Extracellular endo-mannanase from Bacillus sp. CFR1601: Economical production using response surface methodology and downstream processing using aqueous two phase system

Bacillus sp. CFR1601, isolated from decaying plant litter, produced an extra-cellular endo-mannanase (198.0 IU/g) under solid state fermentation (SSF) using defatted coconut residue as the prime solid substrate. In order to enhance endo-mannanase production, three component, five level central composite design (CCD) of response surface methodology (RSM) was used. Based on contour plots and variance analysis, optimum conditions for endo-mannanase production from Bacillus sp. CFR1601 were attained when defatted coconut residue was supplemented with sesame oil meal (10.0, w/w), Tween-80 (0.2%, v/v) and inoculated with bacterial cells from log phase (12 h old; OD_600 nm ≈ 3.6). The empirical model developed through RSM brought about 4.04–4.39-fold (800.0–870.0 IU/g) improvement in endo-mannanase yield as compared to un-optimized growth conditions. Downstream processing of endo-mannanase from SSF media was carried out for the first time using polyethylene glycol (PEG)/salt aqueous two phase system (ATPS). ATPS system consisting of a combination of PEG 3350 12.0% (w/w), Na₂SO₄ 12.0% (w/w), protein load 10.0% (w/w) and pH 5.0 resulted in one-sided partitioning of endo-mannanase towards bottom phase with 3.8-fold purification and 95.4% recovery. Second stage ATPS with fresh top phase further improved purification of endo-mannanase to 12.32-fold. Our overall results suggest a cost-effective and integrated process for production and downstream processing of endo-mannanase.     

Partitioning of lysozyme in aqueous two-phase systems containing ethylene oxide-propylene oxide copolymer and potassium phosphates

Lysozyme partitioning in EO₅₀PO₅₀/potassium phosphate aqueous two-phase systems (ATPS) was studied. In the work, the influence of EO₅₀PO₅₀, potassium phosphate and sodium chloride concentration in the ATPS on lysozyme partition coefficient and separation yield was examined. In addition, the influence of the pH of potassium phosphate solution was also investigated. A Box–Behnken design was defined, and response surface models for the partition coefficient K and percentage yield of the enzyme in the top phase Y were calculated. Among the examined factors, the NaCl concentration had the highest influence on lysozyme separation parameters. This influence can be explained mainly by the hydrophobic interactions between the protein and the phase-forming components. A maximum partition coefficient K_L1, yield Y_L1 and Y_L2 were predicted for EO₅₀PO₅₀, potassium phosphate and NaCl concentrations of 17.40, 22.67% and 0.85 mol/l, respectively, and for pH 9.0. A good agreement was obtained between the experimental and the predicted results.     

Synthesis,characterization, and drug‐release behavior of novel PEGylated bovine serum albumin as a carrier for anticancer agents

To develop a novel pH‐sensitive PEGylated carrier for protein‐based anticancer agents, we modified bovine serum albumin (BSA) with poly(ethylene glycol) citrate ester (PEG–CA) through amidation with its amino groups. Increasing the mixing ratio of albumin from 3 to 6 with respect to PEG–CA resulted in a 2‐fold increase in the degree of albumin modification. Adriamycin (ADR)‐loaded PEG–CA–BSA hydrogels and microparticles were prepared, and the cumulative amounts of ADR released from the PEG–CA–BSA hydrogels (phosphate‐buffered saline, pH 7.4) showed that all the PEG‐CA‐BSA_(x) (x represents degree of substitution of PEG to amino group of albumin, i.e. 26%, 28%, 31% and 49%) hydrogels had lower ADR release rates with a slight initial burst release. During the first 24 h, the cumulative releases were 15.5% for PEG–CA–BSA₍₄₉₎, 24% for PEG–CA–BSA₍₃₁₎, 31% for PEG–CA–BSA₍₂₈₎, and 38% for PEG–CA–BSA₍₂₆₎. Afterward, all the release rates slowed, and they were almost in the following order: PEG–CA–BSA₍₂₆₎ > PEG–CA–BSA₍₂₈₎ > PEG–CA–BSA₍₃₁₎ > PEG–CA–BSA₍₄₉₎. The release rates of ADR from the microparticles were dependent on the amount of glutaraldehyde. According to our findings, a higher PEG–CA/BSA molar ratio led to a reduced cumulative amount of ADR released from the hydrogels, whereas higher release rates were observed for microparticles with a lower amount of BSA in the conjugates in a pH‐dependent manner. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

