UV-B-induced Oxidative Damage and Protective Role of Exopolysaccharides in Desert Cyanobacterium Microcoleus vaginatus

UV-B-induced oxidative damage and the protective effect of exopolysaccharides (EPS) in Microcoleus vaginatus, a cyanobacterium isolated from desert crust, were investigated. After being irradiated with UV-B radiation, photosynthetic activity (Fv/Fm), cellular total carbohydrates, EPS and sucrose production of irradiated cells decreased, while reducing sugars, reactive oxygen species (ROS) generation, malondialdehyde (MDA) production and DNA strand breaks increased significantly. However, when pretreated with 100 mg/L exogenous EPS, EPS production in the culture medium of UV-B stressed cells decreased significantly; Fv/Fm, cellular total carbohydrates, reducing sugars and sucrose synthase (SS) activity of irradiated cells increased significantly, while ROS generation, MDA production and DNA strand breaks of irradiated cells decreased significantly. The results suggested that EPS exhibited a significant protective effect on DNA strand breaks and lipid peroxidation by effectively eliminating ROS induced by UV-B radiation in M. vaginatus.

Structural characterization of Helicobacter Pylori cell-surface lipopolysaccharides and exopolysaccharides

Doutoramento em Bioquímica

Engineering of bacterial exopolysaccharides: from synthesis to properties

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Role of Exopolysaccharides and Monosaccharides in Erwinia amylovora Resistance to Bacteriophages

Fire blight is a disease caused by the phytopathogenic bacterium Erwinia amylovora, an economically important pathogen in the commercial production of apples and pears. Bacteriophages have been proposed as a commercial biopesticide to relieve the pressures on apple and pear production and provide alternatives to existing biological control options. This work reports on the investigation of host resistance in the development of a phage biopesticide. Exopolysaccharide (EPS) deficient bacterial mutants were generated through recombineering to investigate the role of EPS in bacteriophage adsorption and infection. The mutants that were deficient in amylovoran production were avirulent and resistant to infection by phages of the Podoviridae and some of the Siphoviridae family. Levan deficient bacterial mutants resulted in reduced phage titers in some phages from the Myoviridae family. Exopolysaccharide mimetic monosaccharides were used to demonstrate that levan and amylovoran play an important role in phage attack of E. amylovora.

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion. which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and RI stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS RI also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10-24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS RI and with the increases in Bacteroides in cultures with both microbial EPS (RI and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids. (C) 2009 Elsevier B.V. All rights reserved.

Three exopolysaccharides of the beta-(1 -> 6)-D-glucan type and a beta-(1 -> 3;1 -> 6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemical and rheological properties of exopolysaccharides produced by four isolates of rhizobia

The rheological, physicochemical properties, emulsification and stability of exopolysaccharides (EPSs) from four rhizobia isolates (LBMP-C01, LBMP-C02, LBMP-C03 and LBMP-C04) were studied. The EPS yields of isolates under these experimental conditions were in the range of 1.5-6.63gL(-1). The LBMP-C04 isolate, which presented the highest EPS production (6.63gL(-1)), was isolated from Arachis pintoi and was identified as a Rhizobium sp. strain that could be explored as a possible potential source for the production of extracellular heteropolysaccharides. All polymers showed a pseudoplastic non-Newtonian fluid behavior or shear thinning property in aqueous solutions. Among the four EPS tested against hydrocarbons, EPS LBMP-C01 was found to be more effective against hexane, olive and soybean oils (89.94%, 82.75% and 81.15%, respectively). Importantly, we found that changes in pH (2-11) and salinity (0-30%) influenced the emulsification of diesel oil by the EPSs. EPSLBMP-C04 presented optimal emulsification capacity at pH 10 (E24=53%) and 30% salinity (E24=27%). These findings contribute to the understanding of the influence of the chemical composition, physical properties and biotechnology applications of rhizobial EPS solutions their bioemulsifying properties.

A convenient sample preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage

Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM) provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving pre-fixation with 75 mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained from stems stood into water. The lysine-based protocol was compared with four other processing protocols of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with CPD. The non-fixed control. FD and the glutaraldehyde fixation with HMDS drying gave poor preservation of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was superior to these three methods in terms of better preserving the EPS. However, this fourth method gave condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for examination of bacterial stem-end blockage in cut ornamentals. (C) 2012 Elsevier B.V. All rights reserved.

