Integration of continuous production and recovery of solvents from whey permeate: use of immobilized cells of Clostridium acetobutylicum in a flutilized bed reactor coupled with gas stripping

An investigation was performed into the operation of an integrated system for continuous production and product recovery of solvents (acetone-butanol-ethanol) from the ABE fermentation process. Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar, and used in a fluidized bed reactor for continuous solvent production from whey permeate. The reactor effluent was stripped of the solvents using nitrogen gas, and was recycled to the reactor. This relieved product inhibition and allowed further sugar utilization. At a dilution rate of 1.37 h^–1 a reactor productivity of 5.1 kg/(m³ · h) was achieved. The solvents in the stripping gas were condensed to give a solution of 53.7 kg/m³. This system has the advantages of relieving product inhibition, and providing a more concentrated solution for recovery by distillation. Residual sugar and non-volatile reaction intermediates are not removed by gas stripping and this contributes to high solvent yields.List of Symbols C kg/m³ Lactose concentration in reactor effluent - C _b kg/m³ Lactose concentration in bleed stream - C _c kg/m³ Lactose concentration in whey permeate feed - C _i kg/m³ Lactose concentration at reactor inlet - C _p kg/m³ Lactose concentration in condensed solvent stream (=0) - C _r kg/m³ Lactose concentration in recycle line (C _b=C _r) - C kg/h Amount of lactose utilized during certain time period - D h¹ Dilution rate of reactor, F _i/D=F/D - F dm³/h, m3/h F _i = Rate of feed flow to the reactor - F _b dm ³/h, m³/h Rate of bleed - F _c dm³/h, m³/h Rate of feed of whey permeate solution - F _p dm³/h, m³/h Rate of concentrated product removal - F _r dm³/h, m³/h Rate of recycle of stripped effluent to the reactor - P _l % Percent lactose utilization - R _l kg/(m³ · h) Overall lactose utilization rate - R _p kg/(m³ · h) Overall reactor (solvent) productivity - R _sl kg/h Rate of solvent loss - S kg/m³ Solvent concentration in reactor effluent - S _b kg/m³ Solvent concentration in bleed - S _c kg/m³ 0; Solvent concentration in concentrated whey permeate solution - S _i kg/m³ Solvent concentration at inlet of reactor - S _p kg/m³ Solvent concentration in concentrated product stream - S _r kg/m³ Solvent concentration in stripped effluent, S _r=S_b - S kg/h Amount of solvent produced from C amount of lactose in a particular time - ds/dt kg/(m³ · h) Rate of accumulation of solvents in the stripper - t h Time - V dm³, m³ Total reactor volume - V ¹ dm³, m³ Liquid volume in stripper - Y _P/S Solvent yield     

Exhaust gas purification using biocatalysts (fixed bacteria monocultures) — the influence of biofilm diffusion rate (O2) on the overall reaction rate

Summary This paper presents results of experiments on the influence of O₂ and substrate (pollutant) concentration on the overall reaction rate of a trickle-bed reactor used for biological waste gas purification. The biocatalyst was a pollutant-specific bacterial monoculture fixed on porous glass carriers. The conversion of acetone and propionaldehyde, as model pollutants that are easily soluble in water, was measured. Under constant hydrodynamic conditions (gas and liquid flow rates) the inlet pollutant concentration was varied. The O₂ partial pressure in the model gas was increased to investigate the influence of O₂ supply on pollutant conversion. At higher pollutant concentrations (>117 mg acetone.m^-3 gas and > 150 mg propionaldehyde.m^-3 gas) higher concentrations of dissolved O₂ led to a significant rise in the maximum degradation capacity of the reactor. This maximum reaction rate was independent of the pollutant mass flow. It seems that the diffusion of O₂ in the biofilm is rate-determining. The reaction rate at lower inlet concentrations was not affected by the improved O₂ supply. Here the external mass transfer through the liquid film limits the reaction rate and the maximum separation efficiency of about 80% at a residence time of 1.2s (space velocity 3000h^-1) is achieved.     

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log P_octanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between ? 1.1 and 8.8 were examined. Organic solvents with log P_octanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL^?1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.     