The optimization of aqueous two‐phase extraction of lysozyme from crude hen egg white using response surface methodology

BACKGROUND: Aqueous two‐phase extraction is a versatile method for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG 4000/potassium citrate aqueous two‐phase system (ATPS) for recovering and purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification of lysozyme from crude hen egg white. RESULTS: Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol 4000 (PEG 4000)/potassium citrate ATPS are established using response surface methodology. Screening experiments using fractional factorial designs show that the pH of the system significantly affects the recovery and purification of lysozyme. An optimized ATPS was proved to be at pH 5.5 and 30 °C and contained 18% (w/w) PEG, 16% (w/w) potassium citrate, 3.75% (w/w) potassium chloride (KCl). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were 31100 U mg^?1, 21.11 and 103%, respectively. CONCLUSION: The PEG 4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme. © 2012 Society of Chemical Industry     

Influence of Yb3+ doping on phase stability and thermophysical properties of (Y1-xYbx)3Al5O12 under high temperature

In this work, Yb³⁺ was selected to replace the Y³⁺ in yttrium aluminum garnet (YAG) in order to reduce its thermal conductivity under high temperature. A series of (Y_1-xYb_x)₃Al₅O₁₂ (x=0, 0.1, 0.2, 0.3, 0.4) ceramics were prepared by solid-state reaction at 1600 °C for 10 h. The microstructure, thermophysical properties and phase stability under high temperature were investigated. The results showed that all the Yb doped (Y_1-xYb_x)₃Al₅O₁₂ ceramics were comprised of a single garnet-type Y₃Al₅O₁₂ phase. The thermal conductivities of (Y_1-xYb_x)₃Al₅O₁₂ ceramics firstly decreased and subsequently increased with Yb ions concentration rising from room temperature to 1200 °C. (Y_0.7Yb_0.3)₃Al₅O₁₂ had the lowest thermal conductivity among investigated specimens, which was about 1.62 W m⁻¹ K⁻¹ at 1000 °C, around 30% lower than that of pure YAG (2.3 W m⁻¹ K⁻¹, 1000 °C). Yb had almost no effect on the coefficients of thermal expansion (CTEs) of (Y_1-xYb_x)₃Al₅O₁₂ ceramics and the CTE was approximate 10.7×10⁻⁶ K⁻¹ at 1200 °C. In addition, (Y_0.7Yb_0.3)₃Al₅O₁₂ ceramic remained good phase stability when heating from room temperature to 1450 °C.     

Dispersion of nano-sized yttria powder using triammonium citrate dispersant for the fabrication of transparent ceramics

In the present work, transparent Y₂O₃ ceramics were prepared via colloidal processing method from nano-sized Y₂O₃ powders. The effects of triammonium citrate (TAC) on the colloid stability of aqueous suspensions of nano-sized Y₂O₃ powders were studied. The surface properties of yttria powders were notably affected by the addition of TAC dispersant. The adsorption of TAC on the particle surface shifts the IEP to lower pH values and increases the absolute zeta potential in alkaline region. Rheological characterization of the investigated system revealed an optimal dispersant concentration of 1 wt%, which correlated well with the saturation adsorption of TAC on Y₂O₃ powder surfaces. The suspensions with solid loadings up to 35 vol% were achieved with further addition of Tetramethylammonium hydroxide (TMAH) into the dispersing system. The consolidated green bodies were treated by cold isostatic pressing to further increase the green density. Transparent Y₂O₃ ceramics were prepared after vacuum-sintering at 1700 °C for 5 h. The transmittances of the sample were 74.5% at 800 nm and 79.8% at 2000 nm, respectively.     

