Polyhydroxybutyrate production from marine source and its application.

The increasing significance of non-degradable plastic wastes is an emerging concern. As a substitute, researches are being endeavoured from existing reserve to yield bioplastics based on their properties of biodegradability. Owing to their cost, now the experts are quest for a substitute source like bacteria, microalgae, actinomycetes, cyanobacteria and plants. PHB is biodegradable, environmental friendly and biocompatible thermoplastics. Varying in toughness and flexibility, depending on their formulation, they are used in various ways similar to many non-biodegradable petrochemical plastics currently in use. Promising strategies contain genetic engineering of microorganisms to introduce production pathways examined for the past two decades. Such kind of researches focusing on the use of unconventional substrates, novel extraction methods, and genetically enhanced species with assessment to make PHB from marine microbes are commercially attractive field. Hence, this biopolymer synthesis may displayed as one of the survival mechanisms of endosymbiotic, macroalgae, or sponge-associated bacteria, which exist in a highly competitive and stressful marine microenvironment. This review throws light on the promising and growing awareness of using marine microbes as PHB source, along with their applications in different fields of aquaculture, medicine, antifouling and tissue engineering.

κ-Carrageenan: An effective drug carrier to deliver curcumin in cancer cells and to induce apoptosis.

The current study is to develop a natural drug carrier with seaweed derived polymers namely κ-Carrageenan (κ-Car) for drug delivery applications. κ-Car is a natural polysaccharide which derived from edible red seaweeds, they are easily available, non-toxic, cost effective, biodegradable and biocompatible nature. Curcumin (Cur) is a yellow-orange polyphenol existing in turmeric, which is predominantly used as spice and food coloring agent. The ultimate use of polymeric composites, especially those composed of natural polymers, has become a very interesting approach in recent drug delivery applications, due to their non-toxicity and biological origin. In this study the primary approach which depends on the loading of Curcumin into κ-Carrageenan was accomplished, and which (κ-Car-Cur) an active drug carrier was developed for drug delivery against selected lung cancer cells (A549). Thus, the κ-Car-Cur was synthesized by solvent evaporation method followed by freeze drying, and it was further characterized. From this study, it has been reported that the high encapsulation efficiency, good stability, and successful release of Cur from the carrier (κ-Car) was achieved. The drug release was more active at acidic pH 5.0 with the cumulative release of 78%, which is the favorable condition present in tumor microenvironments. The in vitro cellular applications studies of κ-Car-Cur demonstrated that, κ-Car-Cur composites induced higher cytotoxicity against selected cancer cells than free Cur and effectively involved to trigger cellular apoptosis in A549 cancer cells. Further, it was also possessed that inhibition of cell growth and changes in metabolic activity of cancer cells are the unique characteristic features of cellular apoptosis, through reactive oxygen species (ROS) generation. It also observed that there was a decrease in mitochondrial membrane potential (ΔψmΔψm) which leads to a cellular apoptosis during treatment with κ-Car-Cur. Hence, the study outcomes may provide the potential outline for the use of κ-Car-Cur as a promising tool to deliver drugs at intracellular level.

Synthesis of chitosan capped copper oxide nanoleaves using high intensity (30kHz) ultrasound sonication and their application in antifouling coatings.

The synthesis of chitosan capped copper oxide nanoleaves (CCCO NLs) was carried out under three different reaction conditions viz. 1) room temperature, 2) 70°C and 3) high intensity ultrasound (30kHz) sonication method and it has been found that the high intensity ultrasound (30kHz) sonication is the best method when compared to other two methods. The advantages of the present synthetic method are: i) easy one step process, ii) lesser reaction time, iii) good yield, iv) reproducible and v) calcination is not required. The resulting chitosan capped copper oxide nanoleaves were characterized by Diffuse Reflectance UV-Visible Spectroscopy (DRS), Fourier Transform Infra-Red Spectroscopy (FT-IR), X-ray Diffraction (XRD), X-ray Photoelectron Spectroscopy (XPS), Field Emission Scanning Electron Microscopy (FESEM), High Resolution Transmission Electron Microscopy (HRTEM) and Thermo gravimetric analysis (TGA). The CCCO NLs were blended with commercial paints such as polyurethane clear, polyurethane white and acrylic emulsion and applied on to three different surfaces (wood, mild steel and cement slab panels). The hydrophilicity of CCCONP coated panels was analyzed by water contact angle measurement and their antifouling behavior was investigated against three different green and marine algae viz. Arthrospira, Chlorella and Amphora. The antifouling efficiency of the CCCO NLs against the algae was found to be 78-92%.

