G. Seghal Kiran

Synthesis of silver nanoparticles by polysaccharide bioflocculant produced from marine Bacillus subtilis MSBN17.

The polysaccharides are emerging as stabilizing and reducing agents for nanoparticles synthesis, however the commercial polysaccharides are not economically viable. Therefore, the exopolysaccharide from microbial origin such as bioflocculants are promising alternate for the synthesis and stabilization of nanoparticles. In this report, a bioflocculant (MSBF17) was produced from marine sponge-associated Bacillus subtilis MSBN17 under submerged fermentation using the economical substrates. The production was statistically optimized with most significant factors such as palm jaggery, NH(4)NO(2), K(2)HPO(4) and NaCl. The maximum bioflocculant production obtained with statistically optimized medium was 13.42 g/l. Based on the biochemical composition and FT-IR analysis, the flocculant compound was predicted as a polysaccharide derivative. The flocculating activity of the MSBF17 was invariably considerable at high salinity and temperature. It was found that the nano-scale silver can be synthesized in reverse micelles using the bioflocculant as stabilizer. The silver nanoparticles (AgNPs) were characterized by UV-spectroscopy, FT-IR and TEM analysis. The AgNPs were spherical shaped (60 nm) and stable for 5 months. Therefore, the bioflocculant-mediated synthesis of nanomaterials can be considered as environmental benign greener approach.

Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans

BackgroundLipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome.ResultsA metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc 53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C β-lactamases. Heterologous expression of lpc 53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3–12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 μmol/min, respectively.ConclusionWe have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.

Optimization of polyhydroxybutyrate production by marine Bacillus megaterium MSBN04 under solid state culture

A marine sponge-associated bacterium Bacillus megaterium MSBN04 was used for the production of polyhydroxybutyrate (PHB) under solid state culture (SSC). A central composite design (CCD) was employed to optimize the production medium and to find out the interactive effects of four independent variables, viz. tapioca industry waste, palm jaggery, horse gram flour and trace element solution on PHB production. The maximum yield of PHB 8.637 mg g(-1) of substrate (tapioca industry waste) was achieved from biomass 15.203 mg g(-1) of substrate, using statistically optimized medium. The horse gram flour (nitrogen source) and trace element solution were found to be critical control factors for PHB synthesis. The (1)H NMR analysis revealed that the polymer was a PHB monomer. PHB obtained from this study having high molecular weight (6.7×10(5) Da) with low polydispersity index (PDI) value (1.71) and produced PHB was used to synthesize PHB polymeric nanoparticles using solvent displacement approach. Therefore, B. megaterium MSBN04 is an ideal candidate that can be exploited biotechnologically for the commercial production of PHB under solid state culture.

A statistical approach for optimization of polyhydroxybutyrate production by marine Bacillus subtilis MSBN17

The important biological macromolecule polyhydroxybutyrate (PHB) producing Bacillus subtilis was isolated from the marine sponge Callyspongia diffusa and identified by means of 16S rRNA analysis. The central composite design (CCD) was used to optimize the PHB production using cheap raw materials such as pulp industry waste (PIW), tamarind kernel powder (TKP), palm jaggery (PJ) and green gram flour (GGF). The extracted polymer was characterized by (1)H NMR analysis. The PIW was fed at three different intervals and the maximum production of PHB (19.08g/L) was attained after a period of 40h of incubation of B. subtilis. Dissolved oxygen, sodium chloride and nitrogen source were found to be the critical control factors that affected the PHB polymer production. The present investigation demonstrates an inexpensive model of producing PHB green thermoplastics in vitro for biomedical applications.

