Implementation of kLa-Based Strategy for Scaling Up Porphyridium purpureum (Red Marine Microalga) to Produce High-Value Phycoerythrin, Fatty Acids, and Proteins.

Porphyridium purpureum is a well-known Rhodophyta that recently has attracted enormous attention because of its capacity to produce many high-value metabolites such as the pigment phycoerythrin and several high-value fatty acids. Phycoerythrin is a fluorescent red protein-pigment commercially relevant with antioxidant, antimicrobial activity, and fluorescent properties. The volumetric mass transfer coefficient (kLa) was kept constant within the different scaling-up stages in the present study. This scaling-up strategy was sought to maintain phycoerythrin production and other high-value metabolites by Porphyridium purpureum, using hanging-bag photobioreactors. The kLa was monitored to ensure the appropriate mixing and CO2 diffusion in the entire culture during the scaling process (16, 80, and 400 L). Then, biomass concentration, proteins, fatty acids, carbohydrates, and phycoerythrin were determined in each step of the scaling-up process. The kLa at 16 L reached a level of 0.0052 s-1, while at 80 L, a value of 0.0024 s-1 was achieved. This work result indicated that at 400 L, 1.22 g L-1 of biomass was obtained, and total carbohydrates (117.24 mg L-1), proteins (240.63 mg L-1), and lipids (17.75% DW) were accumulated. Regarding fatty acids production, 46.03% palmitic, 8.03% linoleic, 22.67% arachidonic, and 2.55% eicosapentaenoic acid were identified, principally. The phycoerythrin production was 20.88 mg L-1 with a purity of 2.75, making it viable for food-related applications. The results of these experiments provide insight into the high-scale production of phycoerythrin via the cultivation of P. purpureum in an inexpensive and straightforward culture system.

Improvement of Unsaturated Fatty Acid Production from Porphyridium cruentum Using a Two-Phase Culture System in a Photobioreactor with Light-Emitting Diodes (LEDs).

In this study, the culture conditions for Porphyridium cruentum were optimized to obtain the maximum biomass and lipid productions. The eicosapentaenoic acid content was increased by pH optimization. P. cruentum was cultured with modified F/2 medium in 14-L photobioreactors using a two-phase culture system, in which the green (520 nm) and red (625 nm) light-emitting diodes (LEDs) were used during the first and second phases for biomass production and lipid production, respectively. Various parameters, including aeration rate, light intensity, photoperiod, and pH were optimized. The maximum biomass concentration of 0.91 g dcw/l was obtained with an aeration rate of 0.75 vvm, a light intensity of 300 µmol m-2s-1, and a photoperiod of 24:0 h. The maximum lipid production of 51.8% (w/w) was obtained with a light intensity of 400 µmol m-2s-1 and a photoperiod of 18:6 h. Additionally, the eicosapentaenoic acid and unsaturated fatty acid contents reached 30.6% to 56.2% at pH 6.0.

Induced cultivation pattern enhanced the phycoerythrin production in red alga Porphyridium purpureum.

Porphyridium purpureum is a rich source for producing phycoerythrin (PE); however, the PE content is greatly affected by culture conditions. Researchers have aimed to optimize the cultivation of P. purpureum for accumulation of PE. When traditional optimized culture conditions were used to cultivate P. purpureum, high PE contents were not usually achieved. In this study, an induced cultivation pattern was applied to P. purpureum for PE biosynthesis (i.e., an incremental approach by altering temperatures, light intensities, and nitrate concentrations). Results revealed that the induced pattern greatly improved the PE biosynthesis. The optimized PE content of 229 mg/L was achieved on the 12th cultivation day, which was a maximum PE content within one cultivation period and accounted for approximately 3.05% of the dry biomass. The induced cultivation pattern was highly suitable for PE synthesis in P. purpureum, which provided an important reference value to the large-scale production of PE.

Light Intensity and Nitrogen Concentration Impact on the Biomass and Phycoerythrin Production by Porphyridium purpureum.

