An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

Different nitrogen sources and growth responses of Spirulina platensis in microenvironments

Spirulina platensis was cultivated, in comparative studies, using several sources of nitrogen. The standard source used (sodium nitrate) was the same as that used in the synthetic medium Zarrouk, whereas the alternative nitrogen sources consisted of ammonium nitrate, urea, ammonium chloride, ammonium sulphate or acid ammonium phosphate. The initial nitrogen concentrations tested were 0.01, 0.03 and 0.05 M in an aerated photobioreactor at 30 °C, with an illuminance of 1900 lux, and 12 h-light/12 h-dark photoperiod over a period of 672 h. Maximum biomass was produced in medium containing sodium nitrate (0.01–0.03–0.05 M), followed by ammonium nitrate (0.01 M) and urea (0.01 M). The final biomass concentrations were 1.992 g l^–1 (0.03 M sodium nitrate), 1.628 g l^–1 (0.05 M sodium nitrate), 1.559 g l^–1 (0.01 M sodium nitrate), 0.993 g l^–1 (0.01 M ammonium nitrate) and 0.910 g l^–1 (0.01 M urea). This suggested that it is possible to utilize nitrogen sources other than sodium nitrate for growing S. platensis, in order to decrease the production costs of scaled up projects.     

Modelling of growth conditions for cyanobacterium Spirulina platensis in microcosms

The influence of cultivation conditions on the growth of the cyanobacterium Spirulina platensis was investigated by using two types of photobioreactors. In a rotative photobioreactor the doubling time (t _d) was 3.54 days. The better value found for t _d in an aerated photobioreactor by changing the initial nitrogen concentration (NaNO₃) at 0.003, 0.015, 0.030 and 0.060M was 2.5 days. A factorial experimental design was performed in order to estimate the contributions of initial nitrogen concentration, inoculum and cultivation time as well as their interactions. All three factors and their interactions proved to be significant in influencing the cellular concentration of S. platensis.     

Effective transformation of the cyanobacterium Spirulina platensis using electroporation

Although Spirulina (Arthrospira) is expected to be a suitableorganism for producing recombinant proteins, a gene transfer system hasnot yet been established, due to a lack of suitable vectors and because Spirulina appears refractory to common genetic manipulations. As theinitial stages of the development of recombinant DNA methodology, weexamined the effects on transformation efficiency of electroporationconditions such as electric-field strength (2, 4, 6, 8, 10, 12kV cm^-1) and time constant (2.5, 5 ms). At a time constant of2.5 ms, few transformants were observed regardless of the field strength.The longer time constant of 5.0 ms reproducibly yielded transformants atthe middle field strength of 4 - 8 kV cm^-1, but gave high killingand no transformation at the higher field strength of 10 - 12kV cm^-1. Chloramphenicol acetyltransferase (CAT) activities wereincreased only in the transformants from 2–6 kV cm^-1 and 5.0 ms.The density of the transformants was significantly correlated with therelative value of CAT activity (r = 0.89, n = 11, p < 0.01),suggesting that the chloramphenicol resistance was due to CAT activity. Weconcluded that transformation of S. platensis was most effective at apulse duration 5.0 ms with an electric field of 4 kV cm^-1, and thatforeign genes can be expressed in this organism.     

Modelling of Spirulina platensis growth in fresh water using response surface methodology

The influence of nutrient addition on the growth rate of Spirulina platensis in the Mangueira Lagoon water was studied in order to investigate the feasibility of using this water for biomass production. The addition of urea and sodium bicarbonate was studied through surface response methodology, over concentration ranges from 0.0 to 0.01170 M, and 0.0–19.70 gl^–1 respectively. The growth of Spirulina platensis in Mangueira Lagoon water with no addition of nutrients was carried out and compared with the biomass growth after nutrient addition. The results indicated that the optimal level of nutrients was 0.00585 M urea and without the addition of sodium bicarbonate. The biomass concentration was 1.4 gl^–1 in 780 h of cultivation and the doubling time (t _d) was 3.85 days. In 300 h, the biomass concentration in the medium without nutrient addition was 0.9 gl^–1, with a doubling time of 3.80 days.     