Non-aqueous stabilized suspensions of BaZr0.85Y0.15O3−δ proton conducting electrolyte powders for thin film preparation

The development of uniform electrolyte thin films minimizes the ohmic loss of ceramic oxide electrolyzers and/or fuel cells, increasing their efficiency. In this work, thin films of BaZr_0.85Y_0.15O_3?δ (BZY) electrolyte have been obtained over a 40 vol% Nickel–BaZr_0.85Y_0.15O_3?δ (Ni–BZY) cermet anode support by spin coating. To facilitate this aim, stable suspensions have been tailored, by methodically determining suitable solvents and additives (surfactants and/or thickeners). Sedimentation studies showed that ethanol and 1-methoxy-2-propanol can produce similar, stable 5% w/w BZY suspensions. The best result was obtained by simultaneously adding 1.5 wt% of ZEPHRYM PD 7000 (a commercial polyoxyalkylene amine derivative surfactant) and 0.3 wt% of polyvinyl butyral (PVB) in 1-methoxy-2-propanol. Suspensions of 1-methoxy-2-propanol with higher powder concentration (25% w/w) also show good stability that was improved by the addition of 1 wt% of ZEPHRYM. Using this system, dense BZY films of around 14 μm thick could be successfully obtained at 1500 °C.     

Ionic conductivity and mechanical properties of Y2O3-doped CeO2 ceramics synthesis by microwave-induced combustion

We developed a new method, i.e. microwave-induced combustion synthesis to produce highly sinterable Y₂O₃-doped CeO₂ nanopowders. The process took only 15 min to yield Y₂O₃-doped CeO₂ powders. We also investigated the conductivity of Y₂O₃-doped CeO₂ ceramics. It was found Y₂O₃ concentration to have a large effect on the morphology, activation energy, ionic conductivity, and mechanical properties of Y₂O₃-doped CeO₂ ceramics. The results revealed that the bulk densities of Y₂O₃-doped CeO₂ ceramics sintered at 1420 °C for 5 h were all higher than 92% of the theoretical densities, and the maximum ionic conductivity, σ_800 °C = 0.023 S/cm at 800 °C, the minimum activation energy, E_a = 0.954 eV determined in the temperature of 300–800 °C and the maximum fracture toughness, K_IC = 1.825 ± 0.188 MPa m^1/2 were found for 9 mol.% Y₂O₃-doped CeO₂ specimen. The grain size of CeO₂ decreases with increasing Y₂O₃ concentration. The fracture toughness was found to increase at increased Y₂O₃ concentration, because of the decrease of CeO₂ grain size.     

Fabrication of transparent yttria ceramics by alcoholic slip-casting

Y₂O₃ transparent ceramics were prepared from alcoholic slurries of Y₂O₃ nanopowders via a slip-casting method to avoid the hydrolysis issue. Polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) and polyethylenimine (PEI) were used as dispersants to improve the rheological properties of the slurries. It was found that PEI is the most effective dispersant in ethanol. The adsorbed amount of PEI was evaluated by infrared absorption and rheology measurements. Y₂O₃ slurry with a solid loading of 20.8 vol% and a viscosity of <0.1 Pa s at the shear rate of 10 s⁻¹ was obtained using 1.5 wt% PEI. The slurry yielded a homogeneous green body, and finally resulted in a high-quality Y₂O₃ ceramic with the in-line transmittance of 80% at 800 nm.     

The effect of Zn addition on the structure and transport properties of BaCe0.9−xZrxY0.1O3−δ

BaCe_0.9−xZr_xY_0.1O_3−δ (0.1 ≤ x ≤ 0.9) are ceramic proton conductors widely investigated for different electrochemical devices, such as Solid Oxide Fuel Cell (SOFC) electrolytes, however, their applications are limited by the high sintering temperatures necessary to achieve densification. Polycrystalline powders of BaCe_0.9−xZr_xY_0.1O_3−δ (BCZ) have been prepared by freeze-drying precursor method at 1000 °C. These powders were mixed with a zinc nitrate solution to decrease the sintering temperature to 1200–1300 °C. The addition of Zn has several effects on the structure and microstructure of BCZ: the densification and grain growth are enhanced at lower temperature and a reduction of the crystallographic symmetry is observed for samples with low Zr-content (x ≤ 0.3). Furthermore, the bulk and specific grain boundary conductivities are only slightly affected by the addition of Zn.     