Mass cultivation of UV-B adapted Arthrospira platensis RRGK under open raceway pond for the production of Poly-ß-hydroxy butyrate.

Six different strains of cyanobacteria were isolated from the freshwater lake, Arakkonam, India. Staining of cells with Nile Red showed the presence of large quantities of PHB granules in the cell cytoplasm of Arthrospira. Molecular identification of the strain was carried out using 16S rRNA analysis and their systematic position was ascertained as Arthrospira platensis RRGK. Studies were carried out on random mutagenesis approach using UV-B radiation for enhancing the production of PHB. Further, Response Surface Methodology was used for optimization of pH, temperature, and sodium bicarbonate for higher biomass and PHB production. Under open raceway pond A. platensis RRGK produced biomass concentration of 2.2±0.13gL-1 and 131±0.36gL-1 of PHB content. It was chemically characterized through FTIR, DSC, TGA and XRD analyses. Hence, PHB can be produced from cyanobacteria by sequestering harmful CO2. It can also be used as a substitute for synthetic polymers in tissue engineering.

Optimization of polyhydroxybutyrate production utilizing waste water as nutrient source by Botryococcus braunii Kütz using response surface methodology.

Investigations have been made to optimize various factors including pH, temperature, and substrate for enhanced polyhydroxybutyrate (PHB) production in Botryococcus braunii which serves as a pioneer for production of bioplastic (PHB). Polyhydroxybutyrate is a natural, decomposable polymers accumulated by the microorganism under different nutritional condition. Strain selection was done by staining method using Sudan black and Nile red dye. Using response surface methodology (RSM), three level- three variables Box Behnken design (BBD), the best potential combination of pH (4-11), temperature (30-50°C) and sewage waste water as substrate fed at different concentrations at 20%-100% for maximum PHB production was investigated. Maximum yield (247±0.42mg/L) of PHB dry weight was achieved from the 60% concentration of sewage waste water as a growth medium at pH 7.5 at 40°C. It was well in close agreement with the value predicted by RSM model yield (246± 0.32mg/L). Thus the study shows the production of PHB by B. braunii along with the basic characterization of PHB by using FTIR and TEM analysis. These preliminary studies indicated that PHB can also be produced by B. braunii utilizing waste water. There is no report on the optimization of PHB production in this microalgae have been documented.

Biosynthesis, purification and characterization of polyhydroxybutyrate from Botryococcus braunii kütz.

Polyhydroxybutyrate (PHB) is completely biodegradable which is metabolised by microorganisms in the soil as their sole food source in few years. The level of PHB up to 10.6% of algal dry weight is of great potential of the eco-friendly nature. Botryococcus braunii is mainly used for the production of biodiesel and is also capable of producing biopolymer polyhydroxy butyrate (PHB). In this study, Botryococcus braunii is used which generally produce PHB to around 20% of the dry weight. Three different microalgae were isolated from the fresh water of Kolavoi lake of Tamil Nadu. They were identified by their morphological features under the light microscope. The primary screening of PHB intracellular granules was made by using Nile red dye under a fluorescent microscope. Among them, Botryococcus braunii showed high accumulation of PHB granules. For authentic confirmation, the chloroform extracted PHB was analysed by FTIR, XRD and DSC-TGA analyses to characterize PHB with commercial biodegradable thermoplastic. This is the first report in B. braunii for its PHB production.

Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions.

