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The production of levan by Bacillus licheniformis NS032 in a medium based on sugar beet molasses was studied. High polysaccharide yields were produced by using diluted molasses (100–140 g/L of total sugars) with the addition of commercial sucrose up to 200 g/L of total sugars, as well as K2HPO4. A levan yield of 53.2 g/L was obtained on a medium optimized by response surface methodology, containing 62.6% of sugar originating from molasses, and 4.66 g/L of phosphate, with initial pH value of 7.2. In comparison to the media with 200 and 400 g/L sucrose, in the molasses optimized medium, the observed bacterial growth was faster, while the maximum production of polysaccharide was achieved over a shorter time interval (48 h). The polysaccharide produced in molasses medium had a weight average molecular weight of 5.82 × 106 Da, degree of branching 12.68%, viscosity of 0.24 dL/g, and based on methylation analysis and NMR data, it did not significantly differ from levan obtained in the medium with 200 g/L sucrose. © 2018 Elsevier B.V. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2943] This is peer reviewed version of the following article: Gojgic-Cvijovic, G. D.; Jakovljevic, D. M.; Loncarevic, B. D.; Todorovic, N. M.; Pergal, M. V.; Ciric, J.; Loos, K.; Beškoski, V.; Vrvić, M. M. Production of Levan by Bacillus Licheniformis NS032 in Sugar Beet Molasses-Based Medium. International Journal of Biological Macromolecules 2019, 121, 142–151. [https://doi.org/10.1016/j.ijbiomac.2018.10.019]

We developed capillary zone electrophoresis (CZE) with indirect UV detection for the determination of fluoride (F-) in seawater using transient isotachophoresis (tITP) as an on-line concentration procedure. A method of correcting sample salinity effects was also proposed so that F- concentrations were obtained using a calibration graph. The proposed method is simple: it requires no sample pretreatment aside from dilution. The following optimum conditions were established: background electrolyte (BGE), 5 mM 2,6-pyridinedicarboxylic acid (PDC) adjusted to pH 3.5 containing 0.03% m/v hydroxypropyl methylcellulose (HPMC); detection wavelength, 200 nm; vacuum (50 kPa) injection period of sample, 5 s (254 nL); and applied voltage, 23 kV with the sample inlet side as the cathode. The limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) for F- reached 0.024 and 0.070 mg/L, respectively. The respective values of the relative standard deviation (RSD) of the peak area, peak height, and migration time for F- were 2.5, 3.4, and 0.30%. The proposed method was applied for the determination of F- in seawater samples collected from coastal waters of western Japan during August 26-28, 2014. Both results obtained using standard addition method and a calibration graph agreed with those obtained using a conventional spectrophotometric method. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3182]

The aim of this paper was to investigate whether changes in the quantity and structure of humic acids (HA) occur during bioremediation of waste mazute (heavy residual fuel oil). The effects of humification on the structure of HA were evaluated by Fourier Transform Infrared Spectroscopy (FT-IR), potentiometric titrations, the ratio of absorbances at 465 and 665 nm (E4/E6 ratio) and the ratio. Also, the quality of humification was monitored by determining parameters which are important for measuring the evolution of humic substances (HS), such as CHA/CFA (the humic acidifulvic acidratio), the rate of extraction (TE) and the index of polymerization (IP). An increase of the content of HA from 0.23% to 0.70% was observed during the bioremediation process. The FT-IR spectra and 04 ratio of HA extracted at the beginning and the end of the process indicate structural changes during the bioremediation process. The groups containing aromatic and carboxylic carbon increased, resulting in HA structures of higher aromaticity. An increase of the B4/E6 ratio during bioremediation treatment implied the enrichment of HA with functional oxygen groups. Potentiometric titrations of HA solution showed increases in the buffering and the redox capacities of HA during the bioremediation process. The results of parameters of humification showed an increase of the compositional carbon in HA at the end, compared to at the start, of bioremediation, while the carbon that belonged to fulvic acids (FA) reduced by 44%. Transformation into HA is the likely cause of this observed reduction. The TE and the IP demonstrated that humification occured during the bioremediation process. According to the available data, this is the first report on synthesis of HS during bioremediation of petroleum hydrocarbons. (C) 2017 Published by Elsevier Ltd.

