Synthesis of octenyl succinic anhydride-modified levan and investigation of its microstructural, physicochemical, and emulsifying properties.

In this study, levan from Bacillus licheniformis NS032 was modified in an aqueous medium by octenyl succinic anhydride (OSA), and the properties of the obtained derivatives were studied. The maximum efficiency in the synthesis reaction was achieved at 40 °C and a polysaccharide slurry concentration of 30 %. Increasing the reagent concentration (2-10 %) led to an increase in the degree of substitution (0.016-0.048). Structures of derivatives were confirmed by FTIR and NMR. Scanning electronic microscopy, thermogravimetry, and dynamic light scattering analyses showed that the derivatives with degrees of substitution of 0.025 and 0.036 retained levan's porous structure and thermostability and showed better colloidal stability than the native polysaccharide. The intrinsic viscosity of derivatives increased upon modification, while the surface tension of the 1 % solution was lowered to 61 mN/m. Oil-in-water emulsions prepared with sunflower oil (10 % and 20 %) by mechanical homogenization and 2 and 10 % derivatives in the continuous phase showed mean oil droplet sizes of 106-195 µm, while the distribution curves exhibited bimodal character. The studied derivatives have a good capacity to stabilize emulsions, as they have a creaming index ranging from 73 % to 94 %. The OSA-modified levans could have potential applications in new formulations of emulsion-based systems.

SR-FTIR spectro-microscopic interaction study of biochemical changes in HeLa cells induced by Levan-C60, Pullulan-C60, and their cholesterol-derivatives.

Objects of the present study are improved fullerene C60 drug carrier properties trough encapsulation by microbial polysaccharides, levan (LEV), pullulan (PUL), and their hydrophobized cholesterol-derivatives (CHL and CHP), that show better interaction with cancer cells. The zeta potential, polydispersity index, and the diameter of particles were determined, and their cytotoxicity against three cancer cell lines were tested. Biochemical changes in HeLa cells are analyzed by synchrotron radiation (SR) FTIR spectro-microscopy combined with the principal component analysis (PCA). The most significant changes occur in HeLa cells treated with LEV-C60 and correspond to the changes in the protein region, i.e. Amide I band, and the changes in the structure of lipid bodies and membrane fluidity are evident. The highest cytotoxicity was also induced by LEV-C60. In HeLa cells, cytotoxicity could not be strictly associated with biochemical changes in lipids, proteins and nucleic acids, but these findings are significant contribution to the study of the mechanism of interaction of C60-based nanoparticles with cellular biomolecules. In conclusion, LEV, PUL, CHL, and CHP enhanced fullerene C60 potential to be used as target drug delivery system with the ability to induce specific intracellular changes in HeLa cancer cells.

Bacillus licheniformis levan as a functional biopolymer in topical drug dosage forms: From basic colloidal considerations to actual pharmaceutical application.

Ongoing demand in sustainable and biocompatible drug dosage forms is reflected in the search for novel pharmaceutical excipients with equal properties. A group of microbial exopolysaccharides offers a variety of biopolymers with many alleged uses and effects. This study aims to assess applicative properties of levan obtained from Bacillus licheniformis NS032, focusing on its potential co-stabilizing and drug release-controlling functions in pertaining emulsion systems. Despite its high molecular weight and partial existence in globular nanometric structures (180-190 nm), levan was successfully incorporated into both tested colloidal systems: those stabilized with synthetic/anionic or natural-origin/non-ionic emulsifiers. In the tested levan concentrations range (0.2-3.0% w/w) the monitored flow and thermal parameters failed to show linear concentration dependence, which prompted us to revisit certain colloidal fundamentals of this biopolymer. Being a part of the external phase of the investigated emulsion systems, levan contributed to formation of a matrix-like environment, offering additional stabilization of the microstructure and rheology modifying properties (hysteresis loop elevation as high as 4167±98 to 20792±3166 Pa•s-1), especially in case of the samples where lamellar liquid crystalline formation occurred. Apart from its good water solubility and considerable conformational flexibility, the investigated homofructan easily saturated the external phase of the samples stabilized with a conventional anionic emulsifier, leading to similar properties of 0.2% and 3.0% levan-containing samples. After closer consideration of thermal and release behavior, this was considered as a favorable property for a novel excipient, offering tailored formulation characteristics even with lower levan concentrations, consequently not compromising the potential cost of the final drug dosage form.