Several factors have the potential to influence microalgae growth. In the present study, nitrogen concentration and light intensity were evaluated in order to obtain high biomass production and high phycoerythrin accumulation from Porphyridium purpureum. The range of nitrogen concentrations evaluated in the culture medium was 0.075-0.450 g L-1 and light intensities ranged between 30 and 100 µmol m-2 s-1. Surprisingly, low nitrogen concentration and high light intensity resulted in high biomass yield and phycoerythrin accumulation. Thus, the best biomass productivity (0.386 g L-1 d-1) and biomass yield (5.403 g L-1) were achieved with NaNO3 at 0.075 g L-1 and 100 µmol m-2 s-1. In addition, phycoerythrin production was improved to obtain a concentration of 14.66 mg L-1 (2.71 mg g-1 of phycoerythrin over dry weight). The results of the present study indicate that it is possible to significantly improve biomass and pigment production in Porphyridium purpureum by limiting nitrogen concentration and light intensity.

Lipid and unsaturated fatty acid productions from three microalgae using nitrate and light-emitting diodes with complementary LED wavelength in a two-phase culture system.

In this study, Pavlova lutheri, Chlorella vulgaris, and Porphyridium cruentum were cultured using modified F/2 media in a 1 L flask culture. Various nitrate concentrations were tested to determine an optimal nitrate concentration for algal growth. Subsequently, the effect of light emitted at a specific wavelength on biomass and lipid production by three microalgae was evaluated using various wavelengths of light-emitting diodes (LED). Biomass production by P. lutheri, C. vulgaris, and P. cruentum were the highest with blue, red, and green LED wavelength with 1.09 g dcw/L, 1.23 g dcw/L, and 1.28 g dcw/L on day 14, respectively. Biomass production was highest at the complementary LED wavelength to the color of microalgae. Lipid production by P. lutheri, C. vulgaris, and P. cruentum were the highest with yellow, green, and red LEDs' wavelength, respectively. Eicosapentaenoic acid production by P. lutheri, C. vulgaris, and P. cruentum was 10.35%, 10.14%, and 14.61%, and those of docosahexaenoic acid were 6.09%, 8.95%, and 11.29%, respectively.

Optimal cultivation towards enhanced biomass and floridean starch production by Porphyridium marinum.

Optimal conditions for maximal biomass and starch production by the marine red microalgae Porphyridium marinum were investigated. Box-Behnken Design was used to model the effect of light intensity, NaNO3 concentration and salinity on the growth of microalgae but also on their starch and protein contents. These three factors increased biomass production by 13.6% in optimized conditions. A maximum starch production (140.21 µg·mL-1), 30.6% higher than that of the control, was attained at a light intensity of 100 µmol photons·m-2·s-1, a NaNO3 concentration of 1 g·L-1 and a NaCl concentration of 20 g·L-1. FT-IR spectroscopy was used to estimate the biochemical composition (carbohydrate accumulation) of P. marinum and revealed significant changes (P < 0.05) depending on culture conditions. FT-IR analysis highlighted also that the culture conditions leading to highest starch production by P. marinum corresponded to lowest sulfated polysaccharide and protein contents.

Growth and Biochemical Composition of Porphyridium purpureum SCS-02 under Different Nitrogen Concentrations.

Microalgae of the genus Porphyridium show great potential for large-scale commercial cultivation, as they accumulate large quantities of B-phycoerythrin (B-PE), long chain polyunsaturated fatty acids (LC-PUFAs) and exopolysaccharide (EPS). The present study aimed to adjust culture nitrogen concentrations to produce Porphyridium biomass rich in B-PE, LC-PUFAs and EPS. Porphyridium purpureum SCS-02 was cultured in ASW culture medium with low nitrogen supply (LN, 3.5 mM), medium nitrogen supply (MN, 5.9 mM) or high nitrogen supply (HN, 17.6 mM). HN significantly enhanced the accumulation of biomass, intracellular protein, B-PE and eicosapentaenoic acid. LN increased the intracellular carbohydrate and arachidonic acid content, and promoted the secretion of EPS. The total lipids content was almost unaffected by nitrogen concentration. Based on these results, a semi-continuous two-step process was proposed, which included the production of biomass rich in B-PE and LC-PUFAs with sufficient nitrogen, and induced EPS excretion with limited nitrogen and strong light.