Environmental pollution is a global problem, while bioremediation technology removes pollutants from the environment using microorganisms. This study was aimed at investigating how a bioremediation process affected soil humification. In soil polluted with petroleum and its derivatives that was submitted to bioremediation, besides the total petroleum hydrocarbons and the number of microorganisms, quantitative and qualitative changes of isolated humic acids were determined during the process. The bioremediation of 150 m3 of polluted soil lasted 150 days. The level of total petroleum hydrocarbons decreased by 86.6 %, while the level of humic acids increased by 26.5 %. The elemental analysis showed the reduction of C and the H/C ratio and the increase of O and the O/C ratio of isolated humic acids during the process. The ratio of absorbencies at 465 and 665 nm also increased. Based on this and the Fourier-transform infrared spectra, it was shown that the humic acids isolated at the end of bioremediation were enriched with oxygen functional groups and aromatic structures. This study provides one of the first insights into the relationship between bioremediation and humification, as well as evidence of how hydrocarbon-degrading microorganisms have a significant influence on changes to humic acid structure during bioremediation. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III 43004]

An efficient encapsulation of the fullerene into two hydrophobized and one native polysaccharide provided water soluble supramolecular hybrids. After covalent modification of polysaccharides by cholesterol, noncovalent hybrids were prepared by a three-step procedure, including mixing of individual aqueous solutions of hydrophobized, as well as native sugar with solution of the fullerene in pyridine, dialysis and lyophilization. Although the degree of the fullerene incorporation into hydrophobized substrates, cholesterol-levan and cholesterol-pullulan, was lower in comparison to the native polysaccharide levan, hydrophobization provided nanoparticles with improved properties. The particle size distribution, studied by dynamic light scattering and scanning electron microscopy revealed formation of moderately polydisperse aggregates, with the diameter contraction in comparison to the corresponding free polysaccharide, especially in the case of hydrophobized substrates. The morphological examination, done by scanning electron microscopy indicated the self-organization of the fullerene-native polysaccharide to round individual structures, while fullerene-hydrophobized polysaccharide hybrids tend to form networks. The antioxidant activity of the synthesized polysaccharide-C60 noncovalent hybrids versus starting polysaccharides was investigated by the DPPH radical scavenging and the β-carotene-linoleic acid bleaching methods. In all three complexes, the radical scavenging ability of the fullerene remained preserved, and a positive effect of levan hydrophobization was observed. This is the peer-reviewed version of the manuscript: Kop, T.J., Jakovljević, D.M., Živković, L.S., Žekić, A., Beškoski, V.P., Milić, D.R., Gojgić-Cvijović, G.D., Bjelaković, M.S., Polysaccharide-fullerene supramolecular hybrids: synthesis, characterization and antioxidant activity, European Polymer Journal (2019), doi: [https://doi.org/10.1016/j.eurpolymj.2019.109461] The published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3803]

One of the main global focuses of mankind today is the required switch to new energy sources. Generating energy from waste is one of the potential solutions that can be achieved using microbial fuel cells (MFCs). Microorganisms, thanks to their ability to degrade organic substrates in contaminated environments, could contribute to solving our pollution challenge. The aim of this study was to investigate the potential of sediment with its natural microbiota from the River Danube to optimize electricity generation using MFCs. 16S rRNA gene analysis identified the main bacterial genera in the river sediment, Clostridium, Bacillus and Tepidibacter, which were isolated and cultured in the laboratory. Addition of these cultured microorganisms to the MFC resulted in current density of 192 mA/m3, while the power density was about 8.80 mW/m3. Our study confirms proper selection and enrichment of the microbial community can optimize the amount of current obtainable from river sediment by MFCs. This is the peer-reviewed version of the article: Joksimović, K.; Žerađanin, A.; Randjelović, D.; Avdalović, J.; Miletić, S. B.; Gojgić-Cvijović, G. D.; Beškoski, V. Optimization of Microbial Fuel Cell Operation Using Danube River Sediment. Journal of Power Sources 2020, 476, 228739. [https://doi.org/10.1016/j.jpowsour.2020.228739] The published version: [http://cer.ihtm.bg.ac.rs/handle/123456789/3617]