Alterations in the structure of phycobilisomes of the cyanobacterium,Spirulina platensis in response to enhanced Na+ level

Incubation of Spirulina platensis at enhanced Na⁺ concentrations resulted in 30% increased intracellular accumulation of Na⁺ ions. This accumulation of Na⁺ ions intracellularly altered the phycobilisome organization as revealed by transmission electron microscopic data and changes in the absorption spectrum. The room temperature emission peak at 638 nm in the control sample was blue-shifted by 7 nm in the treated samples suggesting phycobilisome disorganization. SDS-PAGE of phycobilisome polypeptides showed a significant increase in a 66.2 kDa polypeptide. This is the first report that the enhanced concentration of intracellular Na⁺ ion alters the structure of the phycobilisome in S. platensis. This revised version was published online in November 2006 with corrections to the Cover Date.     

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     

The response of the filamentous cyanobacterium Spirulina platensis to salt stress

The responses of the filamentous cyanobacterium Spirulina platensis to increased NaCl concentrations (0.25–1.0 M) in addition to the concentration of sodium in the growth medium were studied. A two stage response to the salt stress was observed. This consisted of a relatively short shock stage, followed by adaptation process. It was shown that upon exposure to high salt concentrations of 0.5 M and above, immediate inhibition of photosynthesis and respiration, and complete cessation of growth occurred. After a time lag, the energy-yielding processes exhibited restored activity. At 0.5 and 1.0M NaCl photosynthesis reached 80% and 50% that of the control, while respiration was enhanced by 140 and 200%, respectively. The time lags were longer when the cells were exposed to higher NaCl concentrations. The resumption of growth and the establishment of new steady state growth rates were found to be correlated to the recovery in respiration. The relationship between the growth rates after adaptation and the increased NaCl concentrations was found to be inversely linear. The cellular sodium content was maintained at a constant low level, regardless of the external NaCl concentration, while potassium content declined linearly vs. the external NaCl concentration. The carbohydrate content of the cells rose exponentially with the increase in NaCl concentration.Publication No. 34 from the Micro-Algal Biotechnology Lab.     

Biochemical evidence for multiple forms of acetohydroxy acid synthase in Spirulina platensis

Two isoforms of acetohydroxy acid synthase (AHAS), the first enzyme of the branched-chain amino acids biosynthetic pathway, were detected in cell-free extracts of the cyanobacterium Spirulina platensis and separated both by ion-exchange chromatography and by hydrophobic interaction. Several biochemical properties of the two putative isozymes were analysed and it was found that they differ for pH optimum, FAD requirement for both activity and stability, and for heat lability. The results were partially confirmed with the characterization of the enzyme extracted from a recombinant Escherichia coli strain transformed with one subcloned S. platensis coli strain transformed with one subcloned S. platensis AHAS gene. The approximate molecular mass of both AHAS activities, estimated by gel filtration, indicates that they are distinct isozymes and not different oligomeric species or aggregates of identical subunits.Abbreviations AHAS acetohydroxy acid synthase - DEAE cellulose diethylaminoethyl cellulose - DTT dithiothreitol - FAD flavin adenine dinucleotide - TPP thiamine pyrophosphate     

Effect of Spirulina platensis biomass on the growth of lactic acid bacteria in milk

The stimulatory effect of aqueous suspensions of Spirulina platensis dry biomass extracted at pH 6.8 and 5.5 was studied on four lactic acid bacteria (LAB) grown in milk. The addition of dry S. platensis to milk (6 mg/ml) stimulated growth of Lactococcus lactis by 27%. The growth of other strains was also promoted. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth inhibition of the cyanobacterium Spirulina (Arthrospira) platensis by 1.7 MHz ultrasonic irradiation

Ultrasonic waves of high frequency (1.7 MHz) and low intensity (0.6 W cm^–2) were employed to prevent cyanobacterial cells from growing fast and the effects of this growth inhibition were investigated. At least five minutes of ultrasonic irradiation was essential for effective inhibition. The growth rate of irradiated cells was reduced to 38.9% of the control during short-term culture. Longer exposure did not significantly enhance the inhibition. For a particular level of energy input, distributed ultrasonic exposure (more short intermittent exposures) was more effective in inhibiting growth than fewer, but longer exposures. For instance, the final biomass decreased to 30.1% of the control after ultrasonic irradiation for 4 minutes every 3 days, whereas it only decreased to 60% of the control with exposure for 12 minutes every 11 days. It is suggested that distributed ultrasonic irradiation is a practical method to prevent cyanobacterial cells from fast growth. A possible explanation for the inhibition is discussed in relation to cell structure, the absorption spectrum of intact cells, chlorophyll level and oxygen evolution.     