Essential oils of Cymbopogon citratus were already reported to have wide ranging medical and industrial applications. However, information on polysaccharides from the plant and their anticancer activities are limited. In the present study, polysaccharides from C. citratus were extracted and fractionated by anion exchange and gel filtration chromatography. Two different polysaccharide fractions such as F1 and F2 were obtained, and these fractions were found to have distinct acidic polysaccharides as characterized by their molecular weight and sugar content. NMR spectral analysis revealed the presence of (1→4) linked b-d-Xylofuranose moiety in these polysaccharides. Using these polysaccharide fractions F1 and F2, anti-inflammatory and anticancer activities were evaluated against cancer cells in vitro and the mechanism of action of the polysaccharides in inducing apoptosis in cancer cells via intrinsic pathway was also proposed. Two different reproductive cancer cells such as Siha and LNCap were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation, changes in mitochondrial membrane potential, and profiles of gene and protein expression in response to treatment of cells by the polysaccharide fractions. These polysaccharide fractions exhibited potential cytotoxic and apoptotic effects on carcinoma cells, and they induced apoptosis in these cells through the events of up-regulation of caspase 3, down-regulation of bcl-2 family genes followed by cytochrome c release.

Induction of ROS-dependent mitochondria-mediated intrinsic apoptosis in MDA-MB-231 cells by glycoprotein from Codium decorticatum.

Marine macroalgae consist of a range of bioactive molecules exhibiting different biological activities, and many of these properties are attributed to sulfated polysaccharides, fucoxanthin, phycobiliproteins, and halogenated compounds. In this study, a glycoprotein (GLP) with a molecular mass of ∼48 kDa was extracted and purified from Codium decorticatum and investigated for its cytotoxic properties against human MDA-MB-231 breast cancer cells. The IC50 values of GLP against MDA-MB-231 and normal breast HBL-100 cells (control) were 75 ± 0.23 µg/mL (IC25), 55 ± 0.32 µg/mL (IC50), and 30 ± 0.43 µg/mL (IC75) and 90 ± 0.57 µg/mL (IC25), 80 ± 0.48 µg/mL (IC50), and 60 ± 0.26 µg/mL (IC75), respectively. Chromatin condensation and poly(ADP-ribose) polymerase (PARP) cleavage studies showed that the GLP inhibited cell viability by inducing apoptosis in MDA-MB-231 cells. Induction of mitochondria-mediated intrinsic apoptotic pathway by GLP was evidenced by the events of loss of mitochondrial membrane potential (ΔΨ(m)), bax/bcl-2 dysregulation, cytochrome c release, and activation of caspases 3 and 9. Apoptosis-associated factors such as reactive oxygen species (ROS) formation and loss of ΔΨ(m) were evaluated by DCFH-DA staining and flow cytometry, respectively. Cell cycle arrest of G2/M phase and expression of apoptosis associated proteins were determined using flow cytometry and Western blotting, respectively.

Further studies and biological activities of macromolecular protein R-Phycoerythrin from Portieria hornemannii.

In the present study, the purified R-Phycoerythrin (R-PE) from a red alga Portieria hornemannii was subjected to the analysis of stability under the influence of different agents. Among the various inhibitors tested on R-PE EDTA at lower concentrations (<1 mM) supported the activity of R-PE. When R-PE was exposed to different organic solvents, ethanol supported the activity at the maximum followed by acetone, ethyl acetate, chloroform and methanol. Citric acid, as a preservative maintained the stability of R-PE both under 0 ± 5 °C and 30 ± 5 °C with 59.34% and 56.23% respectively, on 30th day. Thermal decomposition of the R-PE began near 60 °C. Maximum weight loss was occurred between 150 °C and 500 °C. Complete weight loss was recorded around 875 °C. Thermal denaturation was observed between 19 °C and 40 °C. Moderate to low antioxidant activities were observed in R-PE in relation to total antioxidant activities. After characterization, R-PE was taken for in vitro anticancer studies against selected cancer cell lines. Further studies involving AO/EB fluorescence staining and phase contrast microscope revealed characteristic apoptotic features like cell shrinkage, membrane blebbing, and nuclear DNA fragmentation, etc. Likewise, FACS analysis revealed the cell cycle distribution pattern of A549 and HepG2 cells.

