Improvement in the Sequential Extraction of Phycobiliproteins from Arthrospira platensis Using Green Technologies.

Arthrospira platensis (commercially known as Spirulina) is an excellent source of phycobiliproteins, especially C-phycocyanin. Phycobiliproteins are significant bioactive compounds with useful biological applications. The extraction process plays a significant role in downstream microalga production and utilisation. The important pigments found in A. platensis include chlorophyll and carotenoids as nonpolar pigments and phycobiliproteins as polar pigments. Supercritical fluid extraction (SFE) as a green extraction technology for the high-value metabolites of microalgae has potential for trends in food and human health. The nonpolar bioactive compounds, chlorophyll and carotenoids of A. platensis, were primarily separated using supercritical carbon dioxide (SC-CO2) solvent-free fluid extraction pressure; the temperature and ethanol as cosolvent conditions were compared. The residue from the A. platensis cells was subjected to phycobiliprotein extraction. The phosphate and water extraction of A. platensis SFE residue were compared to evaluate phycobiliprotein extraction. The SFE results exhibited higher pressure (350 bar) and temperature extraction (50 °C) with ethanol-free extraction and increased nonpolar pigment. Phycobiliprotein yield was obtained from A. platensis SFE residue by ethanol-free buffer extraction as a suitable process with antioxidant properties. The C-phycocyanin was isolated and enhanced to 0.7 purity as food grade. This developed method can be used as a guideline and applied as a sustainable process for important pigment extraction from Arthrospira microalgae.

Arthrospira platensis Mutagenesis for Protein and C-Phycocyanin Improvement and Proteomics Approaches.

Arthrospira (Spirulina) platensis is known for its use as a food supplement, with reported therapeutic properties including antiviral, anti-inflammatory and antioxidant activity. Arthrospira is also an excellent source of proteins and C-phycocyanin. The latter is a light-harvesting pigment-protein complex in cyanobacteria, located on the outer surface of the thylakoid membrane and comprising 40 to 60% of the total soluble protein in cells. Random mutagenesis is a useful tool as a non-genetically modified mutation method that has been widely used to generate mutants of different microorganisms. Exposure of microalgae or cyanobacteria to chemical stimuli affects their growth and many biological processes. Chemicals influence several proteins, including those involved in carbohydrate and energy metabolisms, photosynthesis and stress-related proteins (oxidative stress-reactive oxygen species (ROS) scavenging enzymes). Signal transduction pathways and ion transportation mechanisms are also impacted by chemical treatment, with changes causing the production of numerous biomolecules and stimulation of defence responses. This study compared the protein contents of A. platensis control and after mutagenesis using diethyl sulphate (DES) under various treatment concentrations for effective mutation of A. platensis. Results identified 1152 peptides using proteomics approaches. The proteins were classified into 23 functional categories. Random mutagenesis of A. platensis by DES was found to be highly effective for C-phycocyanin and protein production.

Efficacy of Eosin Dye Removal by Peanut Shell Agrowaste Adsorbent

Abstract Peanut shell (PS) which is an excessive waste-product from agricultural processes, it can be recycled to a natural adsorbent for example it uses as removal dyes. Synthetic dye effluent without improperly discharged from industries to the river cause wastewater and damage to living organisms, especially, anionic dyes are difficult removed by conventional treatments such as biological, chemical, oxidation, and physical-filtration. However, an adsorption treatment is widely used for decolorization of dyes and give the best results for removal of various types of dissolved coloring materials. This research was used Eosin Y (EO) for the anionic model of dyeing wastewater and used PS for agrowaste adsorbent. The purpose of this study was investigated the efficiency adsorption of EO removal by PS. This efficiency adsorption was measured by different PS dosages, contact times, adsorbate concentration and equilibrium data. The results can be concluded that the PS had the efficiency adsorption of EO removal due to the equilibrium adsorption capacity (qe) and the highest dose of PS were balanced to adsorption of dye. The highest EO removal percentage was found in 87.7%, the qe was 0.351 mg g-1 and can adsorb from 10 mg L-1 to 1.23 mg L-1 in 25 g L-1 of PS dose at 30 minutes. In addition, the PS structure was found in multi-layer and many porous which is suitable for adsorbent. The morphological examination of PS was shown before and after adsorption that not changed. Therefore, PS might be an alternative choice for removal dye, and be used for the recycle adsorbent agrowaste as a commercial product for adding their values.

Enhanced Microencapsulation of C-Phycocyanin from Arthrospira by Freeze-Drying with Different Wall Materials.

