Journal of Environmental Management (03014797)325
The adverse effects of high strength wastewaters on the microbial activities have created a challenge to biological treatments. Microbial fuel cell has been considered as a promising process because the electrical potential generation can stimulate microorganisms and overcome the inhibitory effect. However, several issues (e.g., scalability, high costs and maintenance) have prevented the process from the industrial applications. Elimination of the proton exchange membrane has been suggested as a remedy to the mentioned problems. In this work, a membrane-less microbial fuel cell was modified by putting the cathode within a thin sand layer (instead of the proton exchange membrane) to treat a high strength wastewater sample. The influences of the feed organic load and time of treatment in the modified system were studied in batch and continuous operations. It was revealed that the batch operation efficiency was higher for the lower feed loadings as a 5-day batch treatment removed 66 ± 4% of the 15,000 ± 500 mg/L initial chemical oxygen demand while the continuous process efficiency with 9-day hydraulic residence time was slightly more than 50%. However, the efficiency of the continuous operation for treatment of higher initial loading values was better than the batch mode with the removal efficiency of 41 ± 2% versus 12 ± 2% for a more concentrated leachate feed (45,000 ± 1000 mg/L). Finally, it was disclosed that the modified membrane-less MFC employed in this work can be effective in treatment of high strength wastewaters in larger scales with lower costs. © 2022 Elsevier Ltd
Bioelectrochemistry (15675394)149
Costly cofactors such as nicotinamide adenine dinucleotide hydrogen (NADH) are essential to have high activity in many redox enzymatic processes. Cofactor regeneration methods have been suggested to improve the economic aspects of the system. Here, we introduce a microbial electrosynthesis process to regenerate NADH in a two-chamber set-up with Enterobacter aerogenes biofilm as the bio-cathode. The effects of several important factors on the regeneration efficiency were studied and the highest NADH regeneration yield was achieved equal to 65 % at the potential of −1.5 V and the initial NAD+ concentration of 1 mM after 8 h of operation. The regenerated cofactor was highly enzymatically active (93 ± 4 %) which was a great merit of the process. Studying the kinetics of regeneration revealed that the electron transfer rate to the biofilm was the limiting factor. We tried to remove the limitation through co-culturing Pseudomonas aeruginosa and producing more electrochemical active compounds in the biofilm. Although, this modification was not effective for the regeneration yield, it showed that the external potential implicitly influenced the regeneration process by changing the internal microbial cell metabolic fluxes. Finally, it can be concluded that the microbial electrosynthesis is a promising green process for NADH regeneration. © 2022 Elsevier B.V.
Microbial Cell Factories (14752859)22(1)
Background: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. Results: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL− 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L− 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g− 1 DCW h− 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)–polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. Conclusions: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones. Graphical abstract: [Figure not available: see fulltext.]. © 2023, The Author(s).
Medical Hypotheses (03069877)167
Finding effective drugs to treat SARS-CoV-2 infection as a complementary step to the extensive vaccination is of the great importance to overcome the current pandemic situation. It has been shown that some bio-active unsaturated fatty acids such as Arachidonic Acid (AA) can reduce the infection severity and even destroy the virus by disintegration of the virus lipid envelope. On the other hand, it has been reported that several designed peptides with an activity similar to the angiotensin converting enzyme 2 (ACE-2), which has a high affinity towards the novel corona virus spike protein, can inhibit the viral infection through concealing the spike proteins from the cell surfaces ACE-2. Binding the mentioned peptides to the bio-active lipids like AA will result in a lipopeptide surfactant molecule with the synergistic effect of both the active moieties in its structure to treat the novel corona infection. In addition, the peptide segment increases the aqueous solubility of the lipid segment and enables the targeted delivery of the surfactant molecule to the virus. The resultant lipopeptide would be a potentially effective drug for SARS-CoV-2 infection treatment with the minimum side effects. © 2022 Elsevier Ltd
Karimi-avargani m., M.,
Bazooyar f., F.,
Biria, D.,
Zamani, A.,
Skrifvars m., M. Chemosphere (00456535)278
It has been suggested that cellulolytic enzymes can be effective on the degradation of PLA samples. The idea was investigated by examining the impact of cellulase on degradation of PLA and PLA-jute (64/36) composite in an aqueous medium. The obtained results demonstrated 55% and 61% thickness reduction in PLA and PLA-jute specimens after four months of treatment, respectively. Gel permeation chromatography (GPC) showed significant decline in the number average molecular weight (Mn) approximately equal to 85% and 80% for PLA and PLA-jute in comparison with their control. The poly dispersity index (PDI) of PLA and PLA-jute declined 41% and 49% that disclosed more homogenous distribution in molecular weight of the polymer after treatment with cellulase. The cellulase promiscuity effect on PLA degradation was further revealed by Fourier-transform infrared spectroscopy (FT-IR) analysis where substantial decrease in the peak intensities of the polymer related functional groups were observed. In addition, PLA biodegradation was studied in more detail by differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) of control and cellulase treated specimens. The obtained results confirmed the promiscuous function of cellulase in the presence or the absence of jute as the specific substrate of cellulase. This can be considered as a major breakthrough to develop effective biodegradation processes for PLA products at the end of their life cycle. © 2021 Elsevier Ltd
Colloids and Surfaces A: Physicochemical and Engineering Aspects (09277757)603
The presence of indigenous microorganisms in the petroleum reservoirs with the capability of biosurfactants production implies the existence of significant amounts of biosurfactants dissolved in the reservoir crude oil. The extraction of these biosurfactants to the aqueous phase can be considered as a promising microbial enhanced oil recovery process in a lower cost without the common limitations and risks related to the microbial systems. Ethylenediamine (EDA) was used to extract the crude oil dissolved biosurfactants (as anionic surfactants) of a crude oil sample to the aqueous phase and form a cat-ionic surfactant complex. The biosurfactant was isolated from the crude oil and preliminarily characterized which was capable of reducing the surface tension to 48 mN/m. Various concentrations of EDA were utilized to extract the crude oil biosurfactants and their effectiveness on the oil recovery was studied in a glass micro-model. Results indicated that an incremental 22 % oil production was obtained by flooding the 10 mM EDA solution. The incremental oil production was related to the formation of the cat-ionic complex surfactant which believed to cause a significant IFT reduction and simultaneously, have a higher efficiency in the wettability alteration than the anionic biosurfactants. It can be concluded that the extraction of the indigenous biosurfactant content of the crude oil and their interaction by EDA can be considered as a low-cost low-risk potential to the enhanced oil recovery processes. © 2020 Elsevier B.V.
