R. Sarada

Phycocyanin from Spirulina sp: influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin ☆

A number of drying methods studied for the processing of Spirulina (crossflow dried, spray dried and oven dried) resulted in approximately 50% loss of phycocyanin. Therefore fresh biomass was suitable for phycocyanin extraction. Of the extraction methods tested, freezing and thawing of cells, homogenisation using a mortar and pestle in the presence of abrasive material and homogenisation using a blender at 10 000 rpm yielded 19.4±0.4 mg phycocyanin per 100 mg dry weight of Spirulina while water extraction was a slow process. Acid treatment also resulted in phycocyanin leaching. Phycocyanin was stable over a pH range of 5–7.5 at 9±1°C, whereas temperature beyond 40°C lead to instability. The pigment phycocyanobilin was separated from the phycocyanin.

Influence of stress on astaxanthin production in Haematococcus pluvialis grown under different culture conditions

The influence of stress was studied on astaxanthin production in Haematococcus pluvialis grown under different culture conditions. High concentrations of NaCl (>1.0% w/v) were lethal and the age of the culture was crucial for stress induced astaxanthin production. Four to-eight-day-old cultures were sensitive to NaCl addition while older cultures (12–16 days) were resistant. Older cells accumulated 8.3–10.69 mg/l astaxanthin compared to 0.95–8.1 mg/l in 4–8-day-old cultures, respectively. Cultures grown with calcium nitrate as nitrogen source showed a significant increase in astaxanthin content and production under stress compared to other nitrogen sources. Similarly, cultures grown at pH 7.0 showed more astaxanthin production than those grown at pHs of 6.0, 8.0 and 9.0.

Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions

Haematococcus pluvialis, a green alga, accumulates carotenoids, predominantly astaxanthin, when exposed to stress conditions. In the present work, changes in the pigment profile and expression of carotenogenic genes under various nutrient stress conditions and their regulation were studied. Nutrient stress and higher light intensity in combination with NaCl/sodium acetate (SA) enhanced total carotenoid and total astaxanthin content to 32.0 and 24.5 mg g(-1) of dry biomass, respectively. Expression of carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene cyclase (LCY), beta-carotene ketolase (BKT), and beta-carotene hydroxylase (CHY) were up-regulated under all the stress conditions studied. However, the extent of expression of carotenogenic genes varied with stress conditions. Nutrient stress and high light intensity induced expression of astaxanthin biosynthetic genes, BKT and CHY, transiently. Enhanced expression of these genes was observed with SA and NaCl/SA, while expression was delayed with NaCl. The maximum content of astaxanthin recorded in cells grown in medium with SA and NaCl/SA correlated with the expression profile of the astaxanthin biosynthetic genes. Studies using various inhibitors indicated that general carotenogenesis and secondary carotenoid induction were regulated at both the transcriptional and the cytoplasmic translational levels. The induction of general carotenoid synthesis genes was independent of cytoplasmic protein synthesis while BKT gene expression was dependent on de novo protein synthesis.

Production of astaxanthin in Haematococcus pluvialis cultured in various media.

Production of astaxanthin by Haematococcus pluvialis in autotrophic (Bold Basal (BBM), Z8 or A9 media), heterotrophic (KM1 medium) and mixotrophic (mixotrophic medium 1 (MM1; BBM + sodium acetate), MM2 (MM1 + L-Asn) and KM2 medium (KM1 without yeast extract)) conditions was examined. Growth of H. pluvialis was faster in heterotrophic (KM1) and mixotrophic (MM2 and KM2) media than in autotrophic media. The highest cell count was observed in KM2 medium. In KM1 medium, total carotenoid content was 181.0 pg/cell (88.67% astaxanthin) after 45 days in liquid medium and 462.7 pg/cell (90.45% astaxanthin) after 32 days on agar medium. Addition of trace elements and B vitamins increased astaxanthin production in all mixotrophic media. Max. astaxanthin concn. (2.2% (w/w) yield) was observed in KM2 medium with added trace elements and B vitamins. It is concluded that addition of sodium acetate, L-Asn, trace elements and B vitamins to autotrophic and mixotrophic media increases biomass and total astaxanthin production by H. pluvialis and should therefore be useful for reducing the cost of large scale astaxanthin production.

Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis.

The anti-ulcer properties of astaxanthin fractions such as total carotenoid and astaxanthin esters from Haematococcus pluvialis were evaluated in ethanol-induced gastric ulcers in rats. Since oxygen radical release is a pathogenic factor of ethanol-induced gastric damage, astaxanthin - a free radical scavenger, was investigated as a potential ulcer preventive agent. Astaxanthin fractions - total carotenoid and astaxanthin esters were orally administered to experimental rats at 100, 250 and 500 microg/kg b.w. prior to ulcer induction. Alcian blue binding assay indicates that, total carotenoid and astaxanthin esters at 500 microg/kg b.w could protect gastric mucin approximately 40% and 67% respectively. Pre-treatment with astaxanthin esters, also resulted in significant increase in antioxidant enzyme levels - catalase, superoxide dismutase, and glutathione peroxidase in stomach homogenate. Histopathological examination substantiated the protective effect of astaxanthin in pre-treated rats. The increased antioxidant potencies such as free radical scavenging activity with an IC(50) of approximately 8 microg/ml and reducing power abilities (59 x 10(3) U/g) in vitro, reveal that H. pluvialis astaxanthin may protect gastric mucosal injury by antioxidative mechanism. In addition, approximately 23 fold increased lipoxygenase-inhibitory property, in comparison with standard astaxanthin and significant H(+), K(+)-ATPase-inhibitory activity of astaxanthin esters, in comparison with known proton pump blocking anti-ulcer drug - omeprazole, may envisage the potential gastroprotective effect by regulating the gastric mucosal injury and gastric acid secretion by the gastric cell during ulcer disease.

Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus pluvialis mutants.

Growing culture of green alga Haematococcus was exposed to mutagens such as UV, ethyl methane sulphonate (EMS) and 1-methyl 3-nitro 1-nitrosoguanidine (NTG), and further screened over herbicide - glufosinate. The survival rate of cells decreased with increasing concentration of mutagens and herbicides. The mutants exhibited 23-59% increase in total carotenoid and astaxanthin contents. The NTG treated glufosinate resistant mutant showed increased (2.2% to 3.8% w/w) astaxanthin content. The transcript levels of phytoene synthase, phytoene desaturase, lycopene cyclase, beta-carotene ketolase and beta-carotene hydroxylase enzymes in the mutant cultures were found to be 13-18, 14-17, 3, 3-22 and 6-20 fold higher respectively compared to wild type. The mutant obtained by UV irradiation showed highest lycopene cyclase activity (458 nmole beta-carotene formed/mg protein/h) followed by NTG mutant (315 nmole beta-carotene formed/mg protein/h) when compared to that of parent strain (105 nmole beta-carotene formed/mg protein/h). Expression analysis of carotenoid biosynthetic genes in the mutants exhibited increase in transcript levels compared to wild type.

Effective inhibition of skin cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis.

Astaxanthin mono- (AXME) and diesters (AXDE) were characterized and examined for anticancer potency with total carotenoids (TC) and astaxanthin (AX) against UV-7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer model in rat. At 200 μg/kg bw, AXDE and AXME reduced UV-DMBA-induced tumor incidences up to 96 and 88%, respectively, when compared to AX (66%) and TC (85%). UV-DMBA has been known to generate high levels of free radicals and tyrosinase enzyme, leading to characteristic symptoms of skin pigmentation and tumor initiation. Intriguingly, ~7-fold increase in tyrosinase and 10-fold decrease in antioxidant levels were normalized by AXDE and AXME as opposed to only ~1.4-2.2-fold by AX and TC, respectively. This result together with the appearance of 72 and 58 ng/mL of retinol in the serum of respective AXE-treated (AXDE + AXME) and AX-treated animals suggested that better anticancer potency of AXEs could be due to increased bioavailability.

Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production

The present study focused on cultivation, seasonal variation in growth, hydrocarbon production, fatty acids profiles of Botryococcus braunii (LB-572 and N-836) in raceway & circular ponds under outdoor conditions. After 18days of cultivation the biomass yield and hydrocarbon contents were increased in both raceway and circular ponds. The fat content was found to be around 24% (w/w) with palmitic and oleic acids as prominent fatty acids. Hydrocarbons of C(20)-C(30) carbon chain length were higher in raceway and circular ponds. Maximum biomass yield (2gL(-1)) and hydrocarbon content (28%) were observed in Nov-Dec. In case of B. braunii (N-836) after 25days of cultivation the biomass yield was 1gL(-1) and hydrocarbon content was 27%. Supplementation of 0.1% NaHCO(3) in the medium resulted in biomass yield of 1.5gL(-1) and hydrocarbon content of 30% compared to control.

Selection and evaluation of CO2 tolerant indigenous microalga Scenedesmus dimorphus for unsaturated fatty acid rich lipid production under different culture conditions

Five indigenous microalgal strains of Scenedesmus, Chlorococcum, Coelastrum, and Ankistrodesmus genera, isolated from Indian fresh water habitats, were studied for carbon-dioxide tolerance and its effect on growth, lipid and fatty acid profile. Scenedesmus dimorphus strain showed maximum growth (1.5 g/L) and lipid content (17.83% w/w) under CO2 supplementation, hence selected for detailed evaluation. The selected strain was alkaline adapted but tolerated (i) wide range of pH (5-11); (ii) elevated salinity levels (up to 100 mM, NaCl) with low biomass yields and increased carotenoids (19.34 mg/g biomass); (iii) elevated CO2 levels up to 15% v/v with enhancement in specific growth rate (0.137 d(-1)), biomass yield (1.57 g/L), lipid content (19.6% w/w) and CO2 biofixation rate (0.174 g L(-1) d(-1)). Unsaturated fatty acid content (alpha linolenic acid) increased with CO2 supplementation in the strain.

Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis

Cultures of Haematococcus pluvialis were exposed to mutagens like u.v. and EMS (ethyl methanesulphonate). The results showed that the survival rate decreased with the increase in u.v. exposure time and increase in EMS concentration. These mutants were further screened using inhibitors of the carotenoid biosynthetic pathway viz. diphenylamine (15–90 μM), nicotine (160–320 μM) and compactin (1.5–3.0 μM). The mutants thus obtained showed early enhanced (2.2–3.2-fold) astaxanthin accumulation and also exhibited higher lycopene cyclase activity.