Han's model parameters for microalgae grown under intermittent illumination: Determined using particle swarm optimization.

This work provides a model and the associated set of parameters allowing for microalgae population growth computation under intermittent lightning. Han's model is coupled with a simple microalgae growth model to yield a relationship between illumination and population growth. The model parameters were obtained by fitting a dataset available in literature using Particle Swarm Optimization method. In their work, authors grew microalgae in excess of nutrients under flashing conditions. Light/dark cycles used for these experimentations are quite close to those found in photobioreactor, i.e. ranging from several seconds to one minute. In this work, in addition to producing the set of parameters, Particle Swarm Optimization robustness was assessed. To do so, two different swarm initialization techniques were used, i.e. uniform and random distribution throughout the search-space. Both yielded the same results. In addition, swarm distribution analysis reveals that the swarm converges to a unique minimum. Thus, the produced set of parameters can be trustfully used to link light intensity to population growth rate. Furthermore, the set is capable to describe photodamages effects on population growth. Hence, accounting for light overexposure effect on algal growth.

Scale-up cultivation enhanced arachidonic acid accumulation by red microalgae Porphyridium purpureum.

The present study attempts to cultivate Porphyridium purpureum under different scale-up conditions for further development and commercialization of microalgae-derived PUFAs such as ARA and EPA. Different temperatures (25, 30, and 35 °C) and light intensities (70, 165, and 280 µmol/m2s) were applied to the 50 L pilot-scale cultivation of P. purpureum in ASW. The cultivation under the light intensity of 280 µmol/m2s at 35 °C obtained biomass concentration up to 9.52 g/L, total fatty acid content to 56.82 mg/g, and ARA content to 22.29 mg/g. While the maximum EPA content of 7.00 mg/g was achieved under the light intensity of 280 µmol/m2s at 25 °C and the highest ratio of UFAs to TFAs of 74.66% was also obtained in this trial. Both biomass concentration and TFAs content were improved by increasing light intensity and temperature. Moreover, the ratio of ARA to EPA was enhanced by increasing cultivation temperature under the light intensity of 280 µmol/m2s. In contrast with flask culture, the conversion of linoleic acid (C18:2) to ARA was enhanced in scale-up culture, leading to more ARA content. Phosphate limitation enhanced the synthesis of lipid and LPUFAs. Moreover, the biomass concentration and biosynthesis of palmitic acid were preferred by sufficient C (NaHCO3).

Feasibility of attached cultivation for polysaccharides production by Porphyridium cruentum.

Porphyridium cruentum is one of the most valued microalgae species able to produce both pigments and exopolysaccharides. Conventional liquid suspended cultivation in ponds and photobioreactors show its disadvantages in lower cultivation efficiency and higher stirring power consumption due to the high viscosity of the medium by the accumulation of polysaccharides. In this work, a new method of culture (called attached cultivation) based on the growth of microalgae using a supporting surface was successfully applied to the cultivation of P. cruentum and the effect of the main influential parameters on its growth rate and polysaccharides production has been investigated. Higher values of these factors resulted in a faster growth rate and, in particular, optimum values of 6.98 g m-2 for initial biomass density, 100 µmol m-2 s-1 for light intensity, continuous illumination, 2.0 % for CO2 concentration, and 0.1 v v-1 min-1 for aeration rate produced the best polysaccharide production of 42 % dry weight. The nutrition profile of P. cruentum obtained in attached and suspended cultivations was similar. Overall these results demonstrate that the attached cultivation is a promising technique which greatly improves the growth rate of P. cruentum as well as its production of polysaccharides and, therefore, it is worth enhancing to be exploited for commercial application.