Journal of Biotechnology (01681656)308pp. 27-34
L-Phenylacetylcarbinol (L-PAC) which is used as a precursor for the production of ephedrine and pseudoephedrine is the first reported biologically produced α-hydroxy ketone compound. L-PAC is commercially produced by the yeast Saccharomyces cerevisiae. Yeast cells transform exogenously added benzaldehyde into L-PAC by using the action of pyruvate decarboxylase (PDC) enzyme. In this work, genome-scale model and flux balance analysis were used to identify novel target genes for the enhancement of L-PAC production in yeast. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene and minimization of metabolic adjustments. Six single gene deletion strains, namely Δrpe1, Δpda1, Δadh3, Δadh1, Δzwf1 and Δpdc1, were predicted in silico and further tested in vivo by using knock-out strains cultivated semi-anaerobically on glucose and benzaldehyde as substrates. Δzwf1 mutant exhibited the highest L-PAC formation (2.48 g/L) by using 2 g/L of benzaldehyde which is equivalent to 88 % of the theoretical yield. © 2019
Karimi-avargani m., M.,
Bazooyar f., F.,
Biria, D.,
Zamani, A.,
Skrifvars m., M. Polymer Degradation and Stability (01413910)179
The biodegradation of PLA and PLA-Jute (64/36) in an aqueous media with Aspergillus flavus CCUG 28296, as well as its cell-free enzyme extract, was investigated through their physical, molecular, and thermal characterization. Results indicated that the thicknesses of the fungal treated PLA and PLA-jute samples during seven months have reduced by 52% and 63%, respectively while for the enzyme-treated samples, 45% and 49% reduction in the thickness has occurred. Moreover, the gel permeation chromatography (GPC) revealed a substantial decrease (about 75%) in the weight average molecular weight (Mw) of PLA and PLA-Jute treated with fungus, which confirmed the effective performance of A. flavus on the biological degradation of PLA. The obtained results were further supported by differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) of the treated and control samples as well. Interestingly, the observed reduction in the Mw of PLA in PLA-Jute was 64% after the enzymatic treatment, while for the unblended PLA, it was just about 32%. These results pointed to the synergistic effect of jute on PLA degradation because of the promiscuous activity of the effective enzymes on jute degradation, which could accelerate the PLA decomposition. © 2020 Elsevier Ltd
Chemical Engineering and Processing - Process Intensification (02552701)140pp. 78-84
The enzymatic production of formate from CO2 with the immobilized NADH-dependent formate dehydrogenase (FDH)on the activated electrospun polystyrene nanofibers (EPSNF)was investigated to develop a sustainable process for CO2 reduction. Direct electrochemical regeneration on a Cu foam electrode was employed to supply the reaction with the reduced cofactor (NADH). The formate production was studied in two modes of batch and semi-continuous operations with the cofactor recycle. Results indicated that the regenerated cofactor concentrations in both systems were nearly identical (0.5 mM)which ensured the desirable activity of the immobilized enzyme. This showed that the electrochemical regeneration system was effective even in the semi-continuous operation. Although the cumulative formate concentration in the batch operation was higher, the total amounts of the produced formate were higher for the semi-continuous mode for more than 42% which was justified by the fact that the lower formate concentration in the semi-continuous mode would be favorable to the progress of the enzymatic CO2 to formate conversion. Finally, it was concluded that the proposed semi-continuous process in this work could be considered as a promising process for the enzymatic CO2 conversion. © 2019 Elsevier B.V.
Waste Management (0956053X)85pp. 264-271
The capability of oxalic acid produced by Aspergillus niger was investigated for bioleaching of platinum from a refinery reforming catalyst. The spent medium mode was selected for bioleaching because of its higher efficiency at favorable pH and temperature conditions. The effects of several important factors such as the pulp density, pH and temperature on platinum recovery were optimized using Box-Behnken design of response surface methodology. The results indicated that pH adjustment during the bioleaching process increases the final platinum recovery significantly. The obtained optimum conditions were 1% for the pulp density, 0.5 for the medium pH, and 70 °C for the temperature which led to 37% platinum recovery. The significance of oxalic acid as the leaching agent in platinum bioleaching was highlighted by investigating the recovery of a blank medium without oxalic acid at the optimum conditions which was just about 13%. The presented method can be utilized in an environmentally friendly process to recover platinum from industrial catalysts. © 2019 Elsevier Ltd
Abolhasani, A.,
Biria, D.,
Abolhasani, H.,
Zarrabi, A.,
Komeili, T. International Journal Of Nanomedicine (11769114)14pp. 9535-9546
Purpose: Glucose decorated PLGA and chitosan nanoparticles (GPNPs and GCNPs) have been developed to examine the possibility of preventing the facilitated glucose transport to the cells through blocking the glucose transporters (Gluts) overexpressed by tumor cells. Methods: The MTT assay was used to assess the cytotoxicity towards human colon tumor (HT-29) cells in 72 hrs. Fluorescence microscopy was employed to confirm the attachment of GPNPs to the cells. Moreover, the GPNPs effects on the apoptotic rate of HT-29 cells were analyzed. Finally, the expression levels of GLUT-1 and GLUT-4 by real-time polymerase chain reaction (RT-PCR) were assayed to investigate the response of HT-29 cells to blocking their Gluts by GPNPs. Results: The stability studies showed that the synthesized complexes were mostly stable (more than 80%) at various temperatures (4 to 40ºC) and pH (5.4 to 7.4) conditions. Results indicated that the survival rate of the cells was decreased to 43% and 46% after treatment with GCNPs and GPNPs, respectively. Also, the apoptosis assay results showed that the percentage of viable cells reduced to 47% after GPNPs treatment. These observations were justified by the specific interactions between the glucose terminals and the cells Gluts which resulted in blocking the entries of nutrients to the cells. It was revealed that the GLUT-1 mRNA expression after the first 24 h of treatment by GPNPs was upregulated to more than 145%, while the direction was reversed after 72 h (expression less than 45%), which coincided with the cells death. In the first 24 h, the glucose deprivation stimulated the expression of Glut-1 while the apoptotic enzymes expression was dominant at the end of 72 h treatment time. Conclusion: Finally, it can be concluded that the glucose-nanoparticle complexes could be considered as promising agents in cancer therapy. © 2019 Abolhasani et al.
