorus were lost by stripping and precipitation, respectively. Carbohydrates was the major component of the biomass followed by proteins and lipids. These results support the possibility to produce microalgae biomass below 0.59 €/kg, useful to produce biofertilizers and animal feed.

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

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

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

Microalgae are an interesting source of natural pigments that have valuable applications. However, further research is necessary to develop processes that allow us to achieve high levels of carotenoid recovery while avoiding degradation. This work presents a comprehensive study on the recovery of carotenoids from several microalgae genera, optimizing carotenoid extraction using alkaline saponification at various temperatures and KOH concentrations. Results show that I. galbana requires a temperature of 60 °C and <10% KOH, N. gaditana and K. veneficum require 60 °C and no saponification, P. reticulatum requires 40 °C and 10% KOH, T. suecica and H. pluvialis require 25 °C and 40% KOH while C. sp. and S. almeriensis require 80 °C and 40% KOH. The influence of the solvent on carotenoid recovery was also studied. In general terms, an ethanol:hexane:water (77:17:6 v/v/v) mixture results in good yields.

Pilot-scale outdoor photobioreactor culture of the marine dinoflagellate Karlodinium veneficum: Production of a karlotoxins-rich extract.

A pilot-scale bioprocess was developed for the production of karlotoxin-enriched extracts of the marine algal dinoflagellate Karlodinium veneficum. A bubble column and a flat-panel photobioreactors (80-281 L) were used for comparative assessment of growth. Flow hydrodynamics and energy dissipation rates (EDR) in the bioreactors were characterized through robust computational fluid dynamic simulations. All cultures were conducted monoseptically outdoors. Bubble column (maximum cell productivity in semicontinuous operation of 58 × 103 cell mL-1 day-1) proved to be a better culture system for this alga. In both reactors, the local EDR near the headspace, and in the sparger zone, were more than one order of magnitude higher than the average value in the whole reactor (=4 × 10-3 W kg-1). Extraction of the culture and further purification resulted in the desired KTXs extracts. Apparently, the alga produced three congeners KTXs: KmTx-10 and its sulfated derivative (sulfo-KmTx-10) and KmTx-12. All congeners possessed hemolytic activity.

Wastewater treatment using microalgae: how realistic a contribution might it be to significant urban wastewater treatment?

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.

Pilot-scale bubble column photobioreactor culture of a marine dinoflagellate microalga illuminated with light emission diodes.

Production of biomass of the shear-sensitive marine algal dinoflagellate Karlodinium veneficum was successfully scaled-up to 80L using a bubble column photobioreactor. The scale factor exceeded 28,500. Light-emission diodes were used as the light source. The diel irradiance profile mimicked the outdoor profile of natural sunlight. The final cell concentration in the absence of nutrient limitation in the scaled-up photobioreactor was nearly 12×10(5)cellsmL(-1), or the same as in laboratory culture systems. The pH-controlled culture (pH=8.5) was always carbon-sufficient. The culture was mixed pneumatically by using a superficial air velocity of 1.9×10(-3)ms(-1) and the temperature was controlled at 21±1°C.

New insights into shear-sensitivity in dinoflagellate microalgae.

A modification of a flow contraction device was used to subject shear-sensitive microalgae to well-defined hydrodynamic forces. The aim of the study was to elucidate if the inhibition of shear-induced growth commonly observed in dinoflagellate microalgae is in effect due to cell fragility that results in cell breakage even at low levels of turbulence. The microalgae assayed did not show any cell breakage even at energy dissipation rates (EDR) around 10(12)Wm(-3), implausible in culture devices. Conversely, animal cells, tested for comparison purposes, showed high physical cell damage at average EDR levels of 10(7)Wm(-3). Besides, very short exposures to high levels of EDR promoted variations in the membrane fluidity of the microalgae assayed, which might trigger mechanosensory cellular mechanisms. Average EDR values of only about 4·10(5)Wm(-3) increased cell membrane fluidity in microalgae whereas, in animal cells, they did not.

An optimisation approach for culturing shear-sensitive dinoflagellate microalgae in bench-scale bubble column photobioreactors.

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.

Selection of native Tunisian microalgae for simultaneous wastewater treatment and biofuel production.

This paper focuses on the selection of native microalgae strains suitable for wastewater treatment and biofuel production. Four Chlorophyceae strains were isolated from North-eastern Tunisia. Their performances were compared in continuous mode at a 0.3 1/day dilution rate. The biomass productivity and nutrient removal capacity of each microalgae strain were studied. The most efficient strain was identified as Scenedesmus sp. and experiments at different dilution rates from 0.2 to 0.8 1/day were carried out. Maximal biomass productivity of 0.9 g/L day was obtained at 0.6 1/day. The removal of chemical oxygen demand (COD), ammonium and phosphorus was in the range of 92-94%, 61-99% and 93-99%, respectively. Carbohydrates were the major biomass fraction followed by lipids and then proteins. The saponifiable fatty acid content was in the 4.9-13.2% dry biomass range, with more than 50% of total fatty acids being composed of saturated and monosaturated fatty acids.

Utilization of secondary-treated wastewater for the production of freshwater microalgae.

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.

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

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

Outdoor pilot-scale production of Nannochloropsis gaditana: influence of culture parameters and lipid production rates in tubular photobioreactors.

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 0.31-0.351/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.

Protein production using the baculovirus-insect cell expression system.

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.