Utilising light-emitting diodes of specific narrow wavelengths for the optimization and co-production of multiple high-value compounds in Porphyridium purpureum.

The effect of specific narrow light-emitting diode (LED) wavelengths (red, green, blue) and a combination of LED wavelengths (red, green and blue - RGB) on biomass composition produced by Porphyridium purpureum is studied. Phycobiliprotein, fatty acids, exopolysaccharides, pigment content, and the main macromolecules composition were analysed to determine the effect of wavelength on multiple compounds of commercial interest. The results demonstrate that green light plays a significant role in the growth of rhodophyta, due to phycobiliproteins being able to harvest green wavelengths where chlorophyll pigments absorb poorly. However, under multi-chromatic LED wavelengths, P. purpureum biomass accumulated the highest yield of valuable products such as eicosapentaenoic acid (∼2.9% DW), zeaxanthin (∼586µgg-1DW), ß-carotene (397µgg-1DW), exopolysaccharides (2.05g/L-1), and phycobiliproteins (∼4.8% DW). This increased accumulation is likely to be the combination of both photo-adaption and photo-protection, under the combined specific wavelengths employed.

Microalgae respond differently to nitrogen availability during culturing.

Variations in the exogenous nitrogen level are known to significantly affect the physiological status and metabolism of microalgae. However, responses of red, green and yellow-green algae to nitrogen (N) availability have not been compared yet. Porphyridium cruentum, Scenedesmus incrassatulus and Trachydiscus minutus were cultured in the absence of N in the medium and subsequent resupply of N to the starved cells. Culture growth and in-gel changes in isoenzyme pattern and activity of glutamate synthase, glutamate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were studied. The results demonstrated that the algae responded to the fully N-depleted and N-replete culture conditions by species-specific metabolic enzyme changes, suggesting differential regulation of both enzyme activity and cellular metabolism. Substantial differences in the activities of the antioxidant enzymes between N-depleted and N-replete cells of each species as well as between the species were also found. In the present work, besides the more general responses, such as adjustment of growth and pigmentation, we report on the involvement of specific metabolic and antioxidant enzymes and their isoforms in the mechanisms operating during N starvation and recovery in P. cruentum, T. minutus and S. incrassatulus.

UPLC-MSE profiling of Phytoplankton metabolites: application to the identification of pigments and structural analysis of metabolites in Porphyridium purpureum.

A fast and high-resolution UPLC-MSE analysis was used to identify phytoplankton pigments in an ethanol extract of Porphyridium purpureum (Pp) devoid of phycobiliproteins. In a first step, 22 standard pigments were analyzed by UPLC-MSE to build a database including retention time and accurate masses of parent and fragment ions. Using this database, seven pigments or derivatives previously reported in Pp were unequivocally identified: ß,ß-carotene, chlorophyll a, zeaxanthin, chlorophyllide a, pheophorbide a, pheophytin a, and cryptoxanthin. Minor amounts of Divinyl chlorophyll a, a chemotaxonomic pigment marker for prochlorophytes, were also unequivocally identified using the database. Additional analysis of ionization and fragmentation patterns indicated the presence of ions that could correspond to hydroxylated derivatives of chlorophyll a and pheophytin a, produced during the ethanolic extraction, as well as previously described galactosyldiacylglycerols, the thylakoid coenzyme plastoquinone, and gracilamide B, a molecule previously reported in the red seaweed Gracillaria asiatica. These data point to UPLC-MSE as an efficient technique to identify phytoplankton pigments for which standards are available, and demonstrate its major interest as a complementary method for the structural elucidation of ionizable marine molecules.

Evaluation of batch and semi-continuous culture of Porphyridium purpureum in a photobioreactor in high latitudes using Fourier Transform Infrared spectroscopy for monitoring biomass composition and metabolites production.