Scientific Reports (20452322)9(1)
Blending polyolefins with certain types of natural polymers like starch can be beneficial to their biodegradation. The impact of alpha-amylase on the biodegradation of low-density polyethylene (LDPE)-starch blend samples in an aqueous solution was investigated through characterizing their physical, mechanical and chemical properties. Results indicated that the weight and tensile strength of the enzyme treated samples were reduced by 48% and 87% respectively. Moreover, differential scanning calorimetry (DSC) showed an increase in fusion enthalpy of degraded samples which means that the crystallinity has been increased. The biodegradation of LLDPE appeared in Fourier-transform infrared spectroscopy (FT-IR) through the reduction in the intensity of the related peaks. This observation was supported by energy dispersive x-ray spectroscopy (EDXS) analysis where decreasing the percentage of carbon atoms in the treated blend was obtained. Likewise, the gel permeation chromatography (GPC) results pointed to a significant reduction in both the molecular weight and viscosity of LDPE more than 70% and 60% respectively. Furthermore, thermal gravimetric analysis (TGA) affirmed the function of amylase in degradation of the blend. On the basis of the obtained results, it can be claimed that the main backbone of the polymer, as well as the side branches, have been scissored by the enzyme activity. In other words, alpha-amylase has a promiscuous cometabolic effect on biodegradation of LDPE in polymer-starch blends. © 2019, The Author(s).
Journal of Environmental Management (10958630)243pp. 116-126
Two species of microalgae (Chlorella vulgaris and Dunaliella tertiolecta)as the biological agents along with ZnO nanoparticles as the photocatalyst were used to investigate the hydrocarbon removal efficiency from oily water samples. Firstly, the toxicities of the photocatalyst, normal paraffine hydrocarbons and their combination towards the microalgae were evaluated in terms of cell growth and chlorophyll content. The capability of algae to absorb the nanoparticles in the aqueous phase was confirmed by FT-IR spectroscopy. Then, the hydrocarbon removal efficiencies of the algae, photocatalyst and the combined photocatalyst-algae system were studied by measuring the residual hydrocarbon content of the samples. Results indicated that despite of the growth inhibitory effects of n-alkanes and nanoparticles on the examined algae, both of them could survive in the system. Dunaliella tertiolecta was more affected by normal paraffins while Chlorella vulgaris was more sensitive to ZnO nanoparticles. Both of the studied species were capable of hydrocarbon removal and the efficiency of Chlorella vulgaris was superior. The combination of algae and nanoparticles was also proved to have a synergistic effect on degradation of the hydrocarbon content of the medium. The obtained removal efficiencies for initial hydrocarbon concentrations of 0.05%, 0.1% and 0.5% (v/v)were 100%, 78% and 42% for Dunaliella tertiolecta-ZnO and 100%, 93% and 88% for Chlorella vulgaris- ZnO system, respectively. It can be concluded that the examined microalgae-nanoparticle system can be considered as a final polishing step in hydrocarbons removal from oily waters. © 2019 Elsevier Ltd
Journal of CO2 Utilization (22129820)28pp. 117-125
Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2. © 2018 Elsevier Ltd. All rights reserved..
Journal of Environmental Chemical Engineering (22133437)6(4)pp. 4144-4150
Biodemulsifier production by Acinetobacter calcoaceticus and its capability to break the stable water in oil emulsions were investigated. The chemical structure of the isolated biodemulsifiers from cultivation on various nitrogen sources were preliminarily analyzed by Fourier-transform infrared spectroscopy (FT-IR). The cultivation conditions such as temperature, pH and carbon to nitrogen source concentration (C/N) ratio were optimized for maximum demulsification activity through response surface methodology. It was disclosed that the composition of the isolated biodemulsifiers was depended on the utilized nitrogen source in the medium and altered from a cyclic lipopeptide (on soybean) to a lipopolysaccharide (on ammonium salts). The obtained optimum conditions for the biodemulsifier produced on ammonium nitrate (as the nitrogen source) were 35°C for temperature, 10 for C/N ratio and 5 for pH. Results indicated that the produced extracellular biodemulsifier can reduce the surface tension to 38.6mN/m and break 95% of a surfactant stabilized emulsion at the optimum conditions. © 2018 Elsevier Ltd.
Advances In Environmental Technology (24764779)4(3)pp. 155-161
This study investigated different methods of controlling the fat, oil and grease (FOG) in sewer systems. A comprehensive control program was developed for the city of Mashhad (Iran) to maintain its sewer system and prevent blockages. The control program consisted of three parts: 1) fat, oil and grease source control, 2) sewer system modification, and 3) preventive maintenance. This program included guidelines for food service establishments, which are the major sources of (FOG). Food service establishments must implement better management practices to reduce (FOG) from entering the facility drain and install grease removal devices. As a part of preventive cleaning, the performance of several surfactants was evaluated as a cleaning agent. A 50:50 mixture (10 v. % in water) of two industrial surfactants, one containing monoethyl amine and sulfonated lauryl alcohol and one containing nonylphenol ethoxylate and potassium hydroxide, had the best performance and removed 80 % of the fat. Response Surface Methodology was used to determine the optimum conditions for the surfactant. The optimum conditions were a contact time of 36 h, shaking rate of 30 rpm and surfactant concentration of 12.5%. The second part of the program consisted of removing dead zones and increasing wastewater velocity in the sewer lines to enhance the hydraulic condition of the sewer system and decrease fat deposition. Finally, a detailed and well-defined control program could solve FOG problems in sewer systems. © 2018, Iranian Research Organization for Science and Technology. All rights reserved.
Electrochimica Acta (00134686)247pp. 1095-1102
Herein, Cu foam and bimetallic Cu foams are introduced as the cost efficient and easy to scale up electrode for efficient electrochemical regeneration of NADH. The porous copper foam was electrochemically deposited at the copper foil. Cu foam was used as a template for preparation of Ag and Pt coated Cu foam by galvanic replacement of Cu with silver and platinum. The nature and structure of the prepared foams were characterized by energy dispersive X-ray spectrometry and scanning electron microscopy. Electrochemical behavior of NAD+ was studied by cyclic voltammetry at the surface of prepared foams. Also regeneration of the enzymatic cofactor NADH in a batch electrochemical reactor using Cu foil, Cu foam, Ag coated and Pt coated Cu foam electrodes has been investigated. The effect of thickness of Cu foam and the reduction potential of NAD+ on the recovery percent of 1,4-NADH was investigated to achieve the most efficient conditions for electrochemical regeneration of NADH. The regeneration yields of enzymatically active NADH were about 80%, 70% and 75% using the Cu foam, Ag and Pt coated Cu foam electrodes, respectively which all of them were more than that observed at Cu foil electrode 54%. © 2017 Elsevier Ltd
Renewable Energy (09601481)104pp. 88-95
Simultaneous biological delignification and saccharification of rice straw by the immobilized Trichoderma viride cells were studied in this work. Response surface methodology as a multiple responses optimization technique was utilized to optimize several important factors such as the biomass content of the medium (w/v), inoculum size and agitation rate. Results indicated that at the obtained optimum conditions the lignin removal efficiency of 74% and sugar concentration equal to 8.52 g/L could be achieved in ten days of pretreatment. In addition, the influence of initial glucose concentration in the medium on both the pretreatment saccharification and the subsequent enzymatic hydrolysis efficiencies was investigated. It was revealed that the higher initial glucose concentration is beneficial to obtain higher total saccharification efficiency from pretreatment and the following enzymatic hydrolysis and a total efficiency equal to 81% was obtained for 15 g/L initial glucose concentration. Accordingly, it can be concluded that the immobilized Trichoderma viride in this work can be considered as a potentially applicable strain to design a promising lignocellulosic materials bio-pretreatment process. © 2016 Elsevier Ltd
Doostmohammadi, M.,
Asadollahi, M.A.,
Nahvi i., I.,
Biria, D.,
Ghezelbash, G.R.,
Kheyrandish, M. Annals of Microbiology (18692044)66(3)pp. 1049-1055
The yeast Saccharomyces cerevisiae is able to biotransform benzaldehyde into L-phenylacetylcarbinol (L-PAC), a key intermediate in the production of ephedrine and pseudoephedrine, by the action of pyruvate decarobxylase (PDC) enzyme. This biotransformation can alternatively be performed by acetohydroxyacid synthase (AHAS) which is a mitochondrial enzyme. In the yeast petite mutants, AHAS accumulates in the cytosol. In the current study, wild-type yeast cells and yeast petite mutants were examined for L-PAC biosynthesis. The results showed higher L-PAC titers in the yeast petite mutants. In addition, the effect of cell immobilization and carbon source (glucose or molasses) on L-PAC production was investigated. It was found that cell immobilization enhances L-PAC formation. The highest L-PAC concentration (2.4 g/l) was obtained at 2 g/l of benzaldehyde using the immobilized petite mutants grown on molasses. © 2016, Springer-Verlag Berlin Heidelberg and the University of Milan.
International Journal of Biological Macromolecules (01418130)82pp. 751-756
Production of xanthan gum using immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii grown on glucose or hydrolyzed starch as carbon sources was investigated. Calcium alginate (CA) and calcium alginate-polyvinyl alcohol-boric acid (CA-PVA) beads were used for the immobilization of cells. Xanthan titers of 8.2 and 9.2. g/L were obtained for X. campestris cells immobilized in CA-PVA beads using glucose and hydrolyzed starch, respectively, whereas those for X. pelargonii were 8 and 7.9. g/L, respectively. Immobilized cells in CA-PVA beads were successfully employed in three consecutive cycles for xanthan production without any noticeable degradation of the beads whereas the CA beads were broken after the first cycle. The results of this study suggested that immobilized cells are advantageous over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells immobilized in CA beads for xanthan production. © 2015 Elsevier B.V.
Chemosphere (00456535)152pp. 166-172
The impact of adding soluble starch on biodegradation of n-alkanes (C10-C14) by Bacillus subtilis TB1 was investigated. Gas chromatography was employed to measure the residual hydrocarbons in the system. It was observed that the efficiency of biodegradation improved with the presence of starch and the obtained residual hydrocarbons in the system were 53% less than the samples without starch. The produced bacterial enzymes were studied through electrophoresis and reverse zymography for explaining the observations. The results indicated that the produced amylase by the bacteria can degrade hydrocarbons and the same was obtained by the application of a commercial alpha amylase sample. In addition, in silico docking of alpha-amylase with n-alkanes with different molecular weights was studied by Molegro virtual docker which showed high negative binding energies and further substantiated the experimental observations. Overall, the findings confirmed the catalytic effect of alpha amylase on n-alkanes degradation. © 2016 Elsevier Ltd.
Journal of Chemical and Engineering Data (00219568)60(9)pp. 2575-2584
Distribution of surfactants in an aqueous/nonaqueous two phase system is of the great importance especially in enhanced oil recovery (EOR) processes. In this paper, the application of genetic algorithm to estimate the binary interaction parameters of the nonrandom two liquid (NRTL) activity coefficient model for a brine/oil/ionic surfactant system has been investigated. The presence of ionic surfactant in the system has been taken into account by employing the modified Debye-Huckel model. Moreover, the binary interaction parameters used in NRTL activity coefficient model were found to be interdependent and related to each other by a set of linear equations known as closure equations. These equations were considered as constraints to the optimization calculations. On the other hand, when experimental data were not available, the LLE data were estimated primarily through the Scatchard-Hildebrand activity coefficient model. These data were used to evaluate the GC-NRTL model parameters and calculate equilibrium mole fractions. The estimated binary interaction parameters using GA (genetic algorithm) showed a proper fitness with experimental values and the application of closure equations exhibited lower root-mean-square deviations. In addition, employing the Scatchard-Hildebrand model predictions for the modified GC-NRTL model calculations resulted in an acceptable accuracy. Accordingly, the presented model in this work can be utilized as a powerful method to study liquid-liquid equilibrium systems including surfactants. © 2015 American Chemical Society.
Journal of Peptide Science (10752617)21(1)pp. 10-16
The world is entering the third decade of the acquired immunodeficiency syndrome (AIDS) pandemic. The primary cause of the disease has known to be human immunodeficiency virus type I (HIV-1). Recently, peptides are shown to have high potency as drugs in the treatment of AIDS. Therefore, in the present study, we have developed a method to predict anti-HIV-1 peptides using support vectormachine (SVM) as a powerful machine learning algorithm. Peptide descriptors were represented based on the concept of Chou's pseudo-amino acid composition (PseAAC). HIV-1 P24-derived peptides were examined to predict anti-HIV-1 activity among them. The efficacy of the prediction was then validated in vitro. The mutagenic effect of validated anti-HIV-1 peptides was further investigated by the Ames test. Computational classification using SVMshowed the accuracy and sensitivity of 96.76% and 98.1%, respectively. Based on SVM classification algorithm, 3 out of 22 P24-derived peptides were predicted to be anti-HIV-1, while the restwere estimated to be inactive. HIV-1 replicationwas inhibited by the three predicted anti-HIV-1 peptides as revealed in vitro, while the results of the same test on two of non-anti-HIV-1 peptides showed complete inactivity. The three anti-HIV-1 peptides were shown to be not mutagenic because of the Ames test results. These data suggest that the proposed computational method is highly efficient for predicting the anti-HIV-1 activity of any unknown peptide having only its amino acid sequence. Moreover, further experimental studies can be performed on the mentioned peptides, which may lead to new anti-HIV-1 peptide therapeutics candidates. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Environmental Technology Reviews (21622523)4(1)pp. 71-89
Microbial fuel cell (MFC) is a novel technology that can be used for electricity generation during oxidization of the organic substances presented in the substrate. To obtain a desirable performance, it is essential to understand the influential factors on the MFC. Among the numerous factors affecting the MFC performance, substrate, microorganisms and their metabolism, electron transfer mechanism in an anodic chamber, electrodes material and the shape of electrodes, type of membrane, operating conditions such as temperature, pH and salinity, electron acceptor in a cathodic chamber and geometric design of the MFC are considered as the most important factors. Among different substrates, wastewater is a sustainable rich medium which can be treated by MFCs. There are various types of exoelectrogenic bacteria presented in wastewaters which can oxidize organic matter and transfer electrons to the anode without using mediators. Like other microbial systems, optimum pH and temperature enhance the bacterial growth which can improve the MFC performance. Despite the negative effect of salt on microbial growth, higher salinity and ionic strength can increase the conductivity of substrate and therefore enhance MFC performance. Scaling up MFC is a controversial issue which needs a comprehensive understanding of these factors. By using new inexpensive materials for electrodes and membrane for manufacturing MFCs, a more cost-effective design for scalable wastewater treatment and high power generation can be achieved. Furthermore, MFC is a suitable candidate for bioremediation of contaminated groundwater. These factors and their impact on the MFC performance have been reviewed in the present survey. © 2015, © 2015 Taylor & Francis.
Ghorbani, F.,
Karimi m., ,
Biria, D.,
Kariminia h.r., ,
Jeihanipour, A. Biochemical Engineering Journal (1369703X)101pp. 77-84
Fungal delignification can be considered as a feasible process to pre-treat lignocellulosic biomass in biofuel production, if its performance is improved in terms of efficiency thorough a few modifications. In this study, Trichoderma viride was utilized to investigate the effect of wet-milling, addition of surfactant (Tween 80) and optimization of operating factors such as temperature, biomass to liquid medium ratio and glucose concentration on biodelignification of rice straw. Next, the enzymatic hydrolysis of pretreated biomass was studied at various pretreatment times. Results revealed that the wet milling and addition of surfactant increases the lignin removal about 15% and 11%, respectively. The optimization of operating factors resulted in high lignin removal efficiency equal to 74% in a 30 day pretreatment. The maximum enzymatic saccharification of pretreated biomass at optimum conditions was obtained equal to 56%. Conversion of cellulose to reducing sugars during the pretreatment stage was 21% which suggests that higher saccharification efficiency could be achieved in a simultaneous pretreatment and saccharification process. © 2015 Elsevier B.V.