Optimized and functionalized paper sludge activated with potassium fluoride for single and binary adsorption of reactive dyes

The yield and adsorption uptake of optimized paper sludge activated carbon (PSAC) prepared using potassium fluoride as alternative chemical activation agent was investigated. The PSAC was functionalized with ethylenediamine (FPSAC) and both adsorbents were used for single and binary adsorption of Reactive orange 16 (RO16) and Reactive blue 19 (RB19). Effect of pH on the adsorption process, equilibrium, kinetics, isotherm and thermodynamic studies were carried out. Optimum PSAC preparation parameters were: activation temperature, X₁ = 810 °C; activation time, X₂ = 105 min; and impregnation ratio, X₃ = 0.95 which gave adsorption uptake of 178 and 158 mg/g for RO16 and RB19, respectively.     

Improvements on the thermal shock behaviour of MgO–spinel composite refractories by incorporation of zircon–3 mol% Y2O3

The effects of incorporations of zircon–3 mol% Y₂O₃ (Y) into MgO–spinel (M–S) compositions to improve mechanical properties and thermal shock behaviour were investigated. Mechanical properties were measured, R ? R_st parameters were calculated, and thermal shock tests were performed. Microstructural features were examined using SEM and XRD. By adding zircon + Y to M–S: (i) up to ～2 and ～3-fold improvements were achieved in mechanical properties and R ? R_st parameters, (ii) there were improvements up to ～2-fold in strength data measured after performing thermal shock tests at 1000 °C. Parameters improving mechanical properties and thermal shock behaviour of M–S–(zircon + Y) materials are given as follows: (i) interlinking and arresting or deviation of microcracks when reaching the ZrO₂–Y₂O₃ grains or pores, increases in (ii) K_Ic, (iii) critical defect size and (iv) bulk density, (v) formation of forsterite phase, (vi) coexistence of intergranular and transgranular fractures, and (vii) reduction in MgO grain size, leading to longer service life.     

Novel amorphous precursor densification to transparent Nd:Y2O3 Ceramics

A novel approach of neodymium ion doped yttrium oxide (Nd:Y₂O₃) amorphous precursor compaction and sintering is being reported for the first time. Precursor of 2 at.% Nd³⁺ doped Y₂O₃ was synthesized by gelation of sol of yttrium and neodymium nitrates with l-alanine at 80 °C for 16 h followed by gel combustion in microwave. A part of microwave precursor was heat treated at 700 °C for 5 h to give the partially crystalline Nd:Y₂O₃ amorphous precursor. Thermogravimetric analysis (TGA) of partially crystalline amorphous precursor of Nd:Y₂O₃ gave 8.5% total weight loss indicating removal of maximum organics. X-Ray diffraction (XRD) showed broad peaks indicating incomplete crystallization of cubic Nd:Y₂O₃. Morphology was found to be close to spherical with particles in size range 17–19 nm by TEM. Another part of microwave precursor on calcination at 1000 °C for 3 h led to formation of fully crystalline Nd:Y₂O₃ with particles in size range of 35–85 nm. Both partially crystalline amorphous precursor and fully crystalline Nd:Y₂O₃ were compacted at 400 MPa by cold isostatic press and sintered at 1750 °C for 10 h under vacuum (10^?5 mbar). The partially crystalline Nd:Y₂O₃ amorphous precursor densified to 99% with 65% transmission at 2500 nm (0.5 mm thickness) compared to 96% densification with 34% transmission for fully crystalline Nd:Y₂O₃ without any sintering aids. Retention of cubic phase purity of Y₂O₃ was observed in both the ceramic pellets post sintering by XRD. Good grain fusion with grain growth to ≤2 μm was observed by scanning electron microscope (SEM) for partially crystalline Nd:Y₂O₃ amorphous precursor. Thus partially crystalline Nd:Y₂O₃ amorphous precursor nanopowders, with homogeneous close to spherical fine particles and high reactivity due to ionic mobility of amorphous phase, led to better densification.     

