Chemical stabilization of the phycocyanin from cyanobacterium Spirulina platensis

Phycocyanin (PC) prepared from a cyanobacterium Spirulina platensis by the DEAE-DE52 cellulose column chromatography that was developed by gradient elution of 50-250 mM phosphate buffer (pH 7.0) was stabilized by its subunits cross-linked covalently with formaldehyde. The single blue band that the chemically stabilized PC showed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the stabilized PC still maintained its trimeric aggregate form even after its incubation at 60 degrees C for 3h and at 100 degrees C for 10 min in the denatured buffer containing 5% (w/v) SDS. Moreover, the stabilized PC exhibited similar spectroscopic properties of absorption and fluorescence to those of the native PC, and showed adequate energy coupling with R-phycoerythrin (R-PE) after it was conjugated with R-PE via glutaraldehyde.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Optimization of phycocyanin extraction from Spirulina platensis using factorial design

Phycocyanin extraction from cyanobacteria Spirulina platensis was optimized using factorial design and response surface techniques. The effects of temperature and biomass-solvent ratio on phycocyanin concentration and extract purity were evaluated to determine the optimum conditions for phycocyanin extraction. The optimum conditions for the extraction of phycocyanin from S. platensis were the highest biomass-solvent ratio, 0.08 gmL(-1), and 25 degrees C. Under these conditions it's possible to obtain an extract of phycocyanin with a concentration of 3.68 mgmL(-1) and purity ratio (A(615)/A(280)) of 0.46.     

Kinetic models for phycocyanin production by high cell density mixotrophic culture of the microalga Spirulina platensis

Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated. The results showed that the cell density in fed-batch culture (10.2 g L⁻¹) was 4.29-fold that in batch culture (2.38 g L⁻¹), and the total phycocyanin production in the fed-batch culture (0.795 g L⁻¹) was 3.05-fold that in the batch culture (0.261 g L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as well as glucose consumption was proposed. The data fitted the models well (r ² > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m⁻² s⁻¹, and the stepwise increase in light intensity can be replaced by a constant light intensity mode. Received 28 July 1998/ Accepted in revised form 8 October 1998     

Rapid and selective extraction of phycocyanin from Spirulina platensis with ultrasonic cell disruption

A study was conducted on the efficiency of phycocyanin extraction from Spirulina platensis (Arthrospira platensis) cells disrupted by ultrasonic irradiation. Extraction followed first-order kinetics with respect to the length of time for irradiation. The first-order rate constant increased linearly with the output of ultrasonic irradiation. In order to extract phycocyanin there was an appropriate range of ultrasonic frequency, f_u. In addition the most important finding is that the purity of phycocyanin in its crude extract depended on f_u. For example, phycocyanin was extracted with higher purity at f_u = 28 kHz than at f_u = 20 kHz. It is suggested that rapid and selective extraction of phycocyanin from S. platensis may be possible if an optimized ultrasonic application is developed for a given suspension.     

Clarification and concentration with membrane technology of a phycocyanin solution extracted from Spirulina platensis

Membrane technologies were investigated with the aim to improve stability of C-Phycocyanin extracts resulting from ultrasonic breakage of Spirulina platensis. Five membranes, ranging from microfiltration to reverse osmosis, were utilized both for clarification and concentration steps. Nanofiltration with tubular organic membranes exhibited good performances: pigment recovery was 100%, mean permeation flux was 85 l h^–1 m^–2 for achieving a concentration factor of 7 with 30×10⁵ Pa pressure and 1.5 m s^–1 tangential velocity (turbulent flow).     

Growth of Spirulina platensis enhanced under intermittent illumination

The growth characteristics of microalgae under different light conditions (continuous or intermittent) are essential information for photobioreactor design and operation. In this study, we constructed a thin-layer (10 mm) flat plate photobioreactor device with a light/dark (L/D) alternation system to investigate the growth of Spirulina platensis under two different light regimes: (1) continuous illumination in a wide range of light intensities (1.00-77.16 mW cm⁻²); (2) intermittent illumination in medium frequency (0.01-20 Hz). Specific growth rate and light efficiency based on biomass production were determined for each round of experiment. Four regions (light limited region, intermediate region, light saturated region and light inhibition region) were recognized according to the results under continuous illumination. Under intermittent illumination, when L/D frequency increased from 0.01 Hz to 20 Hz, specific growth rate and light efficiency were enhanced. However, the enhancement was different, depending on the applied light intensity and light fraction. The higher the light intensity, the greater the enhancement would be when L/D frequency increased from 0.01 Hz to 20 Hz; and the higher the light intensity, the lower the light fractions is needed to maintain light efficiency as high as that under continuous illumination in light limited region.     

