Biostimulant Potential of Scenedesmus obliquus Grown in Brewery Wastewater.

Microalgae are microorganisms with the capacity to contribute to the sustainable and healthy food production, in addition to wastewater treatment. The subject of this work was to determine the potential of Scenedesmus obliquus microalga grown in brewery wastewater to act as a plant biostimulant. The germination index of watercress seeds, as well as the auxin-like activity in mung bean and cucumber, and in the cytokinin-like activity in cucumber bioassays were used to evaluate the biostimulant potential. Several biomass processes were studied, such as centrifugation, ultrasonication and enzymatic hydrolysis, as well as the final concentration of microalgal extracts to determine their influence in the biostimulant activity of the Scenedesmus biomass. The results showed an increase of 40% on the germination index when using the biomass at 0.1 g/L, without any pre-treatment. For auxin-like activity, the best results (up to 60% with respect to control) were obtained at 0.5 g/L of biomass extract, after a combination of cell disruption, enzymatic hydrolysis and centrifugation. For cytokinin-like activity, the best results (up to 187.5% with respect to control) were achieved without cell disruption, after enzymatic hydrolysis and centrifugation at a biomass extract concentration of 2 g/L.

Utilization of centrate from urban wastewater plants for the production of Scenedesmus sp. in a raceway-simulating reactor.

This work investigates the production of the native microalgae strain Scenedesmus sp. in semi-continuous mode at lab scale in open raceway-simulating reactors and using centrate as the culture medium. The biomass productivity and nutrient removal capacity of Scenedesmus sp. at different dilution rates were investigated indoors as well as its tolerance to centrate as the culture medium at different concentrations. A biomass productivity of 7.80 g/m2 day was obtained at 200 µE/m2 s, 5 cm culture depth, 0.30 1/day of dilution rate and 60% centrate while nitrogen and phosphorus removal rates were 1.50 g/m2 day and 0.15 g/m2 day, respectively. The produced biomass characterization under these conditions showed a lipid content of 12.60% d wt. along with a favorable fatty acids profile with 57.70% of total fatty acids composed of saturated and monounsaturated fatty acids. Subsequently, the effect of light intensity and culture depth on biomass productivity and nutrient uptake as well as the biochemical composition and fatty acids profile was studied using two irradiance levels (200 and 1000 µE/m2 s) and four culture depths (5 cm, 10 cm, 15 cm and 20 cm). Under optimal conditions of 1000 µE/m2 s, 60% centrate, 0.30 1/day dilution rate and 15 cm culture depth, a maximum biomass productivity of 22.20 g/m2 day was obtained. Nitrogen and phosphorus removal rates of 2.00 gN/m2 day and 0.40 gP/m2 day, respectively, were recorded. An amount of 11.70% d wt. of lipids was determined along with a suitable fatty acids profile for biofuel production.

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.

Production of astaxanthin by Haematococcus pluvialis: taking the one-step system outdoors.

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.

Efficiency assessment of the one-step production of astaxanthin by the microalga Haematococcus pluvialis.

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/g day, 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/g day and an average irradiance range of 77-110 microE/m(2) 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.

Pilot-scale outdoor production of Scenedesmus sp. in raceways using flue gases and centrate from anaerobic digestion as the sole culture medium.

This work investigated the production of Scenedesmus sp. in semi-continuous mode in three pilot-scale outdoor raceways (7.2 m2) using flue gas for CO2 supply and centrate from the anaerobic digestion of urban wastewater as the sole nutrient source. Experiments were performed at different culture depths, 5, 10 and 15 cm, while evaluating two centrate concentrations (30% and 45%) at dilution rates of 0.2 and 0.3 d-1. Under optimal conditions of 30% centrate, 0.3 d-1 dilution rate and a 15 cm culture depth, a maximum biomass productivity of 22.9 g m-2 d-1 was obtained. The optical properties of the cultures were studied and the results showed a photosynthetic efficiency of up to 2.0% and a quantum yield of 0.3 g biomass E-1. Nitrogen and phosphorus removal rates of 3 g N m-2 d-1 and 0.6 g P m-2 d-1 were recorded, respectively. Lipid productivity of 2.3 g m-2 d-1 was determined possessing a suitable fatty acids profile for biofuel production.

Modeling of photosynthesis and respiration rate for Isochrysis galbana (T-Iso) and its influence on the production of this strain.

Isochrysis galbana is a widely-used strain in aquaculture in spite of its low productivity. To maximize the productivity of processes based on this microalgae strain, we have developed a model considering the influence of irradiance, temperature, pH and dissolved oxygen concentration on the photosynthesis and respiration rate. Results demonstrate that this strain tolerates temperatures up to 35 °C but it is highly sensitive to irradiances higher than 500 µE m(-2)s(-1) and dissolved oxygen concentrations higher than 11 mgl(-1). We have validated the developed model using data from an industrial-scale outdoor tubular photobioreactor demonstrating that inadequate temperature and dissolved oxygen concentrations reduce productivity to half that which is maximal, according to light availability under real outdoor conditions. The developed model is a useful tool for managing working processes, but especially in the development of new processes based on this strain and to take decisions regarding optimal control strategies.

Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography.

B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae. Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics. Tedious methodologies for B-PE purification have been published. In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum. Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid-sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h(-1). After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer. In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid-sodium acetate buffer, pH 5.5. With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies. The B-PE purity was tested using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopic characterization.

Development of a process for large-scale purification of C-phycocyanin from Synechocystis aquatilis using expanded bed adsorption chromatography.

In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90 mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90-93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.

Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture.

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 microE/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.