Physical Chemistry Chemical Physics (14639084)17pp. 12561-12574
Bio-electrosynthesis is one of the significant developments in reverse microbial fuel cell technology which is potentially capable of creating organic compounds by combining CO2 with H2O. Accordingly, the main objective in the current study was to present a model of microbial electrosynthesis for producing organic compounds (acetate) based on direct conduction of electrons in biofilms. The proposed model enjoys a high degree of rigor because it can predict variations in the substrate concentration, electrical potential, current density and the thickness of the biofilm. Additionally, coulombic efficiency was investigated as a function of substrate concentration and cathode potential. For a system containing CO2 as the substrate and Sporomusa ovata as the biofilm forming microorganism, an increase in the substrate concentration at a constant potential can lead to a decrease in coulombic efficiency as well as an increase in current density and biofilm thickness. On the other hand, an increase in the surface cathodic voltage at a constant substrate concentration may result in an increase in the coulombic efficiency and a decrease in the current density. The maximum coulombic efficiency was revealed to be 75% at a substrate concentration of 0.025 mmol cm-3 and 55% at a surface cathodic voltage of -0.3 V producing a high range of acetate production by creating an optimal state in the concentration and potential intervals. Finally, the validity of the model was verified by comparing the obtained results with related experimental findings. © the Owner Societies 2015.
Food Science and Biotechnology (20926456)24(2)pp. 453-460
Cheese whey lactose was used as a carbon source for xanthan gum production with Xanthomonas campestris and Xanthomonas pelargonii. Proteins were precipitated and removed from whey prior to fermentation. Box-Behnken response surface methodology was used for optimization of the carbon, magnesium, and phosphate source concentrations in the culture medium to maximize xanthan gum production. After 48 h of fermentation using X. campestris, the highest xanthan concentration (16.4 g/L) was achieved at 65.2 g/L of cheese whey (39.1 g/L of lactose), 14.8 g/L of phosphate (K H2PO4), and 1.1 g/L of magnesium (MgSO4·7H2O). The corresponding optimum cheese whey, phosphate, and magnesium concentrations in cultures of X. pelargonii were 80.0, 6.7, and 0.8 g/L, respectively, which resulted in a xanthan production of 12.8 g/L. The xanthan gum yield (g of xanthan/g of lactose) was 0.42 for X. campestris and 0.27 for X. pelargonii. © 2015, The Korean Society of Food Science and Technology and Springer Science+Business Media Dordrecht.
Savari, M.,
Zarkesh-esfahani, H.,
Edalati, M.,
Biria, D. Protein Expression and Purification (10465928)114pp. 128-135
Abstract Human growth hormone (hGH) is synthesized and stored by somatotroph cells of the anterior pituitary gland and can effect on body metabolism. This protein can be used to treat hGH deficiency, Prader-Willi syndrome and Turner syndrome. The limitations in current technology for soluble recombinant protein production, such as inclusion body formation, decrease its usage for therapeutic purposes. To achieve high levels of soluble form of recombinant human growth hormone (rhGH) we used suitable host strain, appropriate induction temperature, induction time and culture media composition. For this purpose, 32 experiments were designed using Taguchi method and the levels of produced proteins in all 32 experiments were evaluated primarily by ELISA and dot blotting and finally the purified rhGH protein products assessed by SDS-PAGE and Western blotting techniques. Our results indicate that media, bacterial strains, temperature and induction time have significant effects on the production of rhGH. The low cultivation temperature of 25 °C, TB media (with 3% ethanol and 0.6 M glycerol), Origami strain and a 10-h induction time increased the solubility of human growth hormone. © 2015 Elsevier Inc.
Microbiology (Russian Federation) (16083237)84(1)pp. 65-72
Dibenzothiophene (DBT) is a recalcitrant organic sulfur compound which remains in the crude oil after hydrodesulfurization (HDS) process and can be removed by biodesulfurization (BDS). The objective of this study was the isolation of novel strain capable more BDS rate and optimization of DBT removal by both growing and resting cells. Response surface Methodology (RSM) was applied for evaluating the interactive effects of three independent factors including DBT concentration, temperature and pH. The three factors Box-Benken design with three center points was performed to generate the optimum condition for DBT removal by growing cells in aqueous medium and resting cells in biphasic medium. Among the isolated bacteria from oil-contaminated soil, a gram-positive, non-spore forming isolate designated PD1 showed the high BDS rate and capable to convert the DBT to 2-hydroxybiphenyl (2-HBP) as the final product. Analysis of variance (ANOVA) demonstrated that all of the studied parameters in the growing cells system showed significant effect on BDS rate, while in the resting cells effect of pH was not significant (P > 0.05). Maximum 2-HBP production (0.21 mM) by growing cells of PD1 strain was obtained at 0.38 mM initial DBT concentration, pH 6.88 and temperature of 27.57°C. For resting cells, maximum BDS activity of PD1 strain was determined as 0.46 μM 2-HBP/min g DCW at optimum pH 6.29, temperature of 26.13°C and DBT concentration of 7.73 mM. The BDS efficiency of Rhodococcus erythropolis PD1 (NCBI Gene Bank Accession no. JX625154) was increased by setting each factor at the optimum level. © 2015, Pleiades Publishing, Ltd.
Chemical Engineering Communications (00986445)201(11)pp. 1514-1525
The power law logistic model was utilized to investigate the growth of a hydrocarbon assimilating bacterium on a water-insoluble substrate. To achieve this end, population dynamics of Bacillus licheniformis MS3 in a medium containing n-decane as the sole carbon source was monitored for 30 h. Different initial biosurfactant concentrations and shaking rates were employed to examine the role of mass transfer in the cell growth and the consequent hydrocarbon biodegradation. The amount of n-decane degraded in the system was detected by gas chromatography at the end of the incubation period. The results revealed that when mass transfer limitations were lessened through addition of an initial biosurfactant concentration and agitation, the bacterial growth increased more than three times and the n-decane biodegradation was enhanced from 6.7 to 15.1 mg/100 mL. Finally, the power law logistic model proved to be highly capable in simulating both the experimental results and various systems with water-insoluble carbon sources. © 2014, Taylor & Francis Group, LLC.
Applied Biochemistry and Biotechnology (02732289)174(1)pp. 437-451
High-mobility group proteins are a superfamily of DNA-binding proteins that bind to the DNA minor groove and bend it, whereas most of the transcription factors such as centromere protein B (CENP-B), octamer (Oct)-1, growth factor independence 1 (Gfi-1), and WRKY bind to the major groove of DNA. Classification of proteins using their DNA-binding features is the aim of this study. Nuclear localization signals play more important roles in entering DNA-binding proteins to nucleus and doing their functions; therefore, they have been considered as a feature which is important for DNA-binding manner in proteins. Nuclear localization signals (NLSs) were predicted by two prediction web servers, and then, their sequence ordered features were extracted by Chou's pseudo amino acid composition (PseAAC) and ProtParam. Multilayer perceptron was used as an artificial neural network for analyzing the features by calculating the correlation coefficient and 30-fold cross-validation. Another used data-analyzing program was principal component analysis of the Minitab software. By calculating the eigenvalues and considering five principal components, the sequence length of NLSs was known as the best feature for classifying DNA-binding proteins. Minimum mean squared error (MSE) (0.1098) and the highest R (2) (0.963) mean that there is a significant difference between the NLS length of the DNA major groove and minor groove binder proteins. Results showed that it is possible to classify DNA major groove and minor groove binder proteins by their NLS sequences as a feature.
International Journal of Environmental Science and Technology (17351472)11(6)pp. 1701-1710
Biodegradation of petroleum hydrocarbons as a decontamination mechanism is a relatively slow process. This study aimed to investigate the impact of a tailored consortium of bacteria with higher capacities in biosurfactant production and biodegradation on the acceleration of the biodecontamination process. To this end, 18 biosurfactant producing bacteria were isolated from the crude oil-contaminated soil samples of Isfahan refinery, and the activity of the produced biosurfactants was measured in terms of surface tension reduction and emulsification E24 test. Then, the isolates screened for the biodegradation of kerosene hydrocarbons and chemical structure of the purified biosurfactants produced by the most efficient isolates were partially characterized. Next, the isolates were sorted based on their surfactant activity and biodegradation efficiency, and the higher ranked bacteria thus selected were utilized to form an efficient consortium removing hydrocarbons from the oil-contaminated soil samples in a slurry phase system. The consortium consisted of Bacillus subtilis tb1 and Pseudomonas aeruginosa species having the highest biodegradation capabilities and surface activities. The results revealed that the hydrocarbon removal efficiency of the consortium was at least 25 % higher than single species, and the final removal efficiency for the consortium could be reached in a considerably shorter time. © 2014 Islamic Azad University (IAU).
Khajepour, H.,
Mahmoodi, M.,
Biria, D.,
Ayatollahi, S. Journal of Petroleum Science and Engineering (09204105)120pp. 10-17
Microbial Enhanced Oil Recovery (MEOR) as a tertiary process employs microorganisms and their metabolites to reduce the residual oil saturation of the reservoir mainly through interfacial tension (IFT) reduction and wettability alteration. In spite of its great potential and the mentioned advantages, application of MEOR has been limited because of the lack of practical convincing experimental results. In this study, the effects of MEOR process on wettability changes and the reduction of residual oil saturation have been examined by providing microscopic visualization of two phase flow in transparent glass micromodels. Biosurfactant producing bacterial strain (Enterobacter cloacae) was utilized to investigate the impacts of both the biofilm and biosurfactant on wettability of the micromodel pore walls by measuring the relative permeabilities before and after microbial treatment. Results indicated that wettability of the pores was altered towards more water wetness which was also supported by visual observation of the oil/water phase saturations in the glass micromodel. Moreover, the oil recovery was increased up to 24.5% of the original oil in place (OOIP) during the MEOR process. At last, Biofilm formation was found to be more responsible for the wettability alteration process. © 2014 Elsevier B.V.
Minerva Biotecnologica (11204826)26(3)pp. 191-197
Aim. The main goal of the present study was to examine the possibility of using starch as the main substrate for microbial production of xanthan gum by Xanthomonas campestris and Xanthomonas pelargonii. Methods. The bacteria were cultivated on starch hydrolyzate and glucose (as a reference) at three different sugar concentrations of 20, 30, and 40 g/L. Samples were taken at different time intervals (24, 48, and 72 h) from cultivation broth and concentration of xanthan was determined. General linear regression method was employed to analyze the results. Results. For starch hydrolyzate and glucose, the highest xanthan concentration of 8.7 and 7.8 g/L, respectively, was achieved at 40 g/l of substrate concentration after 48 h for Xanthomonas campestris. For all substrate concentrations, maximum xanthan production was obtained after 48 h while prolonged cultivation time (72 h) reduced the concentration significantly. Generally, differences between product yields obtained in cultures containing glucose or starch hydrolyzate were not significant. Conclusion. This study showed that pure glucose can be replaced by inexpensive starch hydrolyzate in the process of fermentative xanthan production.
International Biodeterioration and Biodegradation (09648305)86pp. 343-348
Organic sulfur components of the petroleum are too resistant to be removed by the conventional desulfurization processes. This study aimed to investigate the removal of dibenzothiophene (DBT) as an organic sulfur compound, from the oily phase by a bioprocess employing the immobilized cells. Rhodococcus erythropolis R1 cells were encapsulated in calcium alginate beads by considering factors such as the alginate concentration, size of the beads, the concentration of surfactants and γ-Al2O3 nano particles for optimizing biodesulfurization (BDS) via Taguchi approach. The impact of two cofactor precursors (nicotinamide and riboflavin) on the long term BDS efficiency was also examined. The results indicated that the optimum factor levels for the bigger is better criterion could be achieved at 20% (w/w) of γ-Al2O3 nano particles, alginate beads size equal to 1.5mm, 1% (w/v) of the alginate and 0.5% (v/v) of span 80. The related statistical analysis showed that the concentration of γ-Al2O3 nano particles was the most significant factor in the BDS process. Moreover, the addition of nicotinamide and riboflavin significantly decreased the biocatalytic inactivation of the immobilized cells system after successive operational steps enhancing the BDS efficiency by more than 30% after four steps. It can be concluded that a combination of the nano γ-Al2O3 particles with alginate immobilized cells could be very effective in biodesulfurization process. © 2013.