RESEARCH BACKGROUND: C-phycocyanin (C-PC), a water-soluble blue pigment, was extracted from microalgae Arthrospira sp. C-PC could be a good substitute for synthetic pigments with high antioxidant activity. However, C-PC is unstable due to sensitivity to temperature, light, pH and oxygen; therefore, applications of C-PC in food and other products are limited. Microencapsulation of C-PC using freeze-drying is a solution to this problem and is considered a suitable method for drying the heat-sensitive pigment. EXPERIMENTAL APPROACH: C-phycocyanin was extracted from Arthrospira platensis. C-PC microcapsules were modified by freeze-drying, with maltodextrin and gum Arabic used as microencapsulation wall materials at different fractions from 0 to 100%. The physical properties including moisture content and water activity, solubility, hygroscopicity, bulk density, colour appearance, particle morphology and size distribution of the produced powders were evaluated. Thermal stability and antioxidant activity of freeze-dried microencapsulated C-PC powders were also assessed. RESULTS AND CONCLUSIONS: Freeze-dried microencapsulated C-PC powders with maltodextrin and gum Arabic as wall materials gave high encapsulation efficiency of around 99%. At higher gum Arabic mass fraction, moisture content decreased and water activity improved. Maltodextrin gave higher solubility of C-PC powders whereas gum Arabic led to a similar colour of C-PC to those without microencapsulation. Freeze-dried microencapsulated C-PC powders were composed of different sized microparticles regardless of the combination of wall materials with amorphous glassy shapes. Thermal stability of encapsulated C-PC increased and also showed high antioxidant properties. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study demonstrates that the freeze-dried microencapsulated C-PC powders have pigment stability with antioxidant properties and are resistant to high temperatures. Therefore, they may have a potential for the development of microencapsulated C-PC as a functional ingredient with improved colour and bioactive properties. Such a product can be applied in food, cosmetic, biotechnology and nutraceutical industries.

Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation.

We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aadA-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atpB promoter region. Efficient recombination between atpB repeats deletes the intervening foreign genes and 1,984 bp of plastid DNA (co-ordinates 57,424-59,317) containing the rbcL gene. Only five foreign bases are present in DeltarbcL plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbcL-related sequences in DeltarbcL plants indicated an extra-plastidic origin. Mutant DeltarbcL plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aadA-based transformation with the wild-type rbcL gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aadA genes. Rescue of the DeltarbcL mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbcL deletion mutant isolated here is readily transformed with the efficient aadA marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.

The tobacco plastid accD gene is essential and is required for leaf development.

Angiosperm plastid genomes typically encode approximately 80 polypeptides, mainly specifying plastid-localized functions such as photosynthesis and gene expression. Plastid protein synthesis and expression of the plastid clpP1 gene are essential for development in tobacco, indicating the presence of one or more plastid genes whose influence extends beyond the plastid compartment. The plastid accD gene encodes the beta-carboxyl transferase subunit of acetyl-CoA carboxylase and is present in the plastids of most flowering plants, including non-photosynthetic parasitic plants. We replaced the wild-type accD gene with an aadA-disrupted mutant allele using homologous recombination. Persistent heteroplasmy in the presence of antibiotics indicated that the wild-type accD allele was essential. The phenotype of the accD knockout was revealed in plastid transformants grown in the absence of antibiotics. Leaves contained pale green sectors and lacked part or all of the leaf lamina due to arrested division or loss of cells. Abnormal structures were present in plastids found in mutant plants, indicating that accD might be required to maintain the plastid compartment. Loss of the plastid compartment would be expected to be lethal. These results provide genetic evidence showing the essential role of plastid ACCase in the pathway leading to the synthesis of products required for the extra-plastidic processes needed for leaf development.

Simple and efficient removal of marker genes from plastids by homologous recombination.

Removal of marker genes improves the design of transgenic plants. Homologous recombination between direct repeats provides a simple method for excising marker genes after transgenic cells and shoots have been isolated. Efficient implementation of the method requires high rates of homologous recombination relative to illegitimate recombination pathways. The procedure works well in plastids where homologous recombination predominates. Marker genes are flanked by engineered direct repeats. The number and length of direct repeats flanking a marker gene influence excision rate. Excision is automatic and loss of the marker gene is controlled by selection alone. After transgenic cells have been isolated selection is removed allowing loss of the marker gene. Excision is a unidirectional process resulting in the rapid accumulation of high levels of marker-free plastid genomes. Cytoplasmic sorting of marker-free plastids from marker-containing plastids leads to the isolation of marker free plants. Marker-free plants can be isolated following vegetative propagation or among the progeny of sexual crosses.