Solvent Effect on the Photolysis of Riboflavin

The kinetics of photolysis of riboflavin (RF) in water (pH 7.0) and in organic solvents (acetonitrile, methanol, ethanol, 1-propanol, 1-butanol, ethyl acetate) has been studied using a multicomponent spectrometric method for the assay of RF and its major photoproducts, formylmethylflavin and lumichrome. The apparent first-order rate constants (k_obs) for the reaction range from 3.19 (ethyl acetate) to 4.61 × 10⁻³ min⁻¹ (water). The values of k_obs have been found to be a linear function of solvent dielectric constant implying the participation of a dipolar intermediate along the reaction pathway. The degradation of this intermediate is promoted by the polarity of the medium. This indicates a greater stabilization of the excited-triplet states of RF with an increase in solvent polarity to facilitate its reduction. The rate constants for the reaction show a linear relation with the solvent acceptor number indicating the degree of solute–solvent interaction in different solvents. It would depend on the electron-donating capacity of RF molecule in organic solvents. The values of k_obs are inversely proportional to the viscosity of the medium as a result of diffusion-controlled processes.KEY WORDS: dielectric constant, kinetics, photolysis, riboflavin, solvent effect, viscosity     

青海湖流域矮嵩草草甸土壤有机碳密度分布特征

通过对青海湖流域不同退化程度矮嵩草草甸土壤容重和有机碳含量的测定,确定了其土壤有机碳密度。结果表明:不同退化程度下矮嵩草草甸土壤有机碳含量和变化特征各有不同。从未退化-重度退化,0—100 cm土壤剖面平均有机碳含量分别为(25.17±4.73)g/kg,(17.51±3.06)g/kg,(20.79±1.30)g/kg和(14.53±1.20)g/kg,即未退化中度退化轻度退化重度退化;0—20 cm土壤平均有机碳含量从(64.47±11.70)g/kg减少为(14.52±1.52)g/kg,减少了77.48%。土壤剖面有机碳密度变化趋势与其有机碳含量变化趋势一致。0—100 cm土壤剖面有机碳密度分别为(18.16±4.12)kg/m3,(14.24±3.52)kg/m3,(18.64±2.82)kg/m3和(13.27±2.28)kg/m3,即中度退化未退化轻度退化重度退化;土壤有机碳集中分布在0—40 cm深度,从未退化到严重退化,该深度有机碳密度分别为(32.06±6.41)kg/m3,(25.10±4.20)kg/m3,(22.68±3.17)kg/m3和(17.10±2.77)kg/m3,比整个剖面有机碳密度高出76.53%,76.25%,21.68%和28.88%。不考虑其他因素,以空间尺度代替时间尺度,这一结果说明矮嵩草草甸的退化导致土壤逐渐释放有机碳,其作为储存碳的功能在减弱,必须加强对矮嵩草草甸生态系统的保护,以防止其碳库变为碳源。     

Hydrophobic Substitution of Surface Residues Affects Lipase Stability in Organic Solvents

A novel lipase has been recently isolated from a local Pseudomonas sp. (GQ243724). In the present study, we have tried to increase the organic solvent stability of this lipase using site-directed mutagenesis. Eight variants N219L, N219I, N219P, N219A, N219R, N219D, S251L, and S251K were designed to change the surface hydrophobicity of this enzyme with respect to the wild-type. Among these variants, the stability of N219L and N219I significantly increased in the presence of all tested organic solvents, whereas two mutants (N219R and N219D) significantly exhibited decreased stabilities in all the organic solvent studied, suggesting that improvement of hydrophobic patches on the enzyme surface enhances the stability in organic media. Furthermore, replacing Ser²⁵¹ with hydrophobic residues on the enzyme surface dramatically diminished its stability in the tested condition. In spite of the distance of the mutated sites from the active site, the values of k _cat and K _m were affected. Finally, structural analysis of the wild-type and mutated variants was carried out in the presence and absence of some organic solvents using circular dichroism and fluorescence spectroscopy.     

Concentric-tube airlift bioreactors

Gas holdup investigations were performed in three concentric-tube airlift reactors of different scales of operation (RIMP: 0.070 m³; RIS-1: 2.5 m³; RIS-2: 5.2 m³; nominal volumes). The influences of the top and bottom clearances and the flow resistances at the downcomer entrance were studied using tap water as liquid phase and air as gaseous phase, at atmospheric pressure. It was found that the gas holdup in the individual zone of the reactor: riser, downcomer and gas-separator, as well as that in the overall reactor is affected by the analyzed geometrical parameters in different ways, depending on their effects on liquid circulation velocity. Gas holdup was satisfactorily correlated with Fr, Ga, bottom spatial ratio (B), top spatial ratio (T), gas separation ratio (Y) and downcomer flow resistance ratio (A _d /A _R ). Correlations are presented for gas holdup in riser, downcomer, gas separator and for the total gas holdup in the reactor. All the above stressed the importance of the geometry in dynamic behaviour of airlift reactors.     

