Optimizing light regimes for neutral lipid accumulation in Dunaliella salina MCC 43: a study on physiological status and carbon allocation.

Dunaliella salina is a favourable source of high lipid feedstock for biofuel and medicinal chemicals. Low biomass output from microalgae is a significant barrier to industrial-scale commercialisation. The current study aimed to determine how photosynthetic efficiency, carbon fixation, macromolecular synthesis, accumulation of neutral lipids, and antioxidative defence (ROS scavenging enzyme activities) of D. salina cells were affected by different light intensities (LI) (50, 100, 200, and 400 µmol m-2 s-1). The cells when exposed to strong light (400 µmol m-2 s-1) led to reduction in chlorophyll a but the carotenoid content increased by 19% in comparison to the control (LI 100). The amount of carbohydrate changed significantly under high light and in spite of stress inflicted on the cells by high irradiation, a considerable increase in activity of carbonic anhydrase and fixation rate of CO2 were recorded, thus, preserving the biomass content. The high light exposed biomass when subjected to nitrogen-deficient medium led to increase in lipid content (59.92% of the dry cell weight). However, neutral lipid made up 78.26% of the total lipid while other lipids like phospholipid and glycolipid content decreased, showing that the lipid was redistributed in these cells under nitrogen deprivation, making the organism more appropriate for biodiesel/jet fuel use. Although D. salina cells had a relatively longer generation time (3.5 d) than other microalgal cells, an economic analysis concluded that the amount of carotenoid they produced and the quality of their lipids made them more suited for commercialization.

Antimicrobial assay and genetic screening of selected freshwater Cyanobacteria and identification of a biomolecule dihydro-2H-pyran-2-one derivative.

AIMS: Explorations of freshwater Cyanobacteria as antimicrobial (bacteria, fungi and methicillin-resistant Staphylococcus aureus (MRSA) strains) drug resource using bioassay, NRPS (non-ribosomal polypeptide synthetase) and PKS (polyketide synthase) genes, as well as in silico approach. METHODS AND RESULTS: We have bioassayed the extracts of Phormidium CCC727, Geitlerinema CCC728, Arthrospira CCC729, Leptolyngbya CCC732, Phormidium CCC730, Phormidium CCC731 against six pathogenic bacteria comprising Gram (+ve): S. aureus including seven clinical MRSA and Enterococcus faecalis, Gram (-ve): Escherichia coli, Salmonella Typhimurium, Klebsiella pneumoniae and Shigella boydii along with non-pathogenic Enterobacter aerogenes as well as fungal strains (Cryptococcus neoformans and Candida albicans, C. krusei, C. tropicalis and Aspergillus niger) exhibiting antimicrobial potential. The NRPS and PKS genes of the target strains were also amplified and sequenced. The putative protein structures were predicted using bioinformatics approach. CONCLUSION: PKS gene expression indicated ß keto-acyl synthase as one of the important active domains in the biomolecules related to antitumour and antifungal group. The simultaneous identification of the biomolecule (dihydro-2H-pyran-2-one derivative) was also inferred spectroscopically. SIGNIFICANCE AND IMPACT OF THE STUDY: Freshwater Cyanobacteria are prolific producers of secondary metabolite(s) that may act as the antimicrobial drug resource in addition to their much explored marine counterpart.

Photo-induced biosynthesis of silver nanoparticles from aqueous extract of Dunaliella salina and their anticancer potential.

The synthesis of silver nanoparticles (AgNPs) via green route, using biological entities is an area of interest, because one of the potential applications in the nanomedicine. In the present study, we have developed photo-induced, ecofriendly, low cost method for biosynthesis of the stable silver nanoparticles using aqueous extract of Dunaliella salina (AED) which act as both reducing as well as stabilizing agent. Biosynthesis of the AgNPs was optimized as: sunlight exposure (30min), AED (5% (v/v)) and AgNO3 (4mM). Biosynthesis of AgNPs was monitored by using UV-Vis spectroscopy which exhibited sharp SPR band at 430nm after 30min of bright sunlight exposure. SEM and TEM analyses confirmed the presence of spherical AgNPs with average size of 15.26nm. Crystalline nature of AgNPs was confirmed by SAED and XRD analyses where Braggs reflection pattern at (111), (200), (220) and (311) corresponded to face centered cubic crystal lattice of metallic silver. FTIR analysis revealed the involvement of various functional groups present in AED. AFM analysis confirmed the average surface roughness of synthesized AgNPs as 8.48nm. AgNPs were also screened for anticancer potential using assay of calcein AM/PI, Annexin/PI and cancer biomarkers against cancer cell line (MCF-7), while normal cell line (MCF-10A) were kept as control. Interestingly, anticancer potential was comparable to the known anticancer drug (Cisplatin), and was not detrimental to the normal cell line. Therefore, such green synthesized AgNPs may be explored as anticancer agent.

Identification and structure elucidation of antimicrobial compounds from Lyngbya aestuarii and Aphanothece bullosa.

Cyanobacteria are known to produce array of compounds. In an earlier report, we reported antibacterial and antifungal activities in methanolic crude extracts of laboratory grown Lyngbya aestuarii and Aphanothece bullosa isolated from Chilka Lake and local paddy field respectively. In this report the same methanolic crude extracts were subjected to TLC purification twice by altering the solvents and UV—illuminated bands bioassayed. Such UV illuminated potent bands obtained after 2nd TLC were subjected to spectroscopic analysis (UV, IR, 1H NMR and LCMS/MS). We have screened malyngolide and dragonamide C from L. aestuarii and a diterpenoid and majusculoic acid from A. bullosa. Dragonamide C and malyngolide were found to be antifungal while majusculoic acid and a diterpenoid as antibacterial. As far as our knowledge goes, this is the first ever report where fresh water A. bullosa was found to be a source of diterpenoid and majusculoic acid. Likewise, L. aestuarii was also established as a source of malyngolide and dragonamide C. This again indicated that cyanobacteria are inherently endowed with the capacity to produce metabolites according to niche and species specific manner emphasising fresh water cyanobacterial strain are as important as marine one.

Nymphaea rubra ameliorates TNF-α-induced insulin resistance via suppression of c-Jun NH2-terminal kinase and nuclear factor-κB in the rat skeletal muscle cells.

In this work, we demonstrated insulin signaling and the anti-inflammatory effects by the chloroform fraction of ethanolic extract of Nymphaea rubra flowers in TNF-α-induced insulin resistance in the rat skeletal muscle cell line (L6 myotubes) to dissect out its anti-hyperglycemic mechanism. N. rubra enhances the GLUT4-mediated glucose transport in a dose dependent manner and also increases the tyrosine phosphorylation of both IR-ß and IRS-1, and the IRS-1 associated PI-3 kinase activity in TNF-α-treated L6 myotubes. Moreover, N. rubra decreases Ser(307) phosphorylation of IRS-1 by the suppression of JNK and NF-κB activation. In conclusion, N. rubra reverses the insulin resistance by the inhibition of c-Jun NH2-Terminal Kinase and Nuclear-κB.

Biohydrogen production from sugarcane bagasse by integrating dark- and photo-fermentation.

Hydrogen production from sugarcane bagasse (SCB) by integrating dark-fermentation by Enterobacter aerogenes MTCC 2822 and photo-fermentation by Rhodopseudomonas BHU 01 was investigated. The SCB was hydrolysed by sulphuric acid and the hydrolysate detoxified by passing through adsorbent resin column (Amberlite XAD-4) to remove the inhibitory furfural, and subjected to dark-fermentation. The cellulosic residue from acid hydrolysis was hydrolysed by the new isolate Cellulomonas fimi to release sugars for H2 production by E. aerogenes, through simultaneous saccharification, filtration and fermentation (SSFF). Cumulative H2 production during dark-fermentation and SSFF was 1000 and 613 ml/L, respectively. The spent media of dark-fermentation and SSFF were utilized for photo-fermentation by Rhodopseudomonas BHU 01. The cumulative H2 production was 755 ml/L for dark-fermentation and 351 ml/L for SSFF spent medium.

Biohydrogen production from cheese whey wastewater in a two-step anaerobic process.

Cheese whey-based biohydrogen production was seen in batch experiments via dark fermentation by free and immobilized Enterobacter aerogenes MTCC 2822 followed by photofermentation of VFAs (mainly acetic and butyric acid) in the spent medium by Rhodopseudomonas BHU 01 strain. E. aerogenes free cells grown on cheese whey diluted to 10 g lactose/L, had maximum lactose consumption (∼79%), high production of acetic acid (1,900 mg/L), butyric acid (537.2 mg/L) and H(2) yield (2.04 mol/mol lactose; rate,1.09 mmol/L/h). The immobilized cells improved lactose consumption (84%), production of acetic acid (2,100 mg/L), butyric acid (718 mg/L) and also H(2) yield (3.50 mol/mol lactose; rate, 1.91 mmol/L/h). E. aerogenes spent medium (10 g lactose/L) when subjected to photofermentation by free Rhodopseudomonas BHU 01 cells, the H(2) yield reached 1.63 mol/mol acetic acid (rate, 0.49 mmol/L/h). By contrast, immobilized Rhodopseudomonas cells improved H(2) yield to 2.69 mol/mol acetic acid (rate, 1.87 mmol/L/h). The cumulative H(2) yield for free and immobilized bacterial cells was 3.40 and 5.88 mol/mol lactose, respectively. Bacterial cells entrapped in alginate, had a sluggish start of H(2) production but outperformed the free cells subsequently. Also, the concomitant COD reduction for free cells (29.5%) could be raised to 36.08% by immobilized cells. The data suggest that two-step fermentative H(2) production from cheese whey involving immobilized bacterial cells, offers greater substrate to- hydrogen conversion efficiency, and the effective removal of organic load from the wastewater in the long-term.

Assessment of systemic interaction between Swertia chirata extract and its Bioactive constituents in rabbits.

The plant Swertia chirata (Gentianaceae) is known for its multifarious medicinal value in the Indian system of medicine (Ayurveda). Its methanol extracts having antidiabetic activity contains mangiferin, amarogentin, amaroswerin, sweroside and swertiamarin as active constituents. The pharmacokinetics of mangiferin and amarogentin have been carried out after intravenous administration of pure standards and extract from S. chirata (CT) in rabbits to assess systemic interaction. The remaining three components were also monitored in plasma for pharmacokinetic estimation based on the ratio analysis method. Mangiferin was characterized by a relative low clearance ( approximately 0.14 L/h/kg) and a lesser volume of distribution ( approximately 0.15 L/kg), while amarogentin exhibited a rapid clearance ( approximately 2.62 L/h/kg) and wide distribution ( approximately 1.08 L/kg) from the systemic circulation. No significant difference was observed in pharmacokinetic parameters of mangiferin and amarogentin either administered alone or as CT formulation in rabbits.

Simultaneous estimation of mangiferin and four secoiridoid glycosides in rat plasma using liquid chromatography tandem mass spectrometry and its application to pharmacokinetic study of herbal preparation.

Extracts from Swertia chirata (family Gentianaceae) have antidiabetics and antioxidant activity, largely attributed to the flavonoids and secoiridoids, which are a major class of functional components in methanolic extracts from aerial part of plants. In order to facilitate analysis of systemic exposure to S. chirata derived products in animals, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of routinely monitoring plasma levels of flavonoids and secoiridoids. An LC-MS/MS-based method has been developed for the simultaneous estimation of two bioactive markers, mangiferin and amarogentin along with three other components, amaroswerin, sweroside and swertiamarin in rat plasma. All the analytes including the internal standard (kutkoside) were chromatographed on RP-18 column (250 mm x 4 mm i.d., 5 microm.) coupled with guard column using acetonitrile: 0.5 mM ammonium acetate buffer, pH approximately 3.0 as mobile phase at a flow rate of 1 ml/min in gradient mode. The final flow to source was splitted in 1:1 ratio. The detection of the analytes was performed on API 4000 LC-MS/MS system in the multiple reaction-monitoring (MRM) mode. The quantitation for analytes other than the pure markers was based on relative concentration. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (Intra- and Inter-day), freeze-thaw stability, peltier stability, dry residue stability and long-term stability. The recoveries from spiked control samples were >90% for all analytes and internal standard except mangiferin where recovery was >60%. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% at low and <10% at other concentrations. The quantitation method was successfully applied to generate pharmacokinetic (PK) profile of markers as well as to detect other components in plasma after intravenous dose administration of herbal preparation in male Sprague-Dawley (SD) rats.

Liquid chromatography/tandem mass spectrometric study and analysis of xanthone and secoiridoid glycoside composition of Swertia chirata, a potent antidiabetic.

Swertia chirata is a bitter plant, used in the Indian system of medicine (Ayurveda) for various human ailments. The bioactive constituents include the xanthone and secoiridoid glycosides consisting of mangiferin, amarogentin, amaroswerin, sweroside and swertiamarin. Methanolic extracts of S. chirata possess constituents with antidiabetic activities, which was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Preliminary HPLC analyses were performed on a reversed-phase C18 column using gradient elution. In the LC/ESI-MS spectra, predominant [M+H]+ and [M+Na]+ ions were observed in positive ion mode and provided molecular mass information. The five components of S. chirata were structurally correlated and confirmed based on the fragmentation characteristics and information available in the literature. The fragmentation behavior of [M+H]+/[M+Na]+ ions of these components were deduced from the collision-induced dissociation (CID) spectra obtained from the selective on-column information-dependant acquisition (IDA) approach. Xanthone-C-glycoside showed characteristic fragment ions due to fragmentation in the C-glycosidic unit while iridoid-O-glycosides showed characteristic fragment ions due to cleavage in the glycoside linkage and retro-Diels-Alder (RDA) cleavage within an iridoid aglycone. Furthermore, on the basis of this information, an analytical assay was developed and validated to determine relative concentrations of mangiferin, amarogentin, amaroswerin, sweroside and swertiamarin. The detection was carried out using multiple reaction monitoring (MRM) in positive ionization mode with a total analysis time of 3.5 min. The method was successfully applied to standardize four different batches of herbal preparation on the basis of relative concentration of five bioactive components.

Gene expression in the cyanobacterium Anabaena sp. PCC7120 under desiccation.

The N2-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K+ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.

Nickel uptake by Pseudomonas aeruginosa: role of modifying factors.

Pseudomonas aeruginosa cells growing in minimal medium were 40-fold more sensitive to Ni2+ than cells growing in enriched medium, suggesting a possible protective role of medium ingredients. Likewise, cells pre-grown in enriched medium showed a high Km (6.15 mM) and increased Ni2+ uptake (950 nmol mg-1 protein, 1h) over cells pre-sown in minimal medium (Km, 0.48 mM; 146 nmol mg-1 protein, 1 h). The overall pattern indicates that cells pre-grown in enriched medium were characterized by having lowered affinity towards Ni2+ than those with minimal medium background. The enhanced Ni2+ uptake by enriched medium-grown cells can be correlated with the improved metabolic state of the cells. Ni2+ uptake was optimum at neutrality (pH 7.0). A major Ni2+ transport system was competitively inhibited by Mg2+, Zn2+, Cd2+, or Co2+ (400 microM each). Noticeably, a minor Ni2+ transport pathway was still operative even in the higher concentration range of Mg2+ (4 mM and 40 mM). The stimulation of Ni2+ uptake monitored in the presence of different carbon sources (0.5% wt/vol, each) showed the sequence: glucose (1.6-fold) > phenol = gallic acid (1.5-fold). Succinate, in comparison, reduced Ni2+ uptake (0.5-fold) possibly because of its acting as a metal chelator as well. Sensitivity of Ni2+ transport towards methyl viologen, azide, 2-4 DNP, and DCCD suggested that transport was energy-linked.

Comparison of Ni-sensitive and Ni-resistant strains of Nostoc muscorum.

A wild-type Ni-sensitive (Ni(s)) strain of Nostoc muscorum ISU spontaneously yielded mutants resistant to inhibition by 40 µM Ni with a frequency of about 10(-7). A Ni-resistant (Ni(r)) mutant was deficient in the activities of urease and uptake hydrogenase. Cellular Ni uptake in the Ni(s) strain was dependent on concentration (40 to 120 µM) and time (0 to 30 min) (Vmax=0.51 nmol/µg protein.min; Km=92 µM). The Ni bioconcentration factor for such cells ranged between 0.95×10(3) and 1.89×10(3). Ni uptake in spheroplast preparations from Ni(s) cells followed almost the same trend as intact cells except that the bioconcentration factor was slightly less [(0.82 to 1.39)×10(3)]. In contrast, Ni uptake in the Ni(r) intact cells was not concentration dependent and also the uptake was saturated, even at 40 µM, within 10 min. Spheroplasts from the Ni(r) strain showed a Ni bioconcentration factor of 1.19×10(3) compared with 4.41×10(3) for intact cells. The invariably lower Ni uptake by spheroplasts was attributed to altered membrane transport properties.

Hypoglycemic effect of swerchirin from the hexane fraction of Swertia chirayita.

A xanthone was isolated from the hexane fraction of the Swertia chirayita plant and identified as 1,8-dihydroxy-3,5-dimethoxyxanthone (swerchirin). It has a very significant blood sugar lowering effect in fasted, fed, glucose loaded, and tolbutamide pretreated albino rat models. The ED50 for 40% blood sugar lowering in CF male albino rats (body weight 140-165 g) is 23.1 mg/kg/oral. The possibility of its application in clinical therapy for diabetes mellitus needs exploration.