Effect of temperature on yield and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors

Outdoor experiments carried out in Florence, Italy (latitude 43.8° N, longitude 11.3° E), using tubular photobioreactors have shown that in summer the average net productivity of a Spirulina platensis culture grown at the optimal temperature of 35 °C was superior by 23% to that observed in a culture grown at 25 °C. The rates of night biomass loss were higher in the culture grown at 25 °C (average 7.6% of total dry weight) than in the one grown at 35 °C (average 5%). Night biomass loss depended on the temperature and light irradiance at which the cultures were grown, since these factors influenced the biomass composition. A net increase in carbohydrate synthesis occurred when the culture was grown at a low biomass concentration under high light irradiance or at the suboptimal temperature of 25 °C. Excess carbohydrate synthesized during the day was only partially utilized for night protein synthesis.     

Differential responses of Nostoc sphaeroides and Arthrospira platensis to solar ultraviolet radiation exposure

During October to December 2003 we carried out experiments to assess the impact of high solar radiation levels (as normally occurring in a tropical region of Southern China) on the cyanobacteria Nostoc sphaeroides and Arthrospira (Spirulina) platensis. Two types of experiments were done: a) Short-term (i.e., 20 min) oxygen production of samples exposed to two radiation treatments (i.e., PAR+UVR—280–700 nm, and PAR only—400–700 nm, PAB and P treatments, respectively), and b) Long-term (i.e., 12 days) evaluation of photosynthetic quantum yield (Y) of samples exposed to three radiation treatments (i.e., PAB; PA (PAR+UV-A, 320–700 nm) and P treatments, respectively). N. sphaeroides was resistant to UVR, with no significant differences (P>0.05) in oxygen production within 20 min of exposure, but with a slight inhibition of Y within hours. A fast recovery of Y was observed after one day even in samples exposed to full solar radiation. A. platensis, on the other hand, was very sensitive to solar radiation (mainly to UV-B), as determined by oxygen production and Y measurements. A. platensis had a circadian rhythm of photosynthetic inhibition, and during the first six days of exposure to solar radiation, it varied between 80 and 100% at local noon, but cells recovered significantly during afternoon hours. There was a significant decrease in photosynthetic inhibition after the first week of exposure with values less than 50% at local noon in samples receiving full solar radiation. Samples exposed to PA and P treatments recovered much faster (within 2–3 days), and there were no significant differences in Y between the three radiation treatments when irradiance was low (late afternoon to early morning). Long-term acclimation seems to be important in A. platensis to cope with high UVR levels however, it is not attained through the synthesis of UV-absorbing compounds but it seems to be mostly related to adaptive morphological changes.     

A new model of phycobilisome in Spirulina platensis

Phycobilisomes (PBS) were isolated from blue-green algaSpirulina platensis. Scanning tunneling microscope was used to investigate the three-dimensional structure of PBS deposited on freshly cleaved highly oriented pyrolytic graphite (HOPG) in ambient condition at room temperature. The results showed that the rods of PBS radiated from the core to different directions in the space other than arrayed in one plane, which was different from the typical hemi-discoidal model structure. The diameter of PBS was up to 70 nm, and the rod was approximately 50 nm in length. Similar results were observed in Langmuir-Blodgett (LB) film of PBS. The dissociated PBS could reaggregate into rod-like structures and easily form two-dimensional membrane while being absorbed on HOPG, however, no intact PBS was observed. The filling-space model structure of PBS inSpirulina platensis with STM from three-dimensional real space at nanometer scale was found, which showed that this new structural model of PBS surely exists in blue-green algae and red algae. The function of this structural model of PBS was also discussed.     

Protoplasts from the cyanobacterium,Spirulina platensis

Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.