Microbial levan and pullulan as potential protective agents for reducing adverse effects of copper on Daphnia magna and Vibrio fischeri.

Microbial polysaccharides, due to their unique physiochemical properties, have found application in the food industry, cosmetics, pharmacy and medicine. In the environment, microbes can use polysaccharides to alleviate the adverse effects of heavy metals in their close proximity. This adaptive property shows interesting potential for bioremediation. Herein, the effects of the exopolysaccharides (EPS) levan, produced by the bacterium Bacillus licheniformis NS032 and pullulan, produced by the fungus Aureobasidium pullulans CH-1 in the presence of copper (Cu2+) have been investigated for the first time on antioxidant enzyme activity, respiration and Cu2+ bioaccumulation of Daphnia magna as well as the bioluminescence of Vibrio fischeri. Both EPS decreased toxicity of Cu2+ in the acute test with D. magna. The activity of catalase (CAT) was significantly diminished after acute exposure to Cu2+ in comparison to treatments with Cu2+ and EPS, while in the prolonged acute exposure the CAT activity did not show statistically significant (P ≤ 0.05) differences between treatments with and without the EPS. According to ICP-MS results, during prolonged acute exposure of neonates, the bioaccumulation of Cu2+ in treatments without the EPS was 52.03 µg/g of biomass (wet), while in treatments with EPS, the bioaccumulation was lower by one order of magnitude. The respiration of neonates during acute exposure to Cu2+ with or without the EPS was monitored using the MicroOxymax respirometer, and the results show the EPS can positively effect the respiration. In the case of bacterial bioluminescence, the toxicity of Cu2+ decreased in treatments with EPS (30 min EC10) from 3.54 mg/L to 140.61 mg/L (levan) and 45.00 mg/L (pullulan). This study demonstrates protective effect of EPS against Cu2+ toxicity on D. magna and V. fischeri, and opens the door for further investigation of potential application of levan and pullulan in bioremediation of heavy metals and mitigation of their adverse effects in the aquatic environment.

High-quality draft genome sequence of Pseudomonas aeruginosa san ai, an environmental isolate resistant to heavy metals.

The strain Pseudomonas aeruginosa san ai, isolated from an extreme environment (industrial mineral cutting oil, pH 10), is able to survive and persist in the presence of a variety of pollutants such as heavy metals and organic chemicals. The genome of P. aeruginosa san ai is 6.98 Mbp long with a GC content of 66.08% and 6485 protein encoding genes. A large number of genes associated with proteins, responsible for microbial resistance to heavy metal ions and involved in catabolism of toxic aromatic organic compounds were identified. P. aeruginosa san ai is a highly cadmium-resistant strain. Proteome analysis of biomass after cadmium exposal confirmed a high tolerance to sublethal concentrations of cadmium (100 mg/L), based on: extracellular biosorption, bioaccumulation, biofilm formation, controlled siderophore production and a pronounced metalloprotein synthesis. Proteins responsible for survival in osmostress conditions during exposure to elevated concentrations of cadmium (200 mg/L) demonstrate a strong genetic potential of P. aeruginosa san ai for survival and adaptation. Sequencing of P. aeruginosa san ai genome provides valuable insights into the evolution and adaptation of this microbe to environmental extremes at the whole-genome level, as well as how to optimally use the strain in bioremediation of chemically polluted sites.

Biodegradation of the aromatic fraction from petroleum diesel fuel by Oerskovia sp. followed by comprehensive GC×GC-TOF MS.

