MORPHOLOGICAL REVERSION OF SPIRULINA (ARTHROSPIRA) PLATENSIS (CYANOPHYTA): FROM LINEAR TO HELICAL1

The cyanobacterium Spirulina Turpin is characterized by its regularly coiled trichomes. Under some conditions, its helical filaments can convert to abnormal morphologies, such as irregularly curved and even linear shapes, that had been considered as a permanent degeneration that could not be reversed. However, here we found that the linear filaments of Spirulina platensis Geitler could spontaneously revert to the helical form with the same morphology as the original filaments. Further studies showed that the ultrastructural, physiological, and biochemical characteristics of linear filaments were different from those of the original filaments, whereas they were the same for the reverted and the original filaments. The SDS‐PAGE analysis revealed at least four proteins or subunits related to Spirulina morphogenesis: The 21.9‐kDa and the 20.3‐kDa proteins were highly expressed in the helical filaments, whereas the 52.0‐kDa and the 31.8‐kDa proteins were highly expressed in the linear filaments. The random amplified polymorphic DNA analysis with 96 random primers showed that the genetic background of the reverted filaments was the same as that of the original filaments but distinct from that of the linear filaments. The results indicated that linear filaments of Spirulina could revert to the original morphology under certain conditions, and their other distinctive traits were regained.     

CULTIVATION OF ARTHROSPIRA (SPIRULINA) PLATENSIS (CYANOPHYCEAE) BY FED‐BATCH ADDITION OF AMMONIUM CHLORIDE AT EXPONENTIALLY INCREASING FEEDING RATES1

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was cultivated under light‐limited conditions in 5‐L open tanks by daily supplying NH₄Cl as nitrogen source. Exponentially increasing feeding rates were adopted to prevent ammonia toxicity. The total feeding time (T) was varied between 12 and 20 days, and the starting (m₀) and total (m_T) quantities of the nitrogen source per unit reactor volume were varied in the ranges 0.19–1.7 mM and 2.3–23.1 mM, respectively. This intermittent addition of the nitrogen source prevented ammonia from reaching inhibitory levels and ensured final cell concentrations (X_m) and cell productivities (P_x) comparable with those of batch runs with KNO₃. Moreover, the lower nitrogen addition due to the use of NH₄Cl rather than KNO₃ allowed for higher nitrogen‐to‐cell conversions (Y_x/n). These results were evaluated using three‐factor, five‐level, central composite experimental planning, combined with the response surface methodology, selecting T, m₀, and m_T as the independent variables and X_m, P_x, and Y_x/n as the response variables. This approach allowed us to identify, through the simultaneous optimization of the variables, T=16 days, m₀=1.7 mM, and m_T=21.5 mM as the best conditions for A. platensis cultivation at 72 μmol photons·m^?2·s^?1. Under these conditions, a maximum cell concentration of 1239 mg ·L^?1 was obtained, which is a value comparable with that obtained using KNO₃ as nitrogen source and nearly coincident with the theoretical one estimated by the response surface methodology.     

A simple method for efficient separation and purification of c-phycocyanin and allophycocyanin from Spirulina platensis

c-Phycocyanin and allophycocyanin were separated and purified from Spirulina platensis by precipitation with ammonium sulphate, ion exchange chromatography and gel filtration chromatography. Pure c-phycocyanin and allophycocyanin were finally obtained with an A₆₂₀/A₂₈₀value of 5.06 and an A₆₅₅/A₂₈₀ value of 5.34, respectively.     

Isolation and Photosynthesis of Spheroplasts in Spirulina platensis

Spirulina platensis is a nonheterocystic filamentous blue-green alga (cyanobacterium). Large quantity of highly qualified spheroplasts were obtained by improved isolation method. The spheroplast has a wrinkled and porous surface. Their diameter ranged from 3.8 btm to 4. 6 μm. The activity of photosynthetic oxygen evolution in the spheroplasts was about 40 % of the intact cell. The absorption spectra of the filaments and spheroplasts at room temperature revealed that they had the same pigments, Chla, PC, PEC and carotenoid. In spheroplasts the relative content of PC and carotenoid decreased, and that of PEC increased. It implicated that the light absorption of Spirulina platensis could be influenced by the cell wall. Some differences existed between the original cells and spheroplasts in the low temperature fluorescence emission spectra. F757 of spheroplasts excited by 436 nm was reduced obviously and that excited by 580 nm was disapeared. F728/F685 and F640/F685 enhanced, and F693/F685 was reduced. F728/F640 was lower than that of the original cells. These results indicated that removing the cell wall may inhibit the PS Ⅱ activity and influence the F695 from core antenna pigment system.     