Optimization and production of pyrrolidone antimicrobial agent from marine sponge-associated Streptomyces sp. MAPS15

Twenty-nine actinobacterial strains were isolated from marine sponge Spongia officinalis and screened for antagonistic activity against various bacterial and fungal pathogens. The active antibiotic producer MAPS15 was identified as Streptomyces sp. using 16S rRNA phylogenetic analysis. The critical control factors were selected from Plackett–Burman (PB) factorial design and the bioprocess medium was optimized by central composite design (CCD) for the production of bioactive metabolite from Streptomyces sp. MAPS15. The maximum biomass and active compound production obtained with optimized medium was 6.13xa0g/L and 62.41xa0mg/L, respectively. The economical carbon source, paddy straw was applied for the enhanced production of bioactive compound. The purified active fraction was characterized and predicted as pyrrolidone derivative which showed broad spectrum of bioactivity towards indicator organisms. The predicted antimicrobial spectra suggested that the Streptomyces sp. MAPS15 can produce a suite of novel antimicrobial drugs.

Process optimization and production of polyhydroxybutyrate using palm jaggery as economical carbon source by marine sponge-associated Bacillus licheniformis MSBN12.

The Polyhydroxybutyrate (PHB) producer, Bacillus licheniformis MSBN12 was isolated from the marine sponge Callyspongia diffusa. The PHB production of B. licheniformis MSBN12 was optimized using a four-factor Box-Behnken design to find the interactive effects of variables such as palm jaggery, wheat bran, seawater, and incubation temperature. The maximum yield of PHB (6.38xa0g/L) was achieved through response surface methodology-based optimization and the optimized conditions were further used for the batch and fed-batch fermentation. Maximum biomass was reached at 48 and 36xa0h of incubation with PHB accumulation of 62.91 and 67.16xa0% (w/w of dry cells) for batch and fed-batch process. The production of PHB under fed-batch process with B. licheniformis MSBN12 was increased threefold over shake flask culture when palm jaggery as sole carbon source. The ¹H NMR data was extrapolated with peaks of the PHB reference standard and confirmed as PHB analog.

Characterization of an exopolysaccharide from probiont Enterobacter faecalis MSI12 and its effect on the disruption of Candida albicans biofilm

Biofilm-forming pathogens are a potential threat to indwelling medical devices and infectious diseases. Management of device-associated Candida infections remains challenging with the existing drug discovery platforms. The available antifungal drugs are effective for the control of free-living pathogens but not effective on biofilm-forming pathogens. Thus an antifungal drug synergized with an antibiofilm agent would be an effective strategy to treat Candida biofilms. Mature C. albicans biofilms are anchored by a complex architecture in terms of distribution of fungal cells stabilized by exocellular polymeric substances. The findings of the present study provide a new insight on the possible development of enterococci probiotics and/or its exopolysaccharide (EPS) as synergistic with existing antifungal drugs to treat biofilm infections. The probiont Enterococcus faecalis MSI12 was picked from 142 seawater isolates screened for EPS production using a congo red plate assay. The probiotic characteristics of the isolate MSI12 were evaluated based on the temperature, pH, acid tolerance, autoaggregation, hydrophobicity and antioxidant activity. The biofilm disruption ability of the lyophilized EPS was determined in a microtitre plate assay using fluconazole as reference drug. Scanning electron microscope and confocal laser scanning microscope images were used for analysis of antibiofilm activity. The cell viability of E. faecalis MSI12 was very high at higher temperature, acidic pH, bile salt and salt concentration when compared to the reference strain Lactobacillus plantarum. Therefore the strain MSI12 might survive in the niche like human gut without prebiotics. The EPS from Enterococcus sp. MSI12 showed significant reduction of the treated Candida biofilm. The antibiofilm potential of EPS was much stronger than the standard antifungal drug fluconazole. This study revealed that biofilm disruption/control using a probiont EPS could deliver a synergistic approach as the probiotic strain can colonize in the host to prevent the formation of Candida biofilms.

Production and characterisation of glycolipid biosurfactant by Halomonas sp. MB-30 for potential application in enhanced oil recovery.

