Characterization of bioemulsifying EPS from Halobacillus trueperi MXM-16, a halophilic adhered bacterial isolate from the mangrove ecosystem.

Exopolymeric substances (EPS) produced by bacterial cells play a crucial role in the interaction of the cells with the surrounding environment. Halobacillus trueperi manxer mangrove-16, an adhered bacterial isolate from the mangrove ecosystem was found to produce EPS that was observed by Alcian blue staining and congo red-coomassie blue agar. The EPS of the bacterial isolate exhibited emulsifying properties. Purification of the EPS by dialysis showed an emulsification index of 80% with hexadecane. Qualitative analysis and Fourier's Infrared spectroscopy (FTIR) revealed that the EPS was a glycoprotein in nature. The EPS showed no surface-active properties. Further exploration of the potential of the EPS interaction with metal solutions showed the ability of the bioemulsifier to cause precipitation in the metal solutions and particularly change the color of the Chromium (VI) solution. The scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS) of the cells and EPS particularly indicated the interaction of the EPS with the (Fe0 ) zerovalent iron nanoparticles and its effect on the cells and EPS of the bacteria. It is therefore concluded that the EPS is a crucial component that anchors the bacteria to particulate matter in the mangrove ecosystem and also plays an important role in interaction with metals and hydrocarbons.

Partial characterization of viscous exopolymer produced by Alkalihalobacillus sp. strain SB-D (KJ372395) with emulsification and adhesive properties.

Twenty-two alkaliphilic bacteria were isolated from spent waters of an agrochemical factory, Goa, India, and screened for exopolymer (EP) production on Horikoshi I/II media. Strain SB-D produced highly viscous cell-bound and extracellular EP quantified by alcian blue adsorption assay. The 16S rRNA gene sequence of strain SB-D showed 97.34% similarity to Alkalihalobacillus lehensis strain MLBT. The strain maximally produced EP (10.3 g/L) at pH 10.3, 25 °C after 16 h growth on shaker, followed by 4 h at static condition with 4% inoculum in the presence of 3% glucose and 0.1% yeast extract. The EP precipitated using isopropanol contained carbohydrates (212 µg/mg), lipids (130 µg/mg), and proteins (86.16 µg/mg) as major components. Further, gas chromatography detected the presence of D-glucose, D-arabinose, D-mannose, D-galactose, D-xylose and D-ribose. Preliminary studies on EP characterization revealed emulsification and adhesive properties of the exopolymer.

Studies on Siderophore and Pigment Produced by an Adhered Bacterial Strain Halobacillus trueperi MXM-16 from the Mangrove Ecosystem of Goa, India.

Mangroves are unique ecosystems in the coastal tropical and subtropical regions of the Earth. The fluctuation in salinity due to tidal action results in a prolific population of adhered halophilic and halotolerant bacteria in this ecosystem. In this study, a pigment producing adhered bacterial strain Halobacillus trueperi MXM-16 was isolated from mangrove plant litter of Goa. This strain was moderately halophilic, Gram positive rod, catalase positive and capable of utilizing sodium benzoate as a source of carbon. H. trueperi MXM-16, produced a siderophore that was hydroxamate in nature. The non-diffusible yellow pigment was a carotenoid and HPLC studies revealed a peak that was indicative of astaxanthin as one of the component. Further studies on the pigment exhibited its ability to chelate iron from the chrome azurol sulphonate medium behaving as an additional mechanism for iron acquisition.

Studies on the effects of zerovalent iron nanoparticles on bacteria from the mangrove ecosystem.

Zerovalent iron (ZVI) nanoparticles are gaining popularity in bioremediation of contaminated ground water and antimicrobial studies. In this study, ZVI nanoparticles were synthesized by borohydride method. The effect of these nanoparticles to alter the cell surface hydrophobicity of mangrove bacteria was studied by bacterial adhesion to hydrocarbon assay. The effect of these nanoparticles on the growth and extracellular polymeric substances (EPS) of a novel bacterial strain Halobacillus trueperi MXM-16 from mangroves was evaluated by growing the culture in the presence of ZVI nanoparticles and SEM. The change in the emulsifying ability of the cell-free supernatant of Halobacillus trueperi MXM-16 when grown in media amended with ZVI nanoparticles was also investigated by spectrophotometric analysis.

Effect of metals on a siderophore producing bacterial isolate and its implications on microbial assisted bioremediation of metal contaminated soils.