Synthesis of BaCeO3 and BaCe0.9Y0.1O3−δ from mixed oxalate precursors

An oxalate precipitation route is proposed for the synthesis of BaCe_1−xY_xO₃ (x = 0 and 0.1) after calcination at 1100 °C. The precipitation temperature (70 °C) was a determinant parameter for producing a pure perovskite phase after calcination at 1100 °C for 1 h. TG/DTA measurements showed that the co-precipitated (Ba, Ce and Y) oxalate had a different thermal behaviour from single oxalates. Despite a simple grinding procedure, sintered BaCe_0.9Y_0.1O_3−δ pellets (1400 °C, 48 h) presented 90.7% of relative density and preliminary impedance measurements showed an overall conductivity of around 2 × 10⁻⁴ S cm⁻¹ at 320 °C.     

Experimental and modeling studies on the enantio-separation of 3,5-dinitrobenzoyl-(R),(S)-leucine by continuous liquid–liquid extraction in a cascade of centrifugal contactor separators

The continuous enantioselective liquid–liquid extraction of aqueous 3,5-dinitrobenzoyl-(R),(S)-leucine (A_R,S) using O-(1-t-butylcarbamoyl)-11-octadecylsulfinyl-10,11-dihydro-quinine (C, a cinchona alkaloid) as extractant in 1,2-dichloroethane (DCE) was studied experimentally in a countercurrently operated pilot scale cascade of six centrifugal contactor separators (CCS) at 294 K. The extractant was efficiently recovered by back-extraction in a single CCS allowing the cascade to be run continuously for 10 h. The steady-state ee of A_R (ee_R) in the raffinate was 42% at a 99% yield, the A_S was obtained with high purity (98% ee_S) and a yield of 55% in the back-extraction raffinate. In total 2.23 g of A_S was obtained at steady-state operation from 8.11 g racemate feed. Deterioration of the ee in time was not observed, demonstrating the robustness of the chemistry. The experiments were modeled using an equilibrium stage approach. The correlation between model and experiment was satisfactory. The model was applied to optimize the production of both enantiomers in >97% ee. At zero reflux, 12 stages are required for 99% ee for both enantiomers. Application of a reflux allows a 25% reduction of the total liquid flow through the system by reduction of the wash feed as well as a reduction in the number of stages from 12 to 11. With a configuration of 12 CINC-V02’s operating at an aqueous feed flow of 360 mL/min, the model predicts that 17.7 kg racemate per week may be separated into both enantiomers with 99% ee using only 60 g of extractant.     

Extraction of microbial transglutaminase from <Emphasis Type="Italic">Amycolatopsis</Emphasis> sp. fermentation broth using aqueous two-phase system

Partitioning of microbial transglutaminase (MTG) from Amycolatopsis sp. in the polyethylene glycol (PEG)/salt-based ATPS was investigated for the first time. The key parameters such as the molecular weight of PEG (PEG 600-6000), the type and concentration of phase-forming salt (ammonium sulfate or phosphates), the pH of system (pH 5.0-8.5), and the concentration of neutral salt (0-6% NaCl, w/w) were determined. The partition coefficient of the enzyme was not linear with PEG molecular weight; PEG1000 gave better yield than others. The concentration of PEG1000, ammonium sulfate and NaCl, and the system pH showed effects with different extents on specific activity (SA) and yield of the enzyme. In the ATPS of 26% w/w PEG 1000 and 19% w/w ammonium sulfate in the presence of 5% w/w NaCl and at pH 6.0, MTG was partitioned into the PEG-rich phase with a maximum yield of 86.51% and SA was increased to 0.83. The results of SDS-PAGE showed the MTG produced by the test strain differed from the enzymes reported before. Thus, this study proves that ATPS can be used as a preliminary step for partial purification of MTG from Amycolatopsis sp. fermentation broth.     