Inhibitory effect of phycocyanin from Spirulina platensis on the growth of human leukemia K562 cells

The effect of phycocyanin from Spirulinaplatensis on the growth of human chronic myelogenousleukemia-blast crisis K562 cells was studied bysemi-solid agar assay and cell viability measurement. Phycocyanin significantly inhibited the growth of K562cells in a dose-dependent manner. The IC50 value ofthe phycocyanin was 72.5 mg L^-1. After the K562cells were cultured with phycocyanin for 6 days, flowcytometric assays showed that more K562 cells wereblocked to progress through S-phase and arrested at G1phase. DNA fragmentation assay indicated that therewas no ladder of DNA fragments of approximately 200-bpmultiples, indicating that apoptosis had not occurred. Western blot analysis showed that Bcl-2 protein wasexpressed, but its level remained unchanged, whereasthe expression level of c-myc increased. Thesefindings suggest that phycocyanin may be able toinhibit the growth of K562 cells by pathways otherthan apoptosis, and that changed a expression patternof the c-myc protein may be involved in such inhibition.     

Intracellular phosphorus pool in a culture of the cyanobacterium Spirulina platensis

An intracellular phosphorus pool in a monoculture of the cyanobacterium Spirulina platensis was assessed using radioactive and nonradioactive phosphorus. The derived dependence of specific growth rate on the intracellular content of mineral phosphorus can be presented in the form of the Droop equation. It was found that the stage of replenishment of the intracellular phosphorus pool may affect the phosphorus turnover estimation in aquatic environments from the results of short-term measurements of phosphorus uptake.     

Osmotic adjustment in Spirulina platensis

Unilateral irradiation of maize (Zea mays L.) seedlings results in a fluence-rate gradient, and hence below saturation, a gradient of the far-red-absorbing form of phytochrome (Pfr). The Pfr-gradients established by blue, red and far-red light were spectrophotometrically measured in the mesocotyl. Based on these Pfr-gradients and the fluence-response curves of phytochrome photoconversion the fluence-rate gradients were calculated. The fluence-rate gradient in the blue (460 nm) was steeper than that in the red (665 nm), which in turn was steeper than that in the far-red light (725 nm). The fluence-rate ratios front to rear were 1:0.06 (460 nm), 1:0.2 (665 nm), and 1:0.33 (725 nm). The assumption that phytochrome-mediated phototropism of maize mesocotyls is caused by local phytochrome-mediated growth inhibition was tested in the following manner. Firstly, the Pfr response curve for growth inhibition was calculated; these calculations were based on measurements of Pfr-gradients and data from red-light-induced phototropism. Secondly, the Pfr response curve for growth inhibition was used as a basis for calculating fluence-response curves for blue-and far-red-light-induced phototropism. Finally, these calculated results were compared with experimental data. It was concluded that the threshold for phytochrome-mediated phototropism of maize mesocotyls reflects the apparent photoconversion cross section of phytochrome whereas the maximal inducable curvature depends on the steepness of the light (Pfr) gradient across the mesocotyl.Abbreviations Pfr far-red-absorbing form of phytochrome - Ptot total phytochrome - Fr far-red light     

In vitro antibacterial activity of laboratory grown culture of Spirulina platensis

The hexane, ethyl acetate, dichloromethane, methanol extracts and spent media (extracellular substances) were tested in vitro for their antibacterial activity for which one Gram-positive bacterium (Staphylococcus aureus) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae) were used as test organisms. The methanol extract showed more potent activity than other organic extracts, spent medium of the culture exhibited little activity against E. coli only. No inhibitory effect was found against Klebsiella pneumoniae.The broth microdilution assay gave minimum inhibitory concentrations (MIC) values ranging from 1 to 512 μg/ml. The MIC of methanol extract against S. aureus and E. coli were 128 μg/ml and 256 μg/ml, respectively.     

Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis: protection against oxidative damage to DNA.