Grape pomace (GP) from Vitis labrusca, the main byproduct from “American table wine” production, is recalcitrant to degradation, and its accumulation is a serious problem with negative environmental impacts. In this work, transformation of grape pomace using a steam pretreatment followed by incubation of GP during a 90-day period with six different fungi were evaluated. Several fungi tested reduced the phytotoxicity of water-soluble fraction (WSFd) from steam-pretreated GP after 90 days’ incubation to lettuce and tomato seeds. U. botrytis caused the largest effective phytotoxicity reduction of WSFd (used in the concentration range of 10–1.25% p/v) and was the only fungus causing the removal of monoaromatic compounds. Therefore, this procedure with U. botrytis effectively reduces the availability of phytotoxic monoaromatic compounds in GP, which opens a way for the development of guidelines for the management of these wastes and their potential use as organic amendments in agricultural soil., Centro de Investigaciones en Fitopatología, Instituto de Botánica "Dr. Carlos Spegazzini", Facultad de Ciencias Agrarias y Forestales

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 gt benzo[b]thiophene derivatives gt naphthalene derivatives gt acenaphthene derivatives gt acenaphthylene/biphenyl derivatives gt fluorene derivatives gt 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. © 2018 Elsevier B.V. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2950] This is the peer review version of the following article: Lješević, M.; Gojgić-Cvijović, G.; Ieda, T.; Hashimoto, S.; Nakano, T.; Bulatović, S.; Ilić, M.; Beškoski, V. Biodegradation of the Aromatic Fraction from Petroleum Diesel Fuel by Oerskovia Sp. Followed by Comprehensive GC×GC-TOF MS. Journal of Hazardous Materials 2019, 363, 227–232. [https://doi.org/10.1016/j.jhazmat.2018.10.005]

The Pseudomonas aeruginosa san ai strain was investigated for its capability to degrade the 2,6-di-tert-butylphenol (2,6-DTBP) plastic additive, a hazardous and toxic substance for aquatic life. This investigation was performed under different parameter values: 2,6-DTBP concentration, inoculum size, pH, and temperature. The GC-MS study showed that P. aeruginosa efficiently degraded 2,6-DTBP in the pH range of 5-8 at higher temperatures. Under exposure to 2,6-DTBP concentrations of 2, 10, and 100 mg L-1, the strain degraded by 100, 100, and 85%, respectively, for 7 days. Crude enzyme preparation from the biomass of P. aeruginosa san ai showed higher efficiency in 2,6-DTBP removal than that shown by whole microbial cells. Gene encoding for the enzymes involved in the degradation of aromatic compounds in P. aeruginosa san ai was identified. To complement the genomic data, a comparative proteomic study of P. aeruginosa san ai grown on 2,6-DTBP or sunflower oil was conducted by means of nanoLC-MS/MS. The presence of aromatic substances resulted in the upregulation of aromatic ring cleavage enzymes, whose activity was confirmed by enzymatic tests; therefore, it could be concluded that 2,6-DTBP might be degraded by ortho-ring cleavage. A comparative proteomics study of P. aeruginosa san ai indicated that the core molecular responses to aromatic substances can be summarized as the upregulation of proteins responsible for amino acid metabolism with emphasized glutamate metabolism and energy production with upregulated enzymes of glyoxylate bypass. P. aeruginosa san ai has a high capacity to efficiently degrade aromatic compounds, and therefore its whole cells or enzymes could be used in the treatment of contaminated areas. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3347]