Stabilization of mitochondrial and microsomal function of fucoidan from Sargassum plagiophyllum in diethylnitrosamine induced hepatocarcinogenesis.

Crude fucoidan from Sargassum plagiophyllum extracted from blade and purified by Q-Sepharose fast flow anion-exchange chromatography and three fucoidan fractions were obtained. Maximum sulphate containing fucoidan fraction was considered as purified fucoidan and purity was checked with agarose gel electrophoresis. The monosaccharides of purified fucoidan analysed by HPLC revealed the presence of the sugars such as fucose as a major sugar were 70.8 mol%. The percentages of other sugars were galactose (13.5%), xylose (2.5%) and mannose (11.2%). GPC was used to analyse molecular weight of purified fucoidan and it was found to be 35 kDa. The levels of ICDH, SDH, MDH, a-KGDH, Phase-I biotransformation enzymes, and Phase-II biotransformation enzymes were decreased in cancer bearing animals which may be due to oxidative stress and mitochondrial damage and fucoidan restored these enzyme activities. The inhibition of carcinogen metabolic activation indicates the anticancer activity of fucoidan in DEN induced liver cancer.

Isolation and characterization of macromolecular protein R-Phycoerythrin from Portieria hornemannii.

R-Phycoerythrin (R-PE) is one of the three phycobiliproteins which are extensively used as fluorescent probes, and it is prepared from red macro-algae. This macromolecular protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy, cosmetics, protein and cell labeling, and analytical processes. In the present investigation, R-PE was isolated and purified from a red alga Portieria hornemannii. R-PE extracted and purified through ammonium sulfate precipitation (55%) followed by Q-Sepharose column chromatography had yielded a maximum purity of 5.2%. R-PE exhibited a typical "three-peak" with absorption maxima at 499, 545 and 565 nm. CD spectrum of R-PE yielded the following secondary structure data: alpha helix (14.30%), beta helix (28.10%), turn helix (19.20%) and random coil helix (38.40%). The molecular mass of R-PE was 240 kDa under Native-PAGE. Three different subunits such as α, ß and γ of 16 kDa, 21 kDa and 39 kDa were segregated under SDS-PAGE. On two dimensional gel electrophoresis, one basic and four acidic subunits were detected. Five different tryptic peptides were assigned under MALDI-TOF. The sequences of N-terminus of R-PE of 10 different amino acids are Met Lys Gln Met Trp Asp Arg Met Val Val. The preparative procedures of the R-PE extraction and purification established based on the experiments exhibit advantages and can offer a reference for R-PE preparation from other marine red macro-alga P. hornemannii.

Mass culture of Botryococcus braunii Kutz. under open raceway pond for biofuel production.

Three different strains of colonial green alga Botryococcus bruanii Kutz. AP103, AP104 and AP105 were isolated from the freshwater lake Kolleru, Andhra Pradesh, India. The morphological features of these three isolates were studied under laboratory conditions. Molecular identification of the strains was carried out using 18S rRNA analysis and their systematic position was confirmed to species level as B. braunii. Among these isolates, B. braunii AP103 recorded highest biomass concentration of 1.7±0.12 g L(-1), 17% lipids, 17% proteins, 32% carbohydrates and 13% hydrocarbons under laboratory condition. Under open raceway pond AP103 produced a biomass concentration of 1.8±0.13 g L(-1), 19% lipids, 33% carbohydrates, 18% proteins and 11% hydrocarbons. The hydrocarbons profile showed the presence of heptadecane (34%) and hexadecane (12.5%). Oleic (25.7%), linolenic (34.26%) and palmitic (9.42%) acids were the major fatty acids present in the lipids extracted from AP103.

Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries.

An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component.

Cardioprotective activity of Cladosiphon okamuranus fucoidan against isoproterenol induced myocardial infarction in rats.