Microbially-induced separation of chalcopyrite and galena

Cells of Paenibacillus polymyxa and their metabolic products such as bioproteins and exopolysaccharides could be effectively used in the separation of galena from chalcopyrite. While interaction with bacterial cells resulted in significant flocculation of both chalcopyrite and galena, treatment with bioproteins selectively flocculated only chalcopyrite, dispersing galena. Microbially-induced selective flocculation after conditioning with cells, bioproteins or exopolysaccharides resulted in efficient separation of chalcopyrite and galena from their mixtures. Prior interaction with bioproteins facilitated enhanced flotation of galena from chalcopyrite. The role of bacterial cells and bioreagents such as proteins and polysaccharides in mineral beneficiation is demonstrated.

Microbially induced separation of quartz from hematite using yeast cells and metabolites

The utility of yeast, Saccharomyces cerevisiae, in the separation of quartz from hematite is demonstrated. Yeast cells; as well as their metabolites, functioned as flotation collectors, depressants or flocculants and dispersants for hematite and quartz. Interaction between yeast and the above minerals resulted in significant surface chemical changes, rendering quartz surfaces hydrophobic and hematite hydrophilic. Mineral-specific extracellular proteins and exopolysaccharides were secreted by yeast cells when grown in the presence of quartz and hematite, respectively. Quartz could be efficiently separated from hematite through microbially induced flotation and selective flocculation.

Microbially-induced pyrite removal from galena using Bacillus subtilis

Cells and metabolic products of Bacillus subtilis were used in microbially-induced flocculation and flotation to separate pyrite from galena. Enhanced selective affinity of bacterial cells towards pyrite was observed when compared to galena through adsorption studies. Both extracellular (EP) and intracellular (IP) bacterial proteins were isolated from B. subtilis before and after interaction with the minerals and their profiles established through SDS-PAGE. Protein fractions exhibited significant surface affinity towards galena when compared to pyrite. Presence of galena during bacterial growth promoted increased generation of extracellular proteins, while that of pyrite resulted in enhanced production of exopolysaccharides. Galena surfaces were rendered hydrophobic after bacterial interaction.

Microbially induced selective flocculation of hematite from kaolinite

Microbially induced selective flocculation of hematite from kaolinite has been demonstrated using Bacillus subtilis. Growth of bacterial cells in the presence of kaolinite resulted in enhanced production of extracellular proteins while that of hematite promoted significant secretion of exopolysaccharides. Bacterial cells were adapted to grow in the presence of the minerals and use of hematite-grown and kaolinite-grown cells and their metabolic products in the selective flocculation of hematite and dispersion of kaolinite illustrated. Bacterial cells and extracellular polysaccharides exhibited higher surface affinity towards hematite, rendering it hydrophilic; while significant protein adsorption enhanced surface hydrophobicity of kaolinite. Bacterial interaction with hematite and kaolinite resulted in significant surface chemical changes on the minerals. Due to higher surface affinity towards extracellular proteins, zeta potentials of kaolinite shifted in the positive direction, while those of hematite shifted in the negative direction due to higher adsorption of extracellular polysaccharides. Bacterial interaction promoted selective flocculation of only hematite, while kaolinite was efficiently dispersed. Mineral-specific stress proteins were generated on growing B. subtilis in the presence of kaolinite. Interfacial aspects of microbe-mineral interactions are illustrated to explain microbially-induced selective flocculation of hematite from kaolinite with relevance to clay and iron ore beneficiation. (C) 2013 Elsevier B.V. All rights reserved.

Flocculation behaviour of hematite-kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides

Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells. (C) 2013 Elsevier B.V. All rights reserved.

Effects of salt stress on carbohydrate metabolism in desert soil alga Microcoleus vaginatus Gom.

The effects of salt stress on carbohydrate metabolism in Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crusts, were investigated in the present study. Extracellular total carbohydrates and exopolysaccharides (EPS) in the culture medium produced by M. vaginatus increased significantly during the growth phase and reached a maximum during the stationary phase. The production of extracellular carbohydrates also significantly increased under higher salt concentrations, which was attributed to an increase in low molecular weight carbohydrates. In the presence of NaCl, the production of cellular total carbohydrates decreased and photosynthetic activity was impaired, whereas cellular reducing sugars, water-soluble sugars and sucrose content and sucrose phosphate synthase activity increased, reaching a maximum in the presence of 200 mmol/L NaCl. These parameters were restored to original levels when the algae were transferred to a non-saline medium. Sodium and K+ concentrations of stressed cells decreased significantly and H+-ATPase activity increased after the addition of exogenous sucrose or EPS. The results suggest that EPS and sucrose are synthesized to maintain the cellular osmotic equilibrium between the intra- and extracellular environment, thus protecting algal cells from osmotic damage, which was attributed to the selective exclusion of cellular Na+ and K+ by H+-ATPase.

Towards the identification of the common features of bacterial biofilm development

UPNa. Instituto de Agrobiotecnología. Laboratorio de Biofilms Microbianos