Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coil can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coil homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH. (C) 2012 Elsevier B.V. All rights reserved.

A stable liquid-liquid extraction system for clavulanic acid using polymer-based aqueous two-phase systems

The partitioning of Clavulanic Acid (CA) in a novel inexpensive and stable aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The aqueous two-phase systems are formed by mixing both polymers with a salt (NaCl or Na2SO4) and an aqueous solution of CA. The stability of CA on the presence of both polymers was investigated and it was observed that these polymers do not degrade the biomolecule. The effect of PEG-molecular size, polymer concentrations on the commercial CA partitioning has been studied, at 25 degrees C. The data showed that commercial CA was preferentially partitioned for the PEG-rich phase with a partition coefficient (K-CA) between 1 and 12 in the PEG/NaPA aqueous two phase systems supplemented with NaCl and Na2SO4. The partition to the PEG phase was increased in the systems with high polymer concentrations. Furthermore, Na2SO4 caused higher CA preference for the PEG-phase than NaCl. The systems having a composition with 10 wt.% of PEG4000, 20 wt.% of NaPA8000 and 6 wt.% of Na2SO4 were selected as the optimal ones in terms of recovery of CA from fermented broth of Streptomyces clavuligerus. The partitioning results (K-CA = 9.15 +/- 1.06) are competitive with commercial extraction methods of CA (K-CA = 11.91 +/- 2.08) which emphasizes that the system PEG/NaPA/Na2SO4 can be used as a new process to CA purification/concentration from fermented broth. (C) 2012 Elsevier B.V. All rights reserved.

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Advanced oxidation process H2O2/UV combined with anaerobic digestion to remove chlorinated organics from bleached kraft pulp mill wastewater

This study investigated the application of an advanced oxidation process combining hydrogen peroxide with ultraviolet radiation (H2O2/UV) to remove recalcitrant compounds from Kraft bleaching effluent. Anaerobic pre-treatment was performed to remove easily degraded organics using a horizontal-flow anaerobic immobilized biomass (HAIB) reactor. Bleaching plant effluent was treated in the HAIB reactor processed over 19 h of hydraulic retention time (HRT), reaching the expected removal efficiencies for COD (61 +/- 3%), TOC (69 +/- 9%), BOD5 (90 +/- 5%) and AOX (55 +/- 14%). However, the anaerobic treatment did not achieve acceptable removal of UV254 compounds. Furthermore, there was an increase of lignin, measured as total phenols. The H2O2/UV post-treatment provided a wide range of removal efficiencies depending on the dosage of hydrogen peroxide and UV irradiation: COD ranged from 0 to 11%, UV254 from 16 to 35%, lignin from 0 to 29% and AOX from 23 to 54%. All peroxide dosages applied in this work promoted an increase in the BOD5/COD ratio of the wastewater. The experiments demonstrate the technical feasibility of using H2O2/UV for post-treatment of bleaching effluents submitted to anaerobic pre-treatment.

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na2SO4, (NH4)(2)SO4, MgSO4, LiSO4, Na2HPO4, sodium phosphate buffer (pH 7.0), NaH2PO4, K2HPO4, potassium phosphate buffer (pH 7.0), KH2PO4, C6H8O7, Na3C6HSO7, KCHO2, NaCHO2, NaCO3, NaHCO3, C2H4O2, sodium acetate buffer (pH 4.5), and NaC2H3O2] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents. (C) 2011 Elsevier Inc. All rights reserved.

Partitioning and extraction of collagenase from Penicillium aurantiogriseum in poly(ethylene glycol)/phosphate aqueous two-phase system

Purification of collagenase produced by Penicillium aurantiogriseum URM4622 was carried using a PEG/phosphate aqueous two-phase system (ATPS). A 2(3)-full experimental design was used to investigate the influence of PEG molar mass, PEG concentration and phosphate concentration on the selected responses, namely partition coefficient, activity yield and purification factor. The ATPS was composed of PEG (molar mass of 550, 1500 and 4000 g/mol) at concentrations of 15.0, 17.5 and 20.0% (w/w) and phosphate at concentrations of 12.5, 15.0 and 17.5% (w/w). The best results of one-step extraction of collagenase from the fermentation broth (partition coefficient of 1.01, activity yield of 242% and purification factor of 23.5) were obtained at pH 6.0 using 20.0% (w/w) PEG 550 and 17.5% (w/w) phosphate. The results of this preliminary study demonstrate that the selected ATPS is satisfactorily selective for the extraction of such a collagenase. (C) 2012 Elsevier B.V. All rights reserved.