Polycyclic aromatic hydrocarbons (PAHs) from petroleum and fossil fuels are one of the most dominant pollutants in the environment. Since aromatic fraction from petroleum diesel fuel is mainly composed of PAHs, it is important to discover new microorganisms that can biodegrade these compounds. This article describes the biodegradation of the aromatic fraction separated from petroleum diesel fuel using the strain Oerskovia sp. CHP-ZH25 isolated from petroleum oil-contaminated soil. The biodegradation was monitored by gravimetry and GC × GC-TOF MS. An innovative method was applied to visualize degraded compounds in the data provided by a GC × GC-TOF MS. It was shown that Oerskovia sp. CHP-ZH25 degraded 77.4% based on gravimetric analysis within 30 days. Average rate of degradation was 14.4 mg/L/day, 10.5 mg/l/day and 4.0 mg/l/day from 0 to 10 day, 10-20 and 20-30 day, respectively. The order of PAH degradation based on decrease in peak volume after 30 days of incubation was as follows: dibenzothiophene derivatives > benzo[b]thiophene derivatives > naphthalene derivatives > acenaphthene derivatives > acenaphthylene/biphenyl derivatives > fluorene derivatives > phenanthrene/anthracene derivatives. Here we demonstrated that Oerskovia sp. CHP-ZH25 could potentially be a suitable candidate for use in bioremediation of environments polluted with different PAHs.

Natural attenuation of petroleum hydrocarbons-a study of biodegradation effects in groundwater (Vitanovac, Serbia).

The role of natural attenuation processes in groundwater contamination by petroleum hydrocarbons is of intense scientific and practical interest. This study provides insight into the biodegradation effects in groundwater at a site contaminated by kerosene (jet fuel) in 1993 (Vitanovac, Serbia). Total petroleum hydrocarbons (TPH), hydrochemical indicators (O2, NO3-, Mn, Fe, SO42-, HCO3-), δ13C of dissolved inorganic carbon (DIC), and other parameters were measured to demonstrate biodegradation effects in groundwater at the contaminated site. Due to different biodegradation mechanisms, the zone of the lowest concentrations of electron acceptors and the zone of the highest concentrations of metabolic products of biodegradation overlap. Based on the analysis of redox-sensitive compounds in groundwater samples, redox processes ranged from strictly anoxic (methanogenesis) to oxic (oxygen reduction) within a short distance. The dependence of groundwater redox conditions on the distance from the source of contamination was observed. δ13C values of DIC ranged from - 15.83 to - 2.75‰, and the most positive values correspond to the zone under anaerobic and methanogenic conditions. Overall, results obtained provide clear evidence on the effects of natural attenuation processes-the activity of biodegradation mechanisms in field conditions.

Cadmium specific proteomic responses of a highly resistant Pseudomonas aeruginosa san ai.

Pseudomonas aeruginosa san ai is a promising candidate for bioremediation of cadmium pollution, as it resists a high concentration of up to 7.2 mM of cadmium. Leaving biomass of P. aeruginosa san ai exposed to cadmium has a large biosorption potential, implying its capacity to extract heavy metal from contaminated medium. In the present study, we investigated tolerance and accumulation of cadmium on protein level by shotgun proteomics approach based on liquid chromatography and tandem mass spectrometry coupled with bioinformatics to identify proteins. Size exclusion chromatography was used for protein prefractionation to preserve native forms of metalloproteins and protein complexes. Using this approach a total of 60 proteins were observed as up-regulated in cadmium-amended culture. Almost a third of the total numbers of up-regulated were metalloproteins. Particularly interesting are denitrification proteins which are over expressed but not active, suggesting their protective role in conditions of heavy metal exposure. P. aeruginosa san ai developed a complex mechanism to adapt to cadmium, based on: extracellular biosorption, bioaccumulation, the formation of biofilm, controlled siderophore production, enhanced respiration and modified protein profile. An increased abundance of proteins involved in: cell energy metabolism, including denitrification proteins; amino acid metabolism; cell motility and posttranslational modifications, primarily based on thiol-disulfide exchange, were observed. Enhanced oxygen consumption of biomass in cadmium-amended culture versus control was found. Our results signify that P. aeruginosa san ai is naturally well equipped to overcome and survive high doses of cadmium and, as such, has a great potential for application in bioremediation of cadmium polluted sites.