Peroxynitrite (ONOO(-)) is known to inactivate important cellular targets and also mediate oxidative damage in DNA. The present study has demonstrated that phycocyanin, a biliprotein from spirulina platensis and its chromophore, phycocyanobilin (PCB), efficiently scavenge ONOO(-), a potent physiological inorganic toxin. Scavenging of ONOO(-) by phycocyanin and PCB was established by studying their interaction with ONOO(-) and quantified by using competition kinetics of pyrogallol red bleaching assay. The relative antioxidant ratio and IC(50) value clearly indicate that phycocyanin is a more efficient ONOO(-) scavenger than PCB. The present study has also shown that PCB significantly inhibits the ONOO(-)-mediated single-strand breaks in supercoiled plasmid DNA in a dose-dependent manner with an IC(50) value of 2.9 +/- 0.6 microM. These results suggest that phycocyanin, has the ability to inhibit the ONOO(-)-mediated deleterious biological effects and hence has the potential to be used as a therapeutic agent.     

The Effect of Selenite Ions on Growth and Selenium Accumulation in Spirulina platensis

Selenium accumulation and the growth of cyanobacterium Spirulina platensis (Nordst.) Geitl. were studied in a culture with sodium selenite-supplemented nutritional medium. Selenite concentrations below 20 mg/l did not inhibit the growth of S. platensis. The addition of 30 mg/l of this salt somewhat decreased the growth rate during the linear growth phase, induced the earlier suspension transition to the steady-state phase, and substantially lowered the highest optical density of the suspension. However, even at 170 mg/l Na₂SeO₃, the culture still demonstrated a capacity for growth. The content of selenium in the cells depended directly on its concentration in the medium, up to the lethal level. At high selenium concentrations (100–170 mg/l), S. platensis reduced Se(IV) up to Se(0). The latter was secreted onto the cell surface and into the cultural medium. The high concentrations of Na₂SeO₃ acidified the cytoplasmic pH as was measured by ³¹P-NMR spectroscopy. At the same time, the content of protein on a dry weight basis decreased and that of carbohydrates and lipids somewhat increased, just as was observed in S. platensis cells under other stress factors. In the presence of 20 mg/l Na₂SeO₃, the selenium content in the biomass increased by 20000 times as compared to that in the control cells, whereas the biochemical composition of biomass did not change. In this case, the selenium was incorporated almost completely in the protein fraction. The selenium concentration in this fraction increased more significantly when the sulfur content was lowered in the medium.     

Nitrate and ammonia: two key nitrogen sources for biomass and phycocyanin production by Arthrospira (Spirulina) platensis

Journal of Applied Phycology - Nitrogen plays a key role in the production of biomass and valuable pigments from Arthrospira platensis. The present research aimed to examine the effect of Zarrouk...     

Factors indicating culture status during cultivation of Spirulina (Arthrospira) platensis

Factors indicating culture status of two Spirulina platensis strains were monitored in a batch mode cultivation for 36 days. Changing mode in all factors showed a common turning point, indicating shift of cell or culture status. Mean biomass productivity was highly sustained until day 22, chlorophyll a concentration peaked on day 22, pH value was >12 on day 22, coil number was abruptly shortened on day 22, and floating activity was sustained at greater than 79% after day 22, indicating that day 22 is a criterion reflecting phase-transfer in cell physiology in a batch culture system. Many of these changes may have been caused by increased pH, suggesting that pH control is essential for mass production of S. platensis. Fluctuations in floating activity were likely induced by the number of cellular gas vacuoles. Consequently, coil number per trichome and floating activity of S. platensis could readily act as simple indicators for determination of culture status or harvesting time of cells.     

Scale-up and design of a pilot-plant photobioreactor for the continuous culture of Spirulina platensis

Scale-up of bioreactors has the intrinsic difficulty of establishing a reliable relationship among physical parameters involved in the design of the new bioreactor and the physiology of the cultured cells. This is more critical in those cases where a more complex operation of the bioreactor is needed, such as in photobioreactors. A key issue in the operation of photobioreactors is establishing a quantification for the interaction between external illumination, internal light distribution and cell growth. In this paper an approach to the scale-up of a photobioreactor for the culture of Spirulina platensis, based on a mathematical model describing this interaction, and the operation of a previous reactor 10 times smaller is presented. The paper describes the approach followed in the scale-up, the analysis of different design constraints, the physical realization of the new bioreactor design, innovative use of plastic material walls to improve reactor safety, and finally the corroboration of its satisfactory operation.     

Accumulation of poly-beta-hydroxybutyrate in Spirulina platensis.

Poly-beta-hydroxybutyrate has been identified in the cyanobacterium Spirulina platensis. The addition of reduced carbon compounds to the growth medium was not required for poly-beta-hydroxybutyrate accumulation. Poly-beta-hydroxybutyrate accumulated during exponential growth to 6% of the total dry weight and then decreased during the stationary phase.