The culture strategy (batch or semi-continuous) was evaluated for biomass and metabolite formation in Porphyridium purpureum cultures in higher latitudes (>50° N). FTIR was used technology to characterise macromolecule biomass composition and the quality of the metabolites produced. Semi-continuous culture was found to be the most feasible strategy to develop microalgal biomass production facilities in higher latitudes, due to their average results in terms of growth rate (0.27 day(-1)), duplication time (2.5-4 days), maximum cell density achieved (1.43*10(7) cells m L(-1)), biomass productivity of 47.04 mg L(-1) day(-1) and an exopolysaccharides production of 2.1 g L(-1). FTIR technology applied to microalgal production is a valuable and reliable tool to determine on a daily basis not just the evolution of macromolecules composition (lipids, carbohydrates and proteins) but also for the characterisation of the metabolites produced such as phycoerythrin or exopolysaccharides in P. purpureum cultures.

Influence of sulphate on the composition and antibacterial and antiviral properties of the exopolysaccharide from Porphyridium cruentum.

AIMS: The influence of two culture media and three different concentrations of sulphate in the medium on the growth of two strains of Porphyridium cruentum and on the production, composition and viscoelastic characteristics, and antimicrobial properties of the sulphated exopolysaccharide (EPS) were studied. MAIN METHODS: A Bohlin C50 rheometer was used to evaluate the viscosity and elasticity of the EPS solutions. HSV virus, types 1 and 2, Vaccinia virus and Vesicular stomatitis virus were used along with two Gram-negative (Escherichia coli and Salmonella enteritidis) and one Gram-positive (Staphylococcus aureus) bacteria, for testing the antimicrobial activity of EPS. KEY-FINDINGS: The growth of microalgae was higher in NTIP medium and the production of EPS was enhanced by sulphate 21mM. The protein content of the EPS was enhanced by the addition of sulphate 52mM and 104mM; this concentration also induced an increase in sulphate content of the EPS. However, neither the contents of EPS in carbohydrates and uronic acids were affected by the culture medium supplementation in sulphate. In general, the EPS from the Spanish strain presented a higher antiviral activity than the EPS from the Israeli strain. All EPS extracts revealed a strong activity against V. stomatitis virus, higher than the activity of all chemical compounds tested. The EPS from the Israeli strain also presented antibacterial activity against S. enteritidis. SIGNIFICANCE: Enrichment of the culture medium with sulphate improved protein and sulphate content of EPS. EPS extracts presented a relevant activity against V. stomatitis virus and S. enteritidis bacterium.

Airlift column photobioreactors for Porphyridium sp. culturing: part I. effects of hydrodynamics and reactor geometry.

Photosynthetic microorganisms have been attracting world attention for their great potential as renewable energy sources in recent years. Cost effective production in large scale, however, remains a major challenge to overcome. It is known to the field that turbulence could help improving the performance of photobioreactors due to the so-called flashing light effects. Better understanding of the multiphase fluid dynamics and the irradiance distribution inside the reactor that cause the flashing light effects, as well as quantifying their impacts on the reactor performance, thus, are crucial for successful design and scale-up of photobioreactors. In this study, a species of red marine microalgae, Porphyridium sp., was grown in three airlift column photobioreactors (i.e., draft tube column, bubble column, and split column). The physical properties of the culture medium, the local fluid dynamics and the photobioreactor performances were investigated and are reported in this part of the manuscript. Results indicate that the presence of microalgae considerably affected the local multiphase flow dynamics in the studied draft tube column. Results also show that the split column reactor works slightly better than the draft tube and the bubble columns due to the spiral flow pattern inside the reactor.

Airlift column photobioreactors for Porphyridium sp. culturing: Part II. verification of dynamic growth rate model for reactor performance evaluation.