Cerium oxide (CeO2) nanoparticles (CONPs) are interesting biomaterials with various applications in biomedicine, cosmetics and the pharmaceutical industry, but with limited practical application because of their low stability in aqueous media. The aim of this study was to obtain CONPs with increased stability by coating the particles. Microbial exopolysaccharides (levan, pullulan) and glucose were used to prepare CONPs under different synthesis conditions. Coating was attempted by adding the carbohydrates during (direct coating) or after (subsequent coating) the synthesis of CONPs. The obtained nanoparticles were characterized by X-Ray diffraction analysis, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The suspension stability of the uncoated and coated CONPs in aqueous media was evaluated by measuring the hydrodynamic size, zeta potential and turbidity. The FT-IR spectra revealed the differences between coated CONPs and showed the success of subsequent coating with carbohydrates. Coating with carbohydrates improved the stability the CONP suspension by decreasing the size of aggregated particles. The suspensions of levan- and glucose-coated CONPs had the best stability. In this study, CONPs were prepared using non-toxic materials, which were completely environmentally friendly. The obtained results open new horizons for CONP synthesis, improving their biological applications. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III45012 and Grant no. III43004]

We investigated the capability of microorganisms isolated from environments polluted with perfluoroalkyl adds (PFAAs) to conduct biotransformation of these emerging pollutants. Two different microbial consortia (chemoorganoheterotrophic bacteria and total yeast and molds) were isolated from two river sediments in Saitama and Osaka, Japan, known for long term pollution with perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). The microbial consortia were incubated in the presence of added PFOS and PFOA, and decreases in concentrations of these compounds were between 46-69% and 16-36%, respectively. Decreases in concentrations were, in part, due to sorption on biomass, but defluorinated PFOS and PFOA products were not detected. However, untargeted analysis suggested the presence of several metabolites found only in samples from consortia with PFOS and PFOA but not in the control samples. Molecular formula candidates were narrowed down to two options, C18H28O5F and C21H27O4. It was assumed that these formulas were associated with unsaturated monofluorinated fatty acids and hydrocarbons with multiple unsaturated bonds or ring structures. (C) 2018 Elsevier B.V. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3114]

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 (O-2 , NO3-, Mn, Fe, SO42-, HCO3- ), delta C-13 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. delta C-13 values of DIC ranged from -15.83 to -2.75 parts per thousand, 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.

Bioremediation is the process of detoxification or elimination of pollutants using microorganisms with different metabolic capabilities. Biodegradation by natural populations of microorganisms is one of the primary mechanisms by which oil and other pollutants of hydrocarbon origin can be removed from the environment and it is also much cheaper than the other remediation technologies.In this study, we analyzed the samples of historical waste from the oil industry, which contained sand, organic materials, heavy fuel oil and catalysts used during the process of hydrodesulfurization (HDS) of oil. The aim was to examine the fate of cobalt and molybdenum, toxic heavy metals present in those catalysts. A consortium of microorganisms isolated from the complex pollutants from the oil industry was added to the samples. During the study, beside the transformation of cobalt and molybdenum forms, we also monitored the biodegradation process of the total petroleum hydrocarbons (TPH).

The biogeochemical activity of microorganisms from sulphide biogeocoenosis in the zones of sulphide copper ores deposits in East Serbia has been Studied by us since 1975. All deposits of copper mineralization were dominantly sulphides and arsenic sulphides with the constant presence of pyrite. The obtained results proved that the biochemical and chemical activity of thionic bacteria have an influence on the global pollution of waters. All the waters were characterized by a low pH value and by high Eh values, mineralization (over 7 gL(-1)) and in total iron (even more than 4 gL(-1)) dominantly as Fe3+ ions. These waters were characterized by toxic concentrations of copper ions, within the limits from about 2 to almost 200 mgL(-1) and arsenic, up to more than 4 mgL(-1). These waters partly arrive in an aquifer and the major part flows into surface water courses gravitating towards the Danube River, which empties into the Black Sea. In a hypothetical case, if maximum concentrations of copper and arsenic and maximum capacity of the springs are considered, under the assumption that the pollutants do not precipitate on the way to the main recipient, the striking information is obtained that the Danube River receives about 31 t of copper and more than 0.6 t of arsenic per annum from one place alone, which undoubtedly points to the contribution of thionic bacteria as "biological agents" to global pollution of surface and ground waters. BIOHYDROMETALLURGY: A MEETING POINT BETWEEN MICROBIAL ECOLOGY, METAL RECOVERY PROCESSES AND ENVIRONM

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 C-13 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 degrees 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. (C) 2016 Elsevier Ltd. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3624]

Investigations of heavy metals from lignite ash, which is a coal combustion by-product, have for years been very interesting from different aspects of environmental protection. In this study, the possibility of microbiological ash leaching, with a strong oxidation agent, bacterially generated Fe2(SO4)3 was investigated in order to minimize the concentration of toxic ash elements. Leaching experiment by the shake flask testing technique was performed during a period of 20 d at a temperature of 28?C. The results obtained show 5- to 22-fold (for Cd and Ni approx. 5, Zn approx. 6, Mn approx. 8, Cr approx. 13 and Cu approx. 22) higher metal leaching efficiency in the suspension with bacterially generated Fe2(SO4)3 than in the control suspension. .

This study provides insight into the enhanced in situ bioremediation applied for remediation of groundwater contaminated by petroleum hydrocarbons. Activities prior to the application of this remediation approach included removal of the source of groundwater contamination—an underground storage tank and contaminated sediments—from the unsaturated zone. The hydraulic feasibility of this remediation approach was proved by hydraulic conductivity characterization of the site. Enhanced in situ bioremediation was performed by a combination of biostimulation and bioaugmentation within the closed bipolar system (one extraction and two injection wells). Biostimulation was conducted by addition of nutrients, and stimulation of oxidation processes by injection of H2O2, into the aquifer. Bioaugmentation/re-inoculation was achieved by injection of a zymogenous consortium of hydrocarbon degrading microorganisms isolated from the contaminated groundwater. The average extraction capacity was 0.5 l/s, with an average injection capacity of 0.25 l/s per well. The efficiency of the remediation approach was measured by changes in the content of total petroleum hydrocarbon (TPH), total chemoorganoheterotrophic (TC) and hydrocarbon degrading (HD) microorganisms. After biostimulation and bioaugmentation, the number of TC and HD microorganisms started to increase. This was followed by a decrease of TPH concentration in the groundwater from an initial 6.8–0.5 mg/l at the end of the bioremediation. The applied remediation approach was highly efficient and very effective in reducing TPH to acceptable levels. Together, these facts provide strong evidence of its potential for remediation of groundwater contaminated by petroleum hydrocarbons.

In this study, levan production by Bacillus licheniformis NS032 isolated from a petroleum sludge sample was investigated. High levan yield was obtained in a wide range of sucrose concentrations (up to 400 g/L) and, contrary to most levan-producing strains, using ammonium chloride as the sole N source. Interaction between sucrose, ammonium chloride, and initial pH of the medium in a low sucrose (60-200 g/L) and a high sucrose (300-400 g/L) system was analyzed by response surface methodology. According to the calculated model in the low sucrose system, maximum predicted levan yield was 47.8 g/L (sucrose 196.8 g/L, ammonium chloride 2.4 g/L, pH 7.0), while in the high sucrose system, levan yield was 99.2 g/L (sucrose 397.6 g/L, ammonium chloride 4.6 g/L, pH 7.4). In addition, protective effect of microbial levan against copper toxicity to Daphnia magna is observed for the first time. The acute toxicity (48 h EC50) of copper decreased from 0.14 to 0.44 mg/L by levan in concentration of 50 ppm. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3376]