Brachybacterium sp. CH-KOV3 isolated from an oil-polluted environment-a new producer of levan.

Various microorganisms isolated from polluted environments, such as Pseudomonas sp. and Micrococcus sp. can synthesize exopolysaccharides (EPSs) which are natural, non-toxic and biodegradable polymers. EPSs play a key role in protection of microbial cells under various external influences. For humans, these substances have potential use in many industries. EPSs can be applied as a flavor or a fragrance carrier, an emulsifier, a stabilizer, a prebiotic, an antioxidant or an antitumor agent. In this study, we characterized an environmental microorganism that produces EPS, optimized EPS production by this strain and characterized the EPS produced. Isolate CH-KOV3 was identified as Brachybacterium paraconglomeratum. The sucrose level in the growth medium greatly influenced EPS production, and the highest yield was when the microorganism was incubated in media with 500g/L of sucrose. The optimal temperature and pH were 28°C and 7.0, respectively. The nuclear magnetic resonance (NMR) results and GC-MS analysis confirmed that the residues were d-fructofuranosyl residues with ß-configuration, where fructose units are linked by ß-2,6-glycosidic bonds, with ß-2,1-linked branches. All these data indicate that the investigated EPS is a levan-type polysaccharide. Thus, it was concluded that Brachybacterium sp. CH-KOV3 could constitute a new source for production of the bioactive polysaccharide, levan.

Scrutinizing microwave effects on glucose uptake in yeast cells.

Taking into account different literature reports on microwave (MW) effects on living organisms, we thoroughly investigated the influence of constant 2.45 GHz MW irradiation on glucose uptake in yeast cells. A Saccharomyces cerevisiae suspension of 2.9 × 108 cells/ml was used in all experiments. A large specific absorption rate of 0.55 W/g of suspension is compensated by efficient external cooling of the reaction vessel, which established a strong non-equilibrium flow of energy through the solution and enabled a constant bulk temperature of 30 °C to within 1 °C during glucose uptake. Comparison of MW effects with control experiments revealed insignificant changes of glucose uptake during the initial stages of the experiment (up to the 10th min). Statistically "notable" differences during the next 20 min of the irradiation were detected corresponding to thermal overheating of 2 °C. Possible specific thermal MW effects may be related to local temperature increase and a large flow of energy throughout the system. The obtained effects show that environmental MW pollution (fortunately) is of too low intensity to provoke metabolic changes in living cells. At the same time, a longer exposure of cells to electromagnetic irradiation may have impacts on biochemical applications and production of valuable biotechnological products.

Synthesis and characterization of a new type of levan-graft-polystyrene copolymer.

Novel macromolecular graft copolymers were synthesized by reaction of the hydroxyl groups of the microbial polysaccharide levan, produced using Bacillus licheniformis, with polystyrene (Lev-g-PS). Synthesis was performed by the free radical reaction using potassium persulfate (PPS) as initiator. The prepared copolymer was characterized by FTIR, SEM, TG/DTA, XRD and (13)C NMR. The influence of the different conditions (reaction temperature, air or nitrogen atmosphere, reaction time, type of amines and ascorbic acid (AA) concentration) on the grafting reaction was investigated. Results showed that maximum percentage of grafting (58.1%) was achieved at a reaction temperature 70°C, in a nitrogen atmosphere and using dimethylethanolamine (DMEA) as the amine activator. On the basis of the obtained results, the likely reaction mechanism was proposed. Synthesized copolymers have better thermal stability in comparison with their initial components. Copolymers such as Lev-g-PS could potentially have many applications, such as compatibilizers and material for membranes.

Interactions of the metal tolerant heterotrophic microorganisms and iron oxidizing autotrophic bacteria from sulphidic mine environment during bioleaching experiments.