Petroleum Science and Technology (15322459)31(12)pp. 1259-1267
To obtain potentially applicable microorganisms to an effective in situ microbial enhanced oil recovery operation, bacteria that were compatible with the harsh conditions of a petroleum reservoir were isolated from a crude oil sample. The application of an oil spreading technique showed that all of the isolates were capable of producing biosurfactants from both the glucose and crude oil as carbon sources. The secreted biosurfactants could at least reduce the surface tension 20 mN/m and for one of the isolates; the surface tension value dropped below 40 mN/m. In addition, the contact angle measurements revealed that the produced biosurfactants could effectively alter the wettability of the oil saturated rock samples. At last, the effect of isolates and their biosurfactants on improving oil production from oil saturated rock samples was investigated. It was observed that the presence of bacteria in the system could increase the amount of produced oil in comparison with the case where cell free biosurfactants were utilized. © 2013 Taylor & Francis Group, LLC.
Colloids and Surfaces A: Physicochemical and Engineering Aspects (09277757)436pp. 542-548
It is generally agreed today that effective parameters such as ionic strength, surfactant concentration and the flow rate of the containing fluid have a significant impact on the bacterial transport through porous media. The present study aimed to investigate whether chemotaxis can equally contribute to bacterial deposition in the same environments. To this end, a series of experiments were designed using Taguchi approach in which saturated glass bead packed columns served as the porous media and naphthalene was used as chemo-attractant. Then the contributory role of the factors involved in bacterial filtration through porous media was carefully measured by employing related statistical analyses. The results revealed that the percentage value of chemotaxis contribution to the process was about 20% which was significant compared with ionic strength (43%), surfactant concentration (32%) and fluid flow rate (3%). Notably, the minimum bacterial transport through the columns was about 0.41 of the initial inlet concentration at optimal conditions. © 2013 Elsevier B.V.
Biria, D.,
Maghsoudi e., ,
Roostaazad, R.,
Dadafarin h., ,
Lotfi, S.,
Amoozegar, M.A. World Journal of Microbiology and Biotechnology (09593993)26(5)pp. 871-878
The physical properties and chemical structure of a new biosurfactant (licheniformin) produced by Bacillus licheniformis MS3 were investigated. The purified biosurfactant was identified as a lipopeptide with amino acid sequence of Gly, Ala, Val, Asp, Ser, Gly, Tyr and a lactone linkage between the carboxyl group of Aspargine and hydroxyl group of Tyrosine residue. The fatty acid moiety was attached to N-terminal amino acid residue through an amide bond. The purified licheniformin could lower the surface tension of water from 72 to 38 mN/m at concentrations higher than 15 μg/mL and its relative emulsion volume (EV%) was equal to 36%. It also showed stable surface activity over a wide range of temperature (45-85°C) and pH (3-11). © Springer Science+Business Media B.V. 2009.
An electroflotation technique was utilized to reclaim the surfactant molecules from oil-in-water emulsions. A two-staged separation mechanism was suggested to describe the process. The surfactant recovery efficiencies were higher than 70% after 30 min treatment. The method could be an effective process in recycling surfactants from the emulsions existing in industrial wastes. This is an abstract of a paper presented at the 8th World Congress of Chemical Engineering (Montreal, Quebec, Canada 8/23-27/2009).
Journal of Membrane Science (03767388)311(1-2)pp. 159-164
To describe immobilized cells in porous microcapsule membranes with straight pores, a novel model called corrugated parallel bundle model (CPBM) was utilized. In this model, a network was developed with 10 main pores each composing 10 pore elements. Cell growth kinetic in the network was examined using non-structural models. Effectiveness factor and pore plugging time were calculated by solving reaction-diffusion equation set via finite difference method. The findings revealed that diffusion coefficient for lower order reactions will create a lesser impact on the reduction of effectiveness factor. These findings also indicated that the use of such supporting carrier for cell immobilization could enhance the operational life of the system. © 2007 Elsevier B.V. All rights reserved.
Biria, D.,
Roostaazad, R.,
Darouneh e., ,
Izadi h., Scientia Iranica (10263098)14(2)pp. 161-168
Rock samples from the Asmary outcrop formation of the Ahwaz oil rich zone with a porosity of 16% and permeability of 1 md and MIS crude oil with an API value of 42.5 and moderate asphaltene content of 3%, were used to study the effect of the incubation time and flow rate of the displacing fluid in MEOR operations. Five species of rod shaped, gram positive, thermophile and facultative bacteria were isolated and purified from the crude. Due to the high sweep efficiency prevailing in the core flooding system, the effect of the displacing brine flow rate on the oil recovery efficiency was found not to be significant. On the other hand, a 100% increase in incubation time from 7 to 14 days resulted in an increase from 3% to 4% in the total cumulative production. Application of a cyclic operation was not effective in promoting the efficiency of the MEOR operation, probably due to the stronger effect of flooding on the removal of valuable metabolites, as compared to undesired ones, before the stationary phase of the microbial kinetics. While qualitative measurements did not show a strong change in the water-rock contact angle, more than a 4-fold increase in capillary number occurred after microbial treatment, implying that reduction of interfacial tension was the stronger mechanism of oil recovery in this work. © Sharif University of Technology, April 2007.
Microbial EOR (MEOR) is receiving renewed interest worldwide. The method is based on microorganisms' activities to reduce residual oil of reservoirs, which is dependent on behavior of inherent microorganisms or injection of bioproduct of external microorganisms. Five bacterial species were taken from MIS crude oil that is one of the aging Persian fractured reservoirs. Visualization experiments were carried out to examine the behavior of MEOR in micromodels designed to resemble the fractured system: static and dynamic. A new surface sample of the MIS oil was taken at the well head and physical specifications were measured at the lab. This was fairly light oil with API gravity of 42.5. The respective specifications were presented. The selected microorganisms initially formed spores. The microorganisms were all classified as bacillus. Forming different colonies were observed after separation process. All of the microorganisms were facultative anaerobic. Reservoir rock wettabilty alteration was observed from oil-wet to water-wet by the microorganisms.