A bacterial isolate producing siderophore under iron limiting conditions, was isolated from mangroves of Goa. Based on morphological, biochemical, chemotaxonomical and 16S rDNA studies, the isolate was identified as Bacillus amyloliquefaciens NAR38.1. Preliminary characterization of the siderophore indicated it to be catecholate type with dihydroxy benzoate as the core component. Optimum siderophore production was observed at pH 7 in mineral salts medium (MSM) without any added iron with glucose as the carbon source. Addition of NaCl in the growth medium showed considerable decrease in siderophore production above 2% NaCl. Fe(+2) and Fe(+3) below 2 µM and 40 µM concentrations respectively, induced siderophore production, above which the production was repressed. Binding studies of the siderophore with Fe(+2) and Fe(+3) indicated its high affinity towards Fe(+3). The siderophore concentration in the extracellular medium was enhanced when MSM was amended with essential metals Zn, Co, Mo and Mn, however, decreased with Cu, while the concentration was reduced with abiotic metals As, Pb, Al and Cd. Significant increase in extracellular siderophore production was observed with Pb and Al at concentrations of 50 µM and above. The effect of metals on siderophore production was completely mitigated in presence of Fe. The results implicate effect of metals on the efficiency of siderophore production by bacteria for potential application in bioremediation of metal contaminated iron deficient soils especially in the microbial assisted phytoremediation processes.

Isolation and characterization of a lipopeptide bioemulsifier produced by Pseudomonas nitroreducens TSB.MJ10 isolated from a mangrove ecosystem.

Pseudomonas nitroreducens TSB.MJ10 exhibiting growth and bioemulsifier production with 0.5% sodium benzoate as the sole carbon source was isolated from a mangrove ecosystem in the vicinity of a petroleum pump. The bioemulsifier is a lipopeptide that is stable over a pH range of 5-11 and a temperature range of 20-90°C and showed emulsifying activity in the presence of relatively high NaCl concentrations (up to 25%). The bioemulsifier formed stable emulsions with aliphatic (hexadecane, n-heptane, cyclohexane), aromatic (xylene, benzene, toluene) and petroleum (gasoline, diesel, kerosene, crude oil) compounds. It exhibited a maximum emulsification activity with weathered crude oil (97%) and was capable of transforming the rheological behavior of the pseudoplastic to a Newtonian fluid. The results reveal the potential of the bioemulsifier for use in bioremediation of hydrocarbons in marine environments and in enhanced oil recovery.

Siderophore-producing bacteria from a sand dune ecosystem and the effect of sodium benzoate on siderophore production by a potential isolate.

Bioremediation in natural ecosystems is dependent upon the availability of micronutrients and cofactors, of which iron is one of the essential elements. Under aerobic and alkaline conditions, iron oxidizes to Fe(+3) creating iron deficiency. To acquire this essential growth-limiting nutrient, bacteria produce low-molecular-weight, high-affinity iron chelators termed siderophores. In this study, siderophore-producing bacteria from rhizosphere and nonrhizosphere areas of coastal sand dunes were isolated using a culture-dependent approach and were assigned to 8 different genera with the predominance of Bacillus sp. Studies on the ability of these isolates to grow on sodium benzoate revealed that a pigmented bacterial culture TMR2.13 identified as Pseudomonas aeruginosa showed growth on mineral salts medium (MSM) with 2% of sodium benzoate and produced a yellowish fluorescent siderophore identified as pyoverdine. This was inhibited above 54 µM of added iron in MSM with glucose without affecting growth, while, in presence of sodium benzoate, siderophore was produced even up to the presence of 108 µM of added iron. Increase in the requirement of iron for metabolism of aromatic compounds in ecosystems where the nutrient deficiencies occur naturally would be one of the regulating factors for the bioremediation process.

Phenyltins in surface sediments of the Visakhapatnam harbour, India.

Twenty-two surface sediment samples were collected from Visakhapatnam harbour, east coast of India, and analysed for monophenyltin (MPT), diphenyltin (DPT), triphenyltin (TPT), total bacteria, and TPT tolerant bacteria. Concentrations of MPT, DPT and TPT varied between 1-26, 3-28, and 0.31-145 ng Sn g(-1) dry wt, respectively. Phenyltin concentrations were influenced by ship related activities, agricultural waste and sewage. These phenyltin concentrations indicate sediments are contaminated. Abundance of TPT tolerant bacteria was strongly influenced by the levels of phenyltins.

Native granule associated short chain length polyhydroxyalkanoate synthase from a marine derived Bacillus sp. NQ-11/A2.

A rapidly growing marine derived Bacillus sp. strain NQ-11/A2, identified as Bacillus megaterium, accumulated 61% polyhydroxyalkanoate by weight. Diverse carbon sources served as substrates for the accumulation of short chain length polyhydroxyalkanoate. Three to nine granules either single or attached as buds could be isolated intact from each cell. Maximum activity of polyhydroxyalkanoate synthase was associated with the granules. Granule-bound polyhydroxyalkanoate synthase had a K(m) of 7.1 x 10(-5) M for DL-beta-hydroxybutyryl-CoA. Temperature and pH optima for maximum activity were 30 degrees C and 7.0, respectively. Sodium ions were required for granule-bound polyhydroxyalkanoate synthase activity and inhibited by potassium. Granule-bound polyhydroxyalkanoate synthase was apparently covalently bound to the polyhydroxyalkanoate-core of the granules and affected by the chaotropic reagent urea. Detergents inhibited the granule-bound polyhydroxyalkanoate synthase drastically whilst glycerol and bovine serum albumin stabilized the synthase.