Iron and sulfur oxidizing chemolithoautotrophic acidophilic bacteria, such as Acidithiobacillus species, hold the dominant role in mine environments characterized by low pH values and high concentrations of reduced sulfur and iron compounds, such as ores, rocks and acid drainage waters from mines. On the other hand, heterotrophic microorganisms, especially their biofilms, from these specific niches are receiving increased attention, but their potential eco-physiological roles have not been fully understood. Biofilms are considered a threat to human health, but biofilms also have beneficial properties as they are deployed in waste recycling and bioremediation systems. We have analyzed interactions of the metal tolerant heterotrophic microorganisms in biofilms with iron oxidizing autotrophic bacteria both from the sulphidic mine environment (copper mine Bor, Serbia). High tolerance to Cu(2+), Cd(2+) and Cr(6+) and the presence of genetic determinants for the respective metal tolerance and biofilm-forming ability was shown for indigenous heterotrophic bacteria that included strains of Staphylococcus and Rhodococcus. Two well characterized bacteria- Pseudomonas aeruginosa PAO1 (known biofilm former) and Cupriavidus metallidurans CH34 (known metal resistant representative) were also included in the study. The interaction and survivability of autotrophic iron oxidizing Acidithiobacillus bacteria and biofilms of heterotrophic bacteria during co-cultivation was revealed. Finally, the effect of heterotrophic biofilms on bioleaching process with indigenous iron oxidizing Acidithiobacillus species was shown not to be inhibitory under in vitro conditions.

Application of non-thermal plasma reactor and Fenton reaction for degradation of ibuprofen.

Pharmaceutical compounds have been detected frequently in surface and ground water. Advanced Oxidation Processes (AOPs) were reported as very efficient for removal of various organic compounds. Nevertheless, due to incomplete degradation, toxic intermediates can induce more severe effects than the parent compound. Therefore, toxicity studies are necessary for the evaluation of possible uses of AOPs. In this study the effectiveness and capacity for environmental application of three different AOPs were estimated. They were applied and evaluated for removal of ibuprofen from water solutions. Therefore, two treatments were performed in a non-thermal plasma reactor with dielectric barrier discharge with and without a homogenous catalyst (Fe(2+)). The third treatment was the Fenton reaction. The degradation rate of ibuprofen was measured by HPLC-DAD and the main degradation products were identified using LC-MS TOF. Twelve degradation products were identified, and there were differences according to the various treatments applied. Toxicity effects were determined with two bioassays: Vibrio fischeri and Artemia salina. The efficiency of AOPs was demonstrated for all treatments, where after 15 min degradation percentage was over 80% accompanied by opening of the aromatic ring. In the treatment with homogenous catalyst degradation reached 99%. V. fischeri toxicity test has shown greater sensitivity to ibuprofen solution after the Fenton treatment in comparison to A. salina.

Perfluorinated compounds in sediment samples from the wastewater canal of Pancevo (Serbia) industrial area.

Perfluoroalkyl sulfonates (PFSAs) and perfluoroalkyl carboxylates (PFCAs) were analyzed in sediment samples from the wastewater canal draining the industrial complex of Pancevo, Serbia (oil refinery, petrochemical plant, and fertilizer factory). The canal is directly connected to Europe's second largest river, the Danube, which drains its water into the Black Sea. Perfluorooctane sulfonate (PFOS) up to 5.7ngg(-1) dry weight (dw) and total Perfluorinated compounds (PFCs) up to 6.3ngg(-1) dw were detected. Compared to other reports, high levels of PFOS were found, even though PFCs are not used in the industrial production associated with this canal. The PFOS concentration in water was recalculated using the adsorption coefficient, KOC from literature. Using the average output of wastewater from the canal, a mass load of 1.38kg PFOS per year discharged in the Danube River has been calculated, which undoubtedly points to the contribution to global persistent organic pollution of surface waters originating from this industrial place.

Ex situ bioremediation of a soil contaminated by mazut (heavy residual fuel oil)--a field experiment.