Two-phase flow patterns and pressure drop inside horizontal tubes containing twisted-tape inserts

This paper presents an experimental study on two-phase flow patterns and pressure drop of R134a inside a 15.9 mm ID tube containing twisted-tape inserts. Experimental results were obtained in a horizontal test section for twisted-tape ratios of 3, 4, 9 and 14, mass velocities ranging from 75 to 250 kg/m(2) s and saturation temperatures of 5 and 15 degrees C. An unprecedented discussion on two-phase flow patterns inside tubes containing twisted-tape inserts is presented and the flow pattern effects on the frictional pressure drop are carefully discussed. Additionally, a new method to predict the frictional pressure drop during two-phase flow inside tubes containing twisted-tape inserts is proposed. (C) 2012 Elsevier Ltd. All rights reserved.

Two-Phase Frictional Pressure Drop and Flow Boiling Heat Transfer for R245fa in a 2.32-mm Tube

Experimental two-phase frictional pressure drop and flow boiling heat transfer results are presented for a horizontal 2.32-mm ID stainless-steel tube using R245fa as working fluid. The frictional pressure drop data was obtained under adiabatic and diabatic conditions. Experiments were performed for mass velocities ranging from 100 to 700 kg m−2 s−1 , heat flux from 0 to 55 kW m−2 , exit saturation temperatures of 31 and 41◦C, and vapor qualities from 0.10 to 0.99. Pressures drop gradients and heat transfer coefficients ranging from 1 to 70 kPa m−1 and from 1 to 7 kW m−2 K−1 were measured. It was found that the heat transfer coefficient is a strong function of the heat flux, mass velocity, and vapor quality. Five frictional pressure drop predictive methods were compared against the experimental database. The Cioncolini et al. (2009) method was found to work the best. Six flow boiling heat transfer predictive methods were also compared against the present database. Liu and Winterton (1991), Zhang et al. (2004), and Saitoh et al. (2007) were ranked as the best methods. They predicted the experimental flow boiling heat transfer data with an average error around 19%.

Hydrogen and Methane Production, Energy Recovery, and Organic Matter Removal from Effluents in a Two-Stage Fermentative Process

This study evaluates the potential for using different effluents for simultaneous H-2 and CH4 production in a two-stage batch fermentation process with mixed microflora. An appreciable amount of H-2 was produced from parboiled rice wastewater (23.9 mL g(-1) chemical oxygen demand [COD]) and vinasse (20.8 mL g(-1) COD), while other effluents supported CH4 generation. The amount of CH4 produced was minimum for sewage (46.3 mL g(-1) COD), followed by parboiled rice wastewater (115.5 mL g(-1) COD) and glycerol (180.1 mL g(-1) COD). The maximum amount of CH4 was observed for vinasse (255.4 mL g(-1) COD). The total energy recovery from vinasse (10.4 kJ g(-1) COD) corresponded to the maximum COD reduction (74.7 %), followed by glycerol (70.38 %, 7.20 kJ g(-1) COD), parboiled rice wastewater (63.91 %, 4.92 kJ g(-1) COD), and sewage (51.11 %, 1.85 kJ g(-1) COD). The relatively high performance of vinasse in such comparisons could be attributed to the elevated concentrations of macronutrients contained in raw vinasse. The observations are based on kinetic parameters of H-2 and CH4 production and global energy recovery of the process. These observations collectively suggest that organic-rich effluents can be deployed for energy recovery with sequential generation of H-2 and CH4.

Effect of Feed Strategy on Methane Production and Performance of an AnSBBR Treating Effluent from Biodiesel Production

The aim of this work was to investigate the effect of different feeding times (2, 4 and 6 h) and applied volumetric organic loads (4.5, 6.0 and 7.5 gCOD L-1 day(-1)) on the performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) treating effluent from biodiesel production. Polyurethane foam cubes were used as inert support in the reactor, and mixing was accomplished by recirculating the liquid phase. The effect of feeding time on reactor performance showed to be more pronounced at higher values of applied volumetric organic loads (AVOLs). Highest organic material removal efficiencies achieved at AVOL of 4.5 gCOD L-1 day(-1) were 87 % at 4-h feeding against 84 % at 2-h and 6-h feeding. At AVOL of 6.0 gCOD L-1 day(-1), highest organic material removal efficiencies achieved with 4-h and 6-h feeding were 84 %, against 71 % at 2-h feeding. At AVOL of 7.5 gCOD L-1 day(-1), organic material removal efficiency achieved with 4-h feeding was 77 %. Hence, longer feeding times favored minimization of total volatile acids concentration during the cycle as well as in the effluent, guaranteeing process stability and safety.