Dynamic growth rate model has been developed to quantify the impact of hydrodynamics on the growth of photosynthetic microorganisms and to predict the photobioreactor performance. Rigorous verification of such reactor models, however, is rare in the literature. In this part of work, verification of a dynamic growth rate model developed in Luo and Al-Dahhan (2004) [Biotech Bioeng 85(4): 382-393] was attempted using the experimental results reported in Part I of this work and results from literature. The irradiance distribution inside the studied reactor was also measured at different optical densities and successfully correlated by the Lambert-Beer Law. When reliable hydrodynamic data were used, the dynamic growth rate model successfully predicted the algae's growth rate obtained in the experiments in both low and high irradiance regime indicating the robustness of this model. The simulation results also indicate the hydrodynamics is significantly different between the real algae culturing system and an air-water system that signifies the importance in using reliable data input for the growth rate model.

A new tool to detect high viscous exopolymers from microalgae.

Microalgae are microorganisms often surrounded by a slime layer made of secreted polymeric substances sometimes including polysaccharides. These polysaccharides, weakly described in the literature, can constitute value-added molecules in several industrial areas. The aim of this article is to show that a new tool, the BioFilm Ring Test®, can be used to detect viscous microalgal exopolymers. Two red microalgal strains (Rhodella violacea and Porphyridium purpureum), one cyanobacterium (Arthrospira platensis) and their excreted polymeric fractions were studied. R. violacea and P. purpureum induced a positive response with the BioFilm Ring Test® contrary to A. platensis. Finally, the understanding of the fractions viscosity involvement in the BRT response was performed by a rheological study.

Lipid production in Porphyridium cruentum grown under different culture conditions.

Autotrophic growth of Porphyridium cruentum under 18:12 h and 12:12 h light:dark cycles showed the maximum cell concentration of 2.1 g-dry wt./L, whereas the specific growth rate, 0.042 (1/h), at 18:6 h is faster than that of 12:12 h, 0.031 (1/h), respectively. The highest lipid accumulation level, 19.3 (%, w/w), was achieved at 12:12 h cycle. Under dark cultivation condition with 10 g/L of glucose, the lipid accumulation in the cell was 10.9 (%, w/w), whereas the heterotrophic growth with glycerol as the carbon resource showed low level of cell concentration and lipid production, compared to that of glucose. The glucose was decided to be a suitable carbon resource for the heterotrophic growth of P. cruentum. The lipids from P. cruentum seemed be feasible for biodiesel production, because over 30% of the lipid was C16-C(18:1). The cultivation time and temperature were important factors to increase the maximum cell concentration. Extending the cultivation time helps maintain the maximum cell concentration, and higher lipid accumulation was achieved at 25 degrees C, compared to 35 degrees C. The fed-batch cultures showed that, under the light condition, the specific production rate was slightly decreased to 0.4% lipid/g-dry wt./day at the later stage, whereas, under the dark condition, the specific production rate was maintained to be a maximum value of 1.1% lipid/g-dry wt./day, even in the later stage of cultivation. The results indicate that the heterotrophic or 12:12 h cyclic mixotrophic growth of P. cruentum could be used for the production of biodiesel in long-term fed-batch cultivation of P. cruentum.

Culture media optimization for growth and phycoerythrin production from Porphyridium purpureum.

Porphyridium spp. is a red micro alga and is gaining importance as a source of valuable products viz., phycobiliproteins (PB), sulfated exopolysaccharides, and polyunsaturated fatty acids with potential applications in the food and pharmaceutical industries. In the present study, the effects of the major media constituents of Porphyridium species were studied using response surface methodology (RSM) on biomass yield, total PB and the production of phycoerythrin (PE). A second order polynomial can be used to predict the PB and PE production in terms of the independent variables. The independent variables such as the concentrations of sodium chloride, magnesium sulfate, sodium nitrate, and dipotassium hydrogen phosphate influenced the total PB and PE production. The optimum conditions showed that total PB was 4.8% at the concentration of sodium chloride 26.1 g/L, magnesium sulfate 5.23 g/L, sodium nitrate 1.56 g/L, and dipotassium hydrogen phosphate 0.034 g/L. In case of optimum PE production (3.3%), the corresponding values are 29.62, 6.11, 1.59, and 0.076 g/L, respectively. PE production depends greatly on the concentrations of chloride, nitrate, and sulfate as well as phosphate of which the former possess the maximum effect.