Fucoidans, sulfated polysaccharides of brown algae, have attracted steady attention in the last few years as readily accessible biopolymers possessing a wide spectrum of biological activities. In this study, cardioprotective activity of fucoidan extracted from Cladosiphon okamuranus was evaluated in isoproterenol induced myocardial infarction in rats. Male Wistar albino rats (180±25 g) were divided in to four groups of six animals each as follows: Group (1) control, Group (2) isoproterenol alone, Group (3) fucoidan alone and Group (4) fucoidan+isoproterenol. To evaluate the efficacy of fucoidan treatment against isoproterenol induced myocardial damage, biochemical parameters and histopathological studies were carried out. Isoproterenol administration produced severe myocardial damage and high lipid peroxidation level. On the contrary, fucoidan treatment reduced myocardial damage, which has been reflected by improvement in parameters such as creatinine phosphokinase (CPK), lactate dehydrogenase (LDH), alanine transaminase (ALT) and aspartate transaminase (AST). In addition, fucoidan improved the antioxidant defence system in treated animals and considerably reduced the oxidative stress exerted by isoproterenol. The reduction in oxidative stress in Group (4) was evident from the lipid peroxidation and antioxidant activities. Furthermore, the increase in the levels of total cholesterol, triglycerides and low density lipoprotein (LDL) and decrease in the levels of high density lipoprotein (HDL) was significantly reversed in Group (4), when compared with Group (2). The histopathological studies also showed that fucoidan treatment significantly minimized the damage induced by isoproterenol. Thus, fucoidan provide cardioprotection against isoproterenol induced myocardial infarction in rats.

Synthesis and antioxidant properties of enone core based dendrimers with carbazole as surface group.

Synthesis of enone core based dendrimers with carbazole as surface group has been achieved. All the synthesized dendrimers showed excellent antioxidant behavior with commercially available 1,1-diphenyl-2-picryl hydrazyl (DPPH).

Synthesis and antibacterial activity of some novel imidazole-based dicationic quinolinophanes.

Synthesis of novel quinoline based dicationic benzimidazolophanes and imidazolophanes incorporating various spacer units is described. Some of the quinolinophanes 1b, 3a,3b and 4a exhibit good antibacterial activity against most of the human pathogenic bacteria in the tested concentrations as compared to the other cyclophanes as well as the test control, streptomycin.

PCR-identification of Dunaliella salina (Volvocales, Chlorophyta) and its growth characteristics.

The saline pond microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's (basal) medium under laboratory conditions was confirmed by amplifying the chromosomal DNA of the microalga by PCR with specific primers MA1 and MA2. Seaweed extracts obtained from Sargassum wightii and Ulva lactuca were amended separately at 1.0%, 1.5%, 2.0% and 2.5% levels to the basal medium in order to assess their potential on the growth and concentration of pigments, viz. Chl a, Chl b and beta-carotene of the alga. beta-Carotene was isolated and visible absorption spectrum was taken at 443 and 475 nm confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene isomers. Maximum yield, highest division rate (mu) and highest pigment concentrations were observed in the cells grown in 1.5% S. wightii and 2.0% U. lactuca amended medium and these cells were subjected to DAPI staining. The results of epifluorescence microscopy and image analysis revealed a significant enhancement of the cell and nuclear area of the microalgae.

Protective effect of Dunaliella salina (Volvocales, Chlorophyta) against experimentally induced fibrosarcoma on wistar rats.

The beta-carotene-yielding microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's medium was harvested and lyophilized. Fibrosarcoma was induced in rats by 20-methylcholanthrene. 0.5 g and 1.0 g of lyophilized D. salina powder was administered to the rats orally through carboxy methyl cellulose. Cisplatin was administered along with vitamin E to compare the protective effect of D. salina against fibrosarcoma. Administration of D. salina decreased the levels of cholesterol and lactate dehydrogenase as well as the activities of catalase, superoxide dismutase, serum aspartate aminotransaminase, serum alanine aminotransferase, when compared to control. A significant reduction in the levels of hepatic and renal RNA and DNA was observed in the sarcoma rats when treated with D. salina powder. Histopathological studies of tumor tissues showed regenerative and regressive changes. beta-carotene globules isolated from the powder of Dunaliella salina confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene.