Improvement in vitamin B12 status of Wistar rats by supplementing the diet with Chlorella vulgaris biomass.

The sources of bioavailable vitamin B12 are limited, and most of them are animal-derived. Chlorella vulgaris, a freshwater microalga, is known for immune system boosting, nutraceutical properties and presence of a natural form of vitamin B12. The present study focused on the in vivo evaluation of the Chlorella biomass as a source of bioavailable vitamin B12 to alleviate the vitamin B12 deficiency status of Wistar rats. Experimental animals were evaluated for the vitamin B12 deficiency-related circulatory marker (serum vitamin B12) and functional markers (plasma homocysteine and urinary methylmalonic acid), haematological and histological changes. The results showed that an increase of 2.4-fold in urinary methylmalonic acid (13.01 ± 0.89 µmoles moles of creatinine-1), 2.6-fold in plasma homocysteine (17.18 ± 3.57 µmole L-1), and 48% decrease in serum vitamin B12 levels (252.69 ± 1.46 pg mL-1) in vitamin B12 deficient group compared to control animals. The Chlorella biomass supplementation in the diet led to the restoration of the functional and circulatory markers, hematological parameters, and vitamin B12 content of kidney and liver to control levels. The Chlorella biomass supplementation increased the erythrocyte precursors and MAST cells in the bone marrow and also normalized the histological features of kidney, liver, and lung tissues. The results suggest that the vitamin B12 from the Chlorella biomass was bioavailable and facilitated the improvement of vitamin B12 status in deficient rats.

Chlorella vulgaris cultivation in airlift photobioreactor with transparent draft tube: effect of hydrodynamics, light and carbon dioxide on biochemical profile particularly ω-6/ω-3 fatty acid ratio.

Chlorella vulgaris is used for food and feed applications due to its nutraceutical, antioxidant and anticancer properties. An airlift photobioreactor comprising transparent draft tube was used for C. vulgaris cultivation. The effect of reactor parameters like hydrodynamics (0.3-1.5 vvm), light intensity (85-400 µmol m-2 s-1), photoperiod (12-24 h) and gas-phase carbon dioxide (CO2) concentration (5-15% v/v) were evaluated on microalgae and associated bacterial growth, biochemical profile; with special emphasis on ω-3, ω-6 fatty acids, and vitamin B12. The optimal growth of C. vulgaris without CO2 supplementation was observed at 1.2 vvm, which was associated with higher algal productivity, chlorophyll, vitamin B12 content, and bacterial load along with 72% of nitrate removal. The higher light intensity (400 µmol m-2 s-1) and photoperiod (24:0) increased biomass productivity and ω-3 fatty acid content (in lipid) up to 2-3 fold. The elevated levels of gas-phase CO2 concentration (15% v/v) enhanced EPA content up to 7% and biomass productivity up to 171 mg L-1 day-1. However, the increase in CO2 concentration lowered vitamin B12 content (up to 30%) and bacterial load (2-3 log). Also, all the cultivation conditions favoured desirable ω-6/ω-3 ratio(in the range of 1-2).

Development of a carotenoid enriched probiotic yogurt from fresh biomass of Spirulina and its characterization.

Incorporation of Spirulina in milk as thermally dried powder has the disadvantages of non-uniform distribution with undesirable odor and flavor. Through homogenization (200 ± 10 bar), complete dispersion of fresh Spirulina biomass (7% w/w) in milk was achieved and thereafter a carotenoid enriched probiotic yogurt was developed. Confocal microscopy revealed porous Spirulina-milk protein matrix integrated with smaller fat globules in the yogurt. Spirulina led to a 29.56% increase in Lactobacillus acidophilus count, a 20% reduction in fermentation time and a total probiotic count of 1.2 × 107 CFU mL-1. The protein, total chlorophyll, total carotenoid and ß-carotene content (on dry w/w basis) were 3.58 ± 0.08 g 100 g-1, 0.407 ± 0.018 mg g-1, 0.235 ± 0.016 mg g-1 and 13.28 ± 0.08 µg g-1, respectively. During storage (18 days at 6-8 °C), the L. acidophilus count reached 8.83 ± 0.11 log CFU mL-1 with 103.03% increase in the viability by day three and the yogurt retained 71.5% carotenoids. The probiotc Spirulina yogurt was found to be acceptable to consumers as evaluated by affective consumer test.

Growth and biochemical characteristics of an indigenous freshwater microalga, Scenedesmus obtusus, cultivated in an airlift photobioreactor: effect of reactor hydrodynamics, light intensity, and photoperiod.

The freshwater green algae, Scenedesmus obtusus, was cultivated in a 3.4 L airlift photobioreactor. The hydrodynamic parameters were estimated at different inlet gas flow rates (1, 2, 3, and 4 LPM) and their subsequent impact on the growth and biochemical characteristics of microalgae was studied. The biomass concentration and productivity increased with an increase in flow rates from 1 to 4 LPM. A maximum of 0.07 g L-1 day-1 productivity of biomass was attained at 3 LPM. An increase of total carbohydrate content from 19.6 to 26.4% was noticed with increment in the inlet flow rate of gas from 1 to 4 LPM. Major variations in total fatty acid content were not observed. The impact of light irradiance on growth and biochemical characteristics of S. obtusus was also evaluated. A maximum biomass productivity of 0.103 g L-1 day-1 was attained at an illumination of 150 µmol m-2 s-1 under continuous light. The major fatty acids reported were palmitic acid (C16:0), α-linolenic acid (C18:3), linoleic acid (C18:2), and oleic acid (C18:1). Biodiesel properties of the microalgae were estimated under various culture conditions. The light profile inside the airlift reactor was experimentally measured and the predictive modelling of light profile was also attempted.

Metabolic engineering of Dunaliella salina for production of ketocarotenoids.

Dunaliella is a commercially important marine alga producing high amount of ß-carotene. The use of Dunaliella as a potential transgenic system for the production of recombinant proteins has been recently recognized. The present study reports for the first time the metabolic engineering of carotenoid biosynthesis in Dunaliella salina for ketocarotenoid production. The pathway modification included the introduction of a bkt gene from H. pluvialis encoding ß-carotene ketolase (4,4'ß-oxygenase) along with chloroplast targeting for the production of ketocarotenoids. The bkt under the control of Dunaliella Rubisco smaller subunit promoter along with its transit peptide sequence was introduced into the alga through standardized Agrobacterium-mediated transformation procedure. The selected transformants were confirmed using GFP and GUS expression, PCR and southern blot analysis. A notable upregulation of the endogenous hydroxylase level of transformants was observed where the BKT expression was higher in nutrient-limiting conditions. Carotenoid analysis of the transformants through HPLC and MS analysis showed the presence of astaxanthin and canthaxanthin with maximum content of 3.5 and 1.9 µg/g DW, respectively. The present study reports the feasibility of using D. salina for the production of ketocarotenoids including astaxanthin.

Evaluation of hepatoprotective and antioxidant activity of astaxanthin and astaxanthin esters from microalga-Haematococcus pluvialis.

Effect of isolated astaxanthin (ASX) and astaxanthin esters (ASXEs) from green microalga-Haematococcus pluvialis on hepatotoxicity and antioxidant activity against carbon tetrachloride (CCl4) induced toxicity in rats was compared with synthetic astaxanthin (SASX). ASX, ASXEs, and SASX, all dissolved in olive oil, fed to rats with 100 and 250 µg/kg b.w for 14 days. They were evaluated for their hepatoprotective and antioxidant activity by measuring appropriate enzymes. Among the treated groups, the SGPT, SGOT and ALP levels were decreased by 2, 2.4, and 1.5 fold in ASXEs treated group at 250 µg/Kg b.w. when compared to toxin group. Further, antioxidant enzymes catalase, glutathione, superoxide dismutase and lipid peroxidase levels were estimated in treated groups, their levels were reduced by 30-50 % in the toxin group, however these levels restored by 136.95 and 238.48 % in ASXEs treated group at 250 µg/kg. The lipid peroxidation was restored by 5.2 and 2.8 fold in ASXEs and ASX treated groups at 250 µg/kg. The total protein, albumin and bilirubin contents were decreased in toxin group, whereas normalized in ASXEs treated group. These results indicates that ASX and ASXEs have better hepatoprotection and antioxidant activity, therefore can be used in pharmaceutical and nutraceutical applications and also extended to use as food colorant.

Stress induced modifications in photosystem II electron transport, oxidative status, and expression pattern of acc D and rbc L genes in an oleaginous microalga Desmodesmus sp.

The study aimed at understanding the biochemical and molecular level modifications in Desmodesmus sp. under lipid inducing stress conditions. The low-temperature (5 °C) incubation and nitrogen starvation reduced the PS II electron transport in microalga with a maximum reduction of 50-57% in ET0/ABS values. The PS II electron transport recovered in UV treated cultures after an initial reduction of 87-93% in ET0/ABS values. A 2.7-4.4 fold increase in ROS and MDA levels was observed under low-temperature incubation, and nitrogen starvation. The UV treatment caused 1.3-2.4 fold higher ROS and MDA levels than control. The low-temperature incubated, nitrogen starved, and UV treated cultures showed 2.4-4 fold higher acc D gene expression. A higher rbc L gene expression was observed under low-temperature stress. The study showed modifications in PS II electron transport, oxidative status, and expression of acc D and rbc L genes under stress conditions.

Enhanced accumulation of alpha-linolenic acid rich lipids in indigenous freshwater microalga Desmodesmus sp.: The effect of low-temperature on nutrient replete, UV treated and nutrient stressed cultures.

The indigenous microalga, Desmodesmus sp. produced alpha-linolenic acid (ALA) rich lipids in response to low temperature and UV treatment. Incubation at 5 °C showed a 1.5 fold increase in lipid content (34% w/w) with 44% ALA fraction of total fatty acids. The UV treatment (UV 60 min) exhibited a 1.4 fold increase in biomass productivity and 1.6 fold increase in lipid content (37% w/w) with ALA fraction as 31% of total fatty acids. The nitrogen stress enhanced the lipid content (39% w/w) with a reduced ALA fraction (18%) of total fatty acids. The UV treated cultures (UV 40 and 60 min) on incubation at 5 °C showed maximum lipid accumulation (59 to 62% w/w) with ALA fraction of total fatty acids as 39 to 42%. The incubation of nutrient-replete and UV treated cultures at low-temperature could therefore be used for the production of ALA-rich lipids in microalgae.

Strategies for enhancement of alpha-linolenic acid rich lipids in Desmodesmus sp. without compromising the biomass production.

The indigenous freshwater microalga Desmodesmus sp. produces ALA rich lipids (about 23%). The phytohormones (DAH and KIN; 0.5 mg L-1) increased the biomass yield and lipid content of microalga by 1.4-1.7 fold. Mixotrophic cultivation (500 mM glucose and 100 mM sodium acetate) enhanced the biomass yield and lipid content by 1.8-2.7 fold. The sodium azide (1.0 mM) led to a 1.5 fold and 1.7 fold enhancement in the lipid content and ALA fraction of total fatty acids, respectively without affecting the biomass yield. The low temperature (5 °C) as the second stage of cultivation enhanced the ALA fraction of total fatty acids by 1.2-1.5 fold for untreated, phytohormone supplemented and mixotrophic cultures, without affecting the biomass yield. These cultivation strategies could, therefore, be used for enhancement of ALA rich lipids in microalgae without compromising the biomass production.

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.

Effect of salinity on growth of green alga Botryococcus braunii and its constituents.

Growth of Botryococcus braunii (race 'A') and production of its constituents viz, hydrocarbon, carbohydrate, fatty acid, and carotenoids were influenced by different levels of salinity. Under salinity at 34 mM and 85 mM, 1.7-2.25-fold increase in the relative proportion of palmitic acid and two fold increase in oleic acid were observed. A twofold increase in carotenoid content was noticed at 85 mM salinity with lutein (75% of total carotenoid) as the major carotenoid followed by beta-carotene. The increase in biomass yields and changes in other constituents indicated the influence of salinity and the organism's adaptability to the tested levels of salinity (17 mM to 85 mM).

Influence of CO2 on growth and hydrocarbon production in Botryococcus braunii.

Botryococcus braunii is a green colonial fresh water microalga and it is recognized as one of the renewable resources for production of liquid hydrocarbons. CFTRI-Bb-1 and CFTRI-Bb-2 have been reported for the first time and their performance with regard to growth and biochemical profile is presented here. The present study focused on effect of carbon dioxide (CO2) on biomass, hydrocarbon, carbohydrate production, fatty acid profile, and carotenoid content in various species of B. braunii (LB-572, SAG 30.81, MCRC-Bb, N-836, CFTRI-Bb-1, and CFTRI-Bb-2) at 0.5, 1.0, and 2.0% (v/v) levels using a two-tier flask. CO2 at 2.0% (v/v) level enhanced growth of the organism, and a two-fold increase in biomass and carotenoid contents was observed in all the B. braunii strains studied compared with control culture (without CO2 supplementation). At 1% and 2% (v/v) CO2 concentrations, palmitic acid and oleic acid levels increased by 2.5 to 3 folds in one of the strains of B. braunii (LB-572). Hydrocarbon content was found to be above 20% at 2% CO2 level in the B. braunii LB-572, CFTRI-Bb-2, CFTRI-Bb-1, and N-836 strains, whereas it was less than 20% in the SAG 30.81 and MCRC-Bb strains compared with control culture. This culture methodology will provide information on CO2 requirement for growth of algae and metabolite production. B. braunii spp. can be grown at the tested levels of CO2 concentration without much influence on culture pH.

An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis.

Haematococcus pluvialis is one of the potent organisms for production of astaxanthin, a high value ketocarotenoid. Astaxanthin is accumulated in thick-walled cyst cells of Haematococcus. The thick cell wall is made up of sporopollenin-like material, algaenan, which hinders solvent extraction of astaxanthin. In the present study, an improved method for extraction of astaxanthin without homogenization of cells is reported. Extractability of astaxanthin from cyst cells was evaluated by treating cells with various solvents and pretreating the cells with organic and mineral acids at 70 degrees C followed by acetone extraction. Hydrochloric acid treatment facilitated 86-94% extractability of astaxanthin. Treatment time, temperature, and concentration of the acid were found to be critical factors for maximum extractability. The treatment did not affect the astaxanthin ester profile and the treated cells can be preserved until further use.

Removal of nutrients and organic pollution load from pulp and paper mill effluent by microalgae in outdoor open pond.

A mixed culture of microalgae, containing two Scenedesmus species, was analysed to determine its potential in coupling of pulp and paper mill effluent treatment and microalgal cultivation. Laboratory studies suggested that 60% concentration of wastewater was optimum for microalgal cultivation. A maximum of 82% and 75% removal of BOD and COD respectively was achieved with microalgal cultivation in outdoor open pond. By the end of the cultivation period, 65% removal of NO3-N and 71.29% removal of PO4-P was observed. The fatty acid composition of mixed microalgal culture cultivated with effluent showed the palmitic acid, oleic acid, linoleic acid and α-linolenic acid as major fatty acids. The results obtained suggest that pulp and paper mill effluent could be used effectively for cultivation of microalgae to minimise the freshwater and nutrient requirements.

Evaluation of indigenous fresh water microalga Scenedesmus obtusus for feed and fuel applications: Effect of carbon dioxide, light and nutrient sources on growth and biochemical characteristics.

Scenedesmus obtusus, a freshwater microalga, was evaluated for its growth and biochemical characteristics under various culture conditions. S. obtusus was tolerant at all tested CO2 concentrations up to 20%. Among the different nitrogen sources, urea showed enhanced biomass productivities up to 2-fold compared to control, where the nitrogen source was sodium nitrate. Light intensity and photoperiod had a significant effect on growth rate and biomass productivity. The growth rate was observed maximum under continuous light exposure at the light intensities, 30µmolm(-2)sec(-1) and 60µmolm(-2)sec(-1) The species was able to tolerate the salinity levels up to 25mM NaCl, where, the increase in the concentration of NaCl suppressed the growth. Ammonium acetate and glycine showed better growth rate and biomass productivity indicating mixotrophic ability of S. obtusus. Supplementation of acetate and bicarbonate significantly enhanced the biomass productivity. Biodiesel properties of S. obtusus cultivated at various culture conditions were estimated.

Digital image processing-an alternate tool for monitoring of pigment levels in cultured cells with special reference to green alga Haematococcus pluvialis.

A method for analyzing carotenoid content in Haematococcus pluvialis, a green alga was developed using digital image processing (DIP) and an artificial neural network (ANN) model. About 90 images of algal cells in various phases of growth were processed with the tools of DIP. A good correlation of R(2)=0.967 was observed between carotenoid content as estimated by analytical method and DIP. Similar correlation was also observed in case of chlorophyll. Since the conventional methods of carotenoid estimation are time consuming and result in loss of pigments during analysis, DIP method was found to be an effective online monitoring method. This method will be useful in measurement of pigments in cultured cells.

Chemical composition, iron bioavailability, and antioxidant activity of Kappaphycus alvarezzi (Doty).

Kappaphycus alvarezzi, an edible seaweed from the west coast of India, was analyzed for its chemical composition. It was found that K. alvarezzi is rich in protein (16.24% w/w) and contains a high amount of fiber (29.40% w/w) and carbohydrates (27.4% w/w). K. alvarezzi showed vitamin A activity of 865 mug retinal equivalents/100 g of sample. It contained a higher quantity of unsaturated fatty acids (44.50% of the total), in which relative percentage of oleic acid was 11%, cis-heptadecanoic acid 13.50%, and linoleic acid 2.3% and 37.0% of saturated fatty acids (mainly heptadecanoic acid). K. alvarezziwas also found to be good source of minerals, viz 0.16% of calcium, 0.033% of iron, and 0.016% of zinc, which are essential for various vital biological activities. Bioavailability of iron by in vitro methods showed a higher efficiency in intestinal conditions than in stomach conditions. Ascorbic acid influenced higher bioavailability of iron. Successive extracts of n-hexane, acetone, ethyl acetate, ethanol, and direct extractables of chloroform/methanol (1:1 and 2:1) were screened for antioxidant activity using a beta-carotene linoleic acid model system (B-CLAMS), DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) model system and hydroxyl radical scavenging activity. The chloroform/methanol (2:1) extract has shown 82.5% scavenging activity at 1000 ppm. Acetone fraction extracts at the 1000 ppm level showed 63.31% antioxidant activity in beta-carotene linoleic acid system. The acetone extract showed 46.04% scavenging activity at 1000 ppm concentration. In the case of hydroxyl radical scavenging activity, all the extracts showed better activity at the concentrations of 25 and 50 ppm, where at the 50 ppm level ethyl acetate extract showed 76.0%, acetone 75.12%, and hexane 71.15% activity, respectively. Results of this study suggest the utility of K. alvarezzi (Eucheuma) for various nutritional products, including antioxidant for use as health food or nutraceutical supplement.

Regulation of astaxanthin and its intermediates through cloning and genetic transformation of ß-carotene ketolase in Haematococcus pluvialis.

Astaxanthin, a high-value ketocarotenoid used in the pharmaceutical and nutraceutical industries is mainly produced from green alga, Haematococcus pluvialis. It is biosynthesized by the action of key enzyme, ß-carotene ketolase (BKT) on ß-carotene through intermediates echinenone and canthaxanthin. In this study, the ß-carotene ketolase (bkt) gene was isolated from H. pluvialis and cloned in a vector pRT100 and further mobilized to a binary vector pCAMBIA 1304. The T-DNA of pCAMBIA 1304, which consists of cloned bkt, was successfully transformed to H. pluvialis through Agrobacterium mediation. The cloning and transformation of bkt in H. pluvialis was confirmed by Southern blotting and also by PCR analysis. Total carotenoids and astaxanthin content in the transformed cells were found to be 2-3-fold higher, while the intermediates like echinenone and canthaxanthin were found to be 8-10-fold higher than in the control cells. The expression level of carotenogenic genes like phytoene synthase (psy), phytoene desaturase (pds), lycopene cyclase (lcy), bkt, and ß-carotene hydroxylase (bkh) were found to be higher in transformed cells compared to the non-transformed (NT) H. pluvialis.

Defatted algal biomass as a non-conventional low-cost adsorbent: surface characterization and methylene blue adsorption characteristics.

The present study investigates the use of defatted algal biomass (DAB) as a non-conventional low cost adsorbent. The maximum adsorption capacity of biomass (raw, defatted and sulfuric acid pretreated DAB) was determined by liquid phase adsorption studies in batch mode for the removal of methylene blue present at various concentrations (1, 2, 3, 4, and 5 mg L(-1)) from aqueous solutions. The data was well fitted with Langmuir and Freundlich isotherms. The maximum adsorption capacity for raw, defatted and sulfuric acid pretreated DAB was found to be 6.0, 7.73 and 7.80 mg g(-1), respectively. The specific surface area of raw, defatted and sulfuric acid pretreated DAB was estimated to be 14.70, 18.94, and 19.10 m(2) g(-1), respectively. To evaluate the kinetic mechanism that controls the adsorption process, pseudo-first order, pseudo-second order, intraparticle diffusion and particle diffusion has been tested. The data fitted quite well with pseudo-second order kinetic model.

Statistical optimization of thermal pretreatment conditions for enhanced biomethane production from defatted algal biomass.

The present study analyzes the effect of thermal pretreatment for enhancing the biomethane potential of defatted algal biomass of Scenedesmus dimorphus through statistically guided experimental design. To this end, defatted microalgal biomass at various concentrations (1, 3 and 5 g L(-1)) was pretreated at elevated temperatures (100, 120 and 150°C) for 20, 40 and 60 min. The solubilised TOC was favourably enhanced up to 71 mg L(-1) after pretreatment at a temperature of 150°C for reaction time of 60 min. The methane yield was substantially enhanced (up to 60%) and could be correlated with an increase in organic matter solubilisation and enhanced biodegradability via thermal pretreatment. The optimisation of the integrated thermal pretreatment-biomethanation process resulted in up to 1.6-fold increase in methane yield.