Electrodeposited hybrid films of polyaniline and manganese oxide in nanofibrous structures for electrochemical supercapacitor

Hybrid films of polyaniline (PANI) and manganese oxide (MnO_x) were obtained through potentiodynamic deposition from solutions of aniline and MnSO₄ at pH 5.6. The hybrid films demonstrated characteristic redox behaviors of PANI in acidic aqueous solution. Characterization of the hybrid films by XRD indicated the amorphous nature of MnO_x in the films in which manganese existed in oxidation states of +2, +3 and +4, based on XPS measurement. Hybrid film of PANI and MnO_x, PM120 obtained from the solution of 0.1 M aniline and 120 mM Mn²⁺ displayed a well opened nanofibrous structure which showed an 44% increase in specific capacitance from that of PANI (408 F g^?1) to 588 F g^?1, measured at 1.0 mA cm^?2 in 1 M NaNO₃ (pH 1). The hybrid film kept more than 90% of its capacitance after 1000 charging-discharging cycles, with a coulombic efficiency of 98%. The specific capacitance of a symmetric capacitor using PM120 as the electrodes is 112 F g^?1.     

Inhibition of grain growth in liquid-phase sintered SiC ceramics by AlN additive and spark plasma sintering

SiC ceramics were prepared from nanosized β-SiC powder with different compositions of AlN and Y₂O₃ sintering additives by spark plasma sintering (SPS) at 1900 °C for 600 s in N₂. The relative density of the sintered SiC specimens increased with increasing amount of AlN, reaching a relative density higher than 99%, while at the same time grain size decreased significantly. The smallest average grain size of 150 nm was observed for SiC sample sintered with 10 vol% of additives consisting of 90 mol% AlN and 10 mol% Y₂O₃. Fully dense nanostructured SiC ceramics with inhibited grain growth were obtained by the AlN additive and SPS technique. The flexural strength of the SiC body containing 70 mol% AlN and 30 mol% Y₂O₃ additives reached the maximum value of 1000 MPa. The SiC bodies prepared with AlN and Y₂O₃ additives had the fracture toughness of around 2.5 MPam^1/2.     

Improved recovery of B‐phycoerythrin produced by the red microalga Porphyridium cruentum

A simplified process for the primary recovery and purification of B‐phycoerythrin (BPE) from Porphyridium cruentum exploiting aqueous two‐phase systems (ATPS) and isoelectric precipitation was developed in order to reduce the number of unit operations and benefit from increased purity and yield of the protein product. Evaluation of the partitioning behaviour of BPE in polyethylene glycol (PEG)/sulphate, PEG/dextran and PEG/phosphate ATPS was carried out to determine under what conditions the BPE and contaminants concentrated into opposite phases. An additional stage of isoelectric precipitation at pH 4.0 after cell disruption resulted in an increase in purity of the target protein from the BPE crude extract and enhanced the performance of the subsequent ATPS. PEG1000/phosphate ATPS proved to be suitable after isoelectric precipitation for the recovery of highly purified (defined as absorbance ratio A_{545 nm}/A_{280 nm} > 4.0) BPE with a potential commercial value as high as US$ 50/mg. An ATPS extraction stage comprising 29.5% (w/w) PEG1000, 9.0% (w/w) phosphate, a volume ratio (V_r) equal to 1.0, a system pH of 7.0 and loaded with 40% (w/w) of the BPE extract generated by precipitation allowed BPE recovery with a purity of 4.1±0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9‐fold after isoelectric precipitation and ATPS. The results reported herein demonstrate the benefits of the practical application of isoelectric precipitation together with ATPS for the recovery and purification of BPE produced by P. cruentum as a first step in the development of a commercial purification process. Copyright © 2006 Society of Chemical Industry