Biosurfactant-producing Halomonas sp. MB-30 was isolated from a marine sponge Callyspongia diffusa, and its potency in crude oil recovery from sand pack column was investigated. The biosurfactant produced by the strain MB-30 reduced the surface tension to 30xa0mNxa0m−1 in both glucose and hydrocarbon-supplemented minimal media. The critical micelle concentration of biosurfactant obtained from glucose-based medium was at 0.25xa0mgxa0ml−1 at critical micelle dilution 1:10. The chemical structure of glycolipid biosurfactant was characterised by infrared spectroscopy and proton magnetic resonance spectroscopy. The emulsification activity of MB-30 biosurfactant was tested with different hydrocarbons, and 93.1xa0% emulsification activity was exhibited with crude oil followed by kerosene (86.6xa0%). The formed emulsion was stable for up to 1xa0month. To identify the effectiveness of biosurfactant for enhanced oil recovery in extreme environments, the interactive effect of pH, temperature and salinity on emulsion stability with crude oil and kerosene was evaluated. The stable emulsion was formed at and above pH 7, temperature >80xa0°C and NaCl concentration up to 10xa0% in response surface central composite orthogonal design model. The partially purified biosurfactant recovered 62xa0% of residual crude oil from sand pack column. Thus, the stable emulsifying biosurfactant produced by Halomonas sp. MB-30 could be used for in situ biosurfactant-mediated enhanced oil recovery process and hydrocarbon bioremediation in extreme environments.

Revealing the ability of a novel polysaccharide bioflocculant in bioremediation of heavy metals sensed in a Vibrio bioluminescence reporter assay.

A bioflocculant-producing bacterial strain, designated MSI021, was isolated from the marine sponge Dendrilla nigra and demonstrated 94% flocculation activity in a kaolin clay suspension. MSI021 was identified as Bacillus cereus based on phylogenetic affiliation and biochemical characteristics. The purified extra-cellular bioflocculant was chemically elucidated as a polysaccharide molecule. The polysaccharide bioflocculant was stable under both acidic and alkaline conditions (pH 2.0-10.0) and temperatures up to 100xa0°C. The purified bioflocculant efficiently nucleated the formation of silver nanoparticles which showed broad spectrum antibacterial activity. The ability of the bioflocculant to remediate heavy metal toxicity was evaluated by measuring the inhibition of bioluminescence expression in Vibrio harveyi. Enrichment of heavy metals such as zinc, mercury and copper at concentrations of 1, 2 and 3xa0mM in culture media showed significant reduction of bioluminescence in Vibrio, whereas media enriched with heavy metals and bioflocculant showed dose dependent improvement in the expression of bioluminescence. The assay results demonstrated that the polysaccharide bioflocculant effectively mitigates heavy metal toxicity, thereby improving the expression of bioluminescence in Vibrio. This bioluminescence reporter assay can be developed into a high-throughput format to monitor and evaluate of heavy metal toxicity. The findings of this study revealed that a novel polysaccharide bioflocculant produced by a marine B.xa0cereus demonstrated strong flocculating performance and was effective in nucleating the formation antibacterial silver nanoparticles and removing heavy metals. These results suggest that the MSI021 polysaccharide bioflocculant can be used to develop greener waste water treatment systems.

Medicinal Plants of India: Implications in Modern Medicine

The linkage between biodiversity and human health is well established. Conserving forest biodiversity by valuing and harnessing it, as medicine, is consistent with poverty reduction and local public health prevention efforts. Global demand for herbal medicines is accompanied by reducing supply of medicinal plants due to overharvesting, habitat loss, and agricultural encroachment. Plants are always considered as a primary source of drugs in traditional and alternative system of medicine in various forms such as crude form, juice, decoction, and crude extracts. About 80% people of the world, particularly in the rural areas of developing countries, continue using traditional resources in healthcare. This chapter deals with the assessment of the richness of ethnomedicinal plant species used by the local tribes in forest areas and a basic information on the ethnomedicinal plants, its conservation strategies, and correlation between the ethnomedical usage of medicinal plants and modern medicines.