Sand aggregation by exopolysaccharide-producing Microbacterium arborescens--AGSB.

In the rhizosphere, exopolymers are also known to be useful to improve the moisture-holding capacity. The ability of the isolates from coastal sand dunes to produce exopolymers was determined. Among which the isolate, showing very high production of exopolysaccharide (EPS), Microbacterium arborescens--AGSB, a facultative alkalophile was further studied for exopolymer production. The isolate a gram-positive non-spore forming, slender rod, catalase positive, oxidase negative, showed growth in 12% sodium chloride. The culture was found to produce exopolymer which showed good aggregation of sand which has an important role in the stabilization of sand dunes. The exopolymer was further analysed. The cold isopropanol precipitation of dialysed supernatants grown in polypeptone yeast extract glucose broth produced partially soluble EPSs with glucose as the sole carbon source. Chemical analysis of the EPS revealed the presence of rhamnose, fucose, arabinose, mannose, galactose and glucose. On optimization of growth parameters (sucrose as carbon source and glycine as nitrogen source), the polymer was found to be a heteropolysaccharide containing mannose as the major component. It was interesting to note that the chemical composition of the exopolymers produced from both unoptimized and optimized culture conditions of Microbacterium arborescens--AGSB is different from those of other species from the same genera. This study shows that marine coastal environments such as coastal sand dunes, are a previously unexplored habitat for EPS-producing bacteria, and that these molecules might be involved in ecological roles protecting the cells against dessication especially in nutrient-limited environments such as the coastal sand dunes more so in the extreme conditions of pH. Such polysaccharides may help the bacteria to adhere to solid substrates and survive during the nutrient limitations.

Industrial potential of organic solvent tolerant bacteria.

Most bacteria and their enzymes are destroyed or inactivated in the presence of organic solvents. Organic solvent tolerant bacteria are a relatively novel group of extremophilic microorganisms that combat these destructive effects and thrive in the presence of high concentrations of organic solvents as a result of various adaptations. These bacteria are being explored for their potential in industrial and environmental biotechnology, since their enzymes retain activity in the presence of toxic solvents. This property could be exploited to carry out bioremediation and biocatalysis in the presence of an organic phase. Because a large number of substrates used in industrial chemistry, such as steroids, are water-insoluble, their bioconversion rates are affected by poor dissolution in water. This problem can be overcome by carrying out the process in a biphasic organic-aqueous fermentation system, wherein the substrate is dissolved in the organic phase and provided to cells present in the aqueous phase. In bioprocessing of fine chemicals such as cis-diols and epoxides using such cultures, organic solvents can be used to extract a toxic product from the aqueous phase, thereby improving the efficiency of the process. Bacterial strains reported to grow on and utilize saturated concentrations of organic solvents such as toluene can revolutionize the removal of such pollutants. It is now known that enzymes display striking new properties in the presence of organic solvents. The role of solvent-stable enzymes in nonaqueous biocatalysis needs to be explored and could result in novel applications.

Isolation of an organic-solvent-tolerant cholesterol-transforming Bacillus species, BC1, from coastal sediment.

Steroid transformation is of great importance in the pharmaceutical industry. The major limiting factor in this process is the extremely poor solubility of steroids in aqueous media, which lowers their transformation rate and increases costs. This problem can be overcome by using organic-solvent-tolerant bacteria (OSTB), which can carry out the desired bioconversions in an organic-solvent-saturated system. OSTB are a relatively novel group of extremophilic microbes that have developed various adaptations to withstand solvent toxicity. The aim of this study was to isolate marine bacteria producing organic-solvent-stable cholesterol-transforming enzymes. A Bacillus species, BC1, isolated from Arabian Sea sediment was found to degrade cholesterol and exhibit excellent solvent tolerance particularly to chloroform. OSTB have tremendous potential in industrial processes involving nonaqueous biocatalysis and transformation in the presence of an organic phase.

Tolerance of bacteria to organic solvents.

Organic-solvent-tolerant bacteria are a relatively novel group of extremophilic microorganisms. They overcome the toxic and destructive effects of organic solvents due to the presence of various adaptive mechanisms. Extensive studies done on the toluene tolerance of certain Pseudomonas strains have led to an understanding of the mechanisms of organic solvent tolerance involving novel adaptations such as the toluene efflux pumps, cis-trans isomerisation of membrane fatty acids, rapid membrane repair mechanisms, etc. Organic-solvent-tolerant mutants of Escherichia coli have been constructed and genes enhancing such tolerance characterised. However, there is practically no information available on the tolerance mechanisms of the reported Gram-positive organic-solvent-tolerant bacterial strains like Bacillus, Rhodococcus and Arthrobacter. This review discusses the general aspects of organic-solvent-tolerant bacteria, their history, biodiversity, mechanisms of tolerance and proposes certain probable adaptations of Gram-positive bacteria in tolerance to organic solvents.