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum and Spirulina platensis under phosphate limitation

Nostoc muscorum and Spirulina platensis were grown under phosphate deficiency in order to investigate the role of internal phosphate pool and activity of alkaline phosphatase on poly-β-hydroxybutyrate (PHB) accumulation. PHB accumulation in N. muscorum increased to 22.7% of dry weight (dw) after 4 day of phosphate deficiency, while the internal phosphate pool reduced to the level of 0.02 μM mg dw⁻¹ at a maximum APase activity of 2.57 nM PNP mg dw⁻¹ h⁻¹. In contrary, S. platensis depicted maxima of 1.39 nM PNP mg dw⁻¹ h⁻¹ on day 30 of incubation, which was about 2 fold lower than the observed value of N. muscorum. PHB content in S. platensis remained low even after prolonged phosphate starvation, and a rise only up to 3.5% of dw was recorded on day 60 of phosphate deficiency. Supplementation of NADPH exogenously to S. platensis cultures grown under phosphate deficiency favoured PHB accumulation in 10, 20 and 30 days old cultures, but not in the cultures grown under phosphate deficiency for 60 days. The possible role of phosphate limitation on PHB accumulation is discussed.     

Higher plant-like fluorescence induction and thermoluminescence characteristics in cyanobacterium, Spirulina mutant defective in PQH2 oxidation by cytb6/f complex

Characterization of the photosynthetic electron transport in a mutant of Spirulina platensis, generated by chemical mutagenesis, demonstrated that the electron transfer from the plastoquinone (PQ) to cytochrome b6/f was slowed. Thermoluminescence (TL) measurements suggested the presence of reversed energy flow via PQ, which resulted in an emergence of the plant-like after-glow TL band at 45 degrees C that could be enhanced by the transthylakoidal pH gradient and could be eliminated by an uncoupler, FCCP. The localization of the changes in the electron transport of the mutant cells measured by various methods revealed that the re-oxidation of the PQ pool is hampered in the mutant compared to the wild-type cells. The reduction in energy migration was localized between PQ and PS I reaction centers.     

Urea increases fed‐batch growth and γ‐linolenic acid production of nutritionally valuable Arthrospira (Spirulina) platensis cyanobacterium

In this article we investigate the simultaneous influence of feeding time and amount of urea added as a nitrogen source on the fed‐batch growth and composition of Arthrospira (Spirulina) platensis. Cultivations were performed in 5‐L minitanks at constant temperature (25°C) and light intensity (42 μmol photons/m²s), using exponentially increasing rate of urea addition, and varying the above independent variables in the ranges 9–15 days and 4.6–12.1 mM, respectively. Special emphasis was placed on the content of added high value fatty acids (e.g., γ‐linolenic acid) of concern for the food industry. To this purpose, a 2²‐plus star central composite design was employed, and maximum cell concentration, cell productivity, yield of biomass on nitrogen added, protein content and fatty acids profile were evaluated by multiple regression analysis. The highest cell concentration (1759 mg/L) was obtained at feeding time of 14 days and amount of urea per unit reactor volume of 5.8 mM, while the highest contents of γ‐linolenic acid (27.5% of the lipid fraction) and proteins (77.2%) were obtained at 10 and 14 days and 5.8 and 10.8 mM, respectively. The results confirm the possibility of using urea as cheap nitrogen source to culture this nutritionally valuable cyanobacterium.     

Separation and nanoencapsulation of antitumor polypeptide from Spirulina platensis

Spirulina platensis is a multicellular edible blue‐green alga with abundant proteins (～60%). No report is available on the antitumor polypeptides from the whole proteins of S. platensis. In this study, for the first time, an antitumor polypeptide Y2 from trypsin digest of S. platensis proteins was obtained by using freeze‐thawing plus ultrasonication extraction, hydrolysis with four enzymes (trypsin, alcalase, papain, and pepsin), and gel filtration chromatography. The results showed that the degree of hydrolysis can be ordered as: trypsin (38.5%) > alcalase (31.2%) > papain (27.8%) > pepsin (7.1%). For MCF‐7 and HepG2 cells, at 250 µg/mL, the maximum inhibitory rate of Y2 was 97%, while standard drug 5‐FU was 55 and 97%, respectively. Furthermore, the nanoencapsulation of Y2 with chitosan (CS) was also investigated. After nanoencapsulation, the maximum encapsulation efficiency and polypeptides contents are 49 and 15%, respectively; and the antitumor activity is basically not lost. These data demonstrated the potential of nanopolypeptides (Y2‐CS) in food and pharmaceutical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1230–1238, 2013     

Organization of the Biosynthetic Gene Cluster for the Polyketide Antitumor Macrolide,Pladienolide, in Streptomyces platensis Mer-11107

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.