Mazut (heavy residual fuel oil)-polluted soil was exposed to bioremediation in an ex situ field-scale (600 m(3)) study. Re-inoculation was performed periodically with biomasses of microbial consortia isolated from the mazut-contaminated soil. Biostimulation was conducted by adding nutritional elements (N, P and K). The biopile (depth 0.4m) was comprised of mechanically mixed polluted soil with softwood sawdust and crude river sand. Aeration was improved by systematic mixing. The biopile was protected from direct external influences by a polyethylene cover. Part (10 m(3)) of the material prepared for bioremediation was set aside uninoculated, and maintained as an untreated control pile (CP). Biostimulation and re-inoculation with zymogenous microorganisms increased the number of hydrocarbon degraders after 50 d by more than 20 times in the treated soil. During the 5 months, the total petroleum hydrocarbon (TPH) content of the contaminated soil was reduced to 6% of the initial value, from 5.2 to 0.3 g kg(-1) dry matter, while TPH reduced to only 90% of the initial value in the CP. After 150 d there were 96%, 97% and 83% reductions for the aliphatic, aromatic, and nitrogen-sulphur-oxygen and asphaltene fractions, respectively. The isoprenoids, pristane and phytane, were more than 55% biodegraded, which indicated that they are not suitable biomarkers for following bioremediation. According to the available data, this is the first field-scale study of the bioremediation of mazut and mazut sediment-polluted soil, and the efficiency achieved was far above that described in the literature to date for heavy fuel oil.

Enzymatic characterization of 30 kDa lipase from Pseudomonas aeruginosa ATCC 27853.

An extracellular lipase from Pseudomonas aeruginosa ATCC 27853 has been purified and its enzymatic characteristics were determined. According to SDS-PAGE and gel filtration molecular mass estimated to be 30 kDa, what classified the lipase in group I.1. Although 14 lipases from P. aeruginosa with similar molecular mass are referred to date, their basic enzymatic properties have not been reported yet. To address the gap we found: the optimal temperature and pH in water solution being 50 degrees C and 9.3, respectively; the lipase was inhibited with Hg2+ ions and sodium dodecylsulphate (SDS), while non-ionic detergent Triton X-100 activated the enzyme; the lipase hydrolyzed more rapidly middle chain triglycerides and it was not regiospecific; the lipase demonstrated naturally occurring stability in different organic solvents with concentrations ranging from 30 to 70%, including good thermal stability in 30% organic solvent solution. Even though strain P. aeruginosa ATCC 27853 was not isolated from extreme environment it showed activity in organic solvent suggesting that this lipase is suitable for variety of applications, including reactions in water restricted medium and bioremediation of contaminations by organic solvents.

Impact of carboxymethylcellulose on morphology and antibiotic production by Streptomyces hygroscopicus.

A chemically defined media consisting of carboxymethylcellulose (CMC) was developed to maximize the production of antibiotics, hexaene H-85 and azalomycine, by Streptomyces hygroscopicus CH-7. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. By adding the polymer to the fermentation medium, the growth was changed from a single large glob to small reproducible pellets, and wall growth was diminished to a minimum. Maximum concentrations of hexaene H-85 (146.7 mg/dm(3)) and azalomycine (188.6 mg/dm(3)) were reached at 3.0% and 1.0% (w/v) CMC, respectively.

Leucine aminopeptidase from Streptomyces hygroscopicus is controlled by a low molecular weight inhibitor.

In culture filtrate of Streptomyces hygroscopicus a producer of polyketide antibiotics, a leucine aminopeptidase and its autogenous inhibitor were detected. The leucine aminopeptidase was purified 4573-fold with yield of 82% by combination of ion exchange and hydrophobic chromatography. The enzyme is monomeric with a molecular mass of 51 kDa determined by gel chromatography and 67 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal activity was at pH 8.0 and 40 degrees C. The pI of leucine aminopeptidase is 8.2. The enzyme is strongly inhibited by 1,10-phenantroline, amastatin and dithiothreitol. Atomic absorption spectrometry indicated 2 mols of ion zinc per mol of enzyme. The enzyme is stable at up to 70 degrees C. Leucine aminopeptidase prefers leucine and methionine as N-terminal amino acids. Activity of leucine aminopeptidase is strongly modulated by an autogenous low-molecular weight inhibitor during fermentation, especially during periods of intensive antibiotic production.