Microbial characterization and removal of anionic surfactant in an expanded granular sludge bed reactor

This study evaluated linear alkylbenzene sulfonate removal in an expanded granular sludge bed reactor with hydraulic retention times of 26 h and 32 h. Sludge bed and separator phase biomass were phylogenetically characterized (sequencing 16S rRNA) and quantified (most probable number) to determine the total anaerobic bacteria and methanogenic Archaea. The reactor was fed with a mineral medium supplemented with 14 mg l(-1) LAS, ethanol and methanol. The stage I-32 h consisted of biomass adaptation (without LAS influent) until reactor stability was achieved (COD removal >97%). In stage II-32 h, LAS removal was 74% due to factors such as dilution, degradation and adsorption. Higher HRT values increased the LAS removal (stage III: 26 h - 48% and stage IV: 32 h - 64%), probably due to increased contact time between the biomass and LAS. The clone libraries were different between samples from the sludge bed (Synergitetes and Proteobacteria) and the separator phase (Firmicutes and Proteobacteria) biomass. (C) 2011 Elsevier Ltd. All rights reserved.

On the stability of the Higher Manganese Silicides

We investigated in this work the stability of the Higher Manganese Silicides (HMS). Several alloys in the composition range 62-66 at.% Si were prepared from their constitutive elements by arc-melting. The prepared alloys were then analysed by in situ X-ray diffraction measurements and Electron Probe Micro-Analyser (EPMA). The whole results allow us to suggest that whatever the composition is, only Mn(27)Si(47) is stable for the temperatures 500 degrees C and 800 degrees C. At higher temperatures, the studied samples undergo two phase transformations which consecutively lead to the formation of Mn(15)Si(26) and Mn(11)Si(19). Mn(4)Si(7) was never evidenced in the present work. It is shown for the first time in this work that Mn(27)Si(47) is the only HMS stable phase at room temperature. (C) 2011 Elsevier B.V. All rights reserved.

alpha-6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of alpha-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of alpha-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 x 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding alpha-6 integrin expression. Flow cytometry analysis demonstrated no differences in the alpha-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to alpha-6 integrin, the expression of GFR alpha-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to alpha-6 integrin expression.

Saturated flow boiling heat transfer and critical heat flux in small horizontal flattened tubes

This paper presents experimental results for flow boiling heat transfer coefficient and critical heat flux (CHF) in small flattened tubes. The tested flattened tubes have the same equivalent internal diameter of 2.2 mm, but different aspect height/width ratios (H/W) of 1/4, 1/2, 2 and 4. The experimental data were compared against results for circular tubes using R134a and R245fa as working fluids at a nominal saturation temperature of 31 degrees C. For mass velocities higher than 200 kg/m(2)s, the flattened and circular tubes presented similar heat transfer coefficients. Such a behavior is related to the fact that stratification effects are negligible under conditions of higher mass velocities. Heat transfer correlations from the literature, usually developed using only circular-channel experimental data, predicted the flattened tube results for mass velocities higher than 200 kg/m(2)s with mean absolute error lower than 20% using the equivalent diameter to account for the geometry effect. Similarly, the critical heat flux results were found to be independent of the tube aspect ratio when the same equivalent length was kept. Equivalent length is a new parameter which takes into account the channel heat transfer area. The CHF correlations for round tubes predicted the flattened tube data relatively well when using the equivalent diameter and length. Furthermore, a new proposed CHF correlation predicted the present flattened tube data with a mean absolute error of 5%. (C) 2012 Elsevier Ltd. All rights reserved.

Water-assisted Flow of Heavy Oil in a Vertical Pipe: Pilot-scale Experiments

The use of the core-annular flow pattern, where a thin fluid surrounds a very viscous one, has been suggested as an attractive artificial-lift method for heavy oils in the current Brazilian ultra-deepwater production scenario. This paper reports the pressure drop measurements and the core-annular flow observed in a 2 7/8-inch and 300 meter deep pilot-scale well conveying a mixture of heavy crude oil (2000 mPa.s and 950 kg/m3 at 35 C) and water at several combinations of the individual flow rates. The two-phase pressure drop data are compared with those of single-phase oil flow to assess the gains due to water injection. Another issue is the handling of the core-annular flow once it has been established. High-frequency pressure-gradient signals were collected and a treatment based on the Gabor transform together with neural networks is proposed as a promising solution for monitoring and control. The preliminary results are encouraging. The pilot-scale tests, including long-term experiments, were conducted in order to investigate the applicability of using water to transport heavy oils in actual wells. It represents an important step towards the full scale application of the proposed artificial-lift technology. The registered improvements in terms of oil production rate and pressure drop reductions are remarkable.