Biological elimination of volatile xenobiotic compounds in biofilters

Biofiltration is a technique which is frequently applied for the odour abatement of waste gases. This technique is based on the ability of microorganisms (generally bacteria, and to a small extent moulds and yeasts) to degrade several organic as well as inorganic compounds to mineral end-products, like water and carbon dioxide. In the case of biofiltration, microorganisms are attached to suited packing materials in the filter, which contain the inorganic nutrients necessary for microbial growth. In order to make biofiltration applicable on a larger scale in process industry, it is necessary to find microorganisms able to eliminate compounds which are strange to life, the so-called xenobiotics. At the Eindhoven University of Technology microorganisms were isolated for the elimination of a number of xenobiotics, e.g. aromatic compounds and chlorinated hydrocarbons.Both the microkinetics of the biodegradation in aqueous batch systems and the macrokinetics of biofilters were studied. Special attention was paid to the influence of the superficial gas velocity and the organic load on the filter bed's elimination capacity. Also the discontinuous operation of biofilters is discussed.Nomenclature C ₁],C _1,0,C _í mol/m³ liquid phase concentration - K _s mol/m³ saturation constant - Y kg/kmol yield coefficient - t, t d time - _md^–1 maximum growth rate - X, X ₀ g/m³ microorganism concentration - P _gPa partial vapour pressure - H Pa m³/mol Henry's law constant - m (-) distribution coefficient - C _g,C _g,max mol/m³ gas phase concentration - R J (mol K) gas constant - T K temperature - (-) gas phase- and liquid phase volume ratio - m³/(m² h) superficial gas velocity - H m height of the filter bed - h m height in the filter bed In memory of Dick van Zuidam     

Effect of salt on the stability of the pH 4.2 rA8 double helix in a series of organic/aqueous mixed solvents: A test of oligoelectrolyte theory

Temperature-dependent uv absorption spectroscopy has been used to investigate the salt dependence of the order–disorder transition for the pH 4.2 rA₈ double helix in 100% aqueous buffer and in a series of organic/aqueous mixed solvents. Melting temperature, T_m, data were obtained for the transitions in the different solvents by analysis of the uv melting curves. For the pure aqueous buffer solvent, the melting temperature was found to exhibit a reduced salt dependence (?t_m/? log Na⁺) when compared to the corresponding polymer. This reduction is explained in terms of end effects and is shown to be consistent with the theoretical treatments of oligoelectrolyte transitions developed by Record and Lohman [Biopolymers, 17 , 159–166 (1978)]. In the mixed solvents, the salt dependence of the melting temperature (?t_m/? log Na⁺) is shown to exhibit a linear dependence on the bulk dielectric constant of the medium for all of the hydroxyl-containing solvents studied. Significantly, N,N-dimethylformamide demonstrated different behavior.     

Hydrodynamics of non-Newtonian liquids in external-loop airlift bioreactors

Gas holdup investigation was performed in two external-loop airlift bioreactors of laboratory (V _L =1.189·10^?3? 1.880·10^?3 m³; H _R =1.16 ? 1.56 m; H _D = 1.10 m; A _D /A _R = 0.111 ? 1.000) and pilot scale (V _L =0.157?0.170 m³; H _R =4.3?4.7 m; H _D =4.0?4.4 m;A _D /A _R =0.04?0.1225), respectively, using as liquid phase non-Newtonian starch solutions of different concentration with K=0.061?3.518 Pa sⁿ and n=0.86?0.39 and fermentation broths of P. chrysogenum, S. griseus, S. erythreus, B. licheniformis and C. acremonium at different hours since inoculation and from different batches. The influence of bioreactor geometry, liquid properties and the amount of introduced compressed air was investigated. The effect of sparger design on gas holdup was found to be negligible. It was found that gas holdup depends on the flow media index, ?_GR decreasing with the increase of liquid pseudoplasticity, A _D /A _R ratio and H _R /H _D ratio. The experimental data are in agreement with those presented in literature by Popovic and Robinson, which take into account liquid properties, geometric parameters and gas superficial velocity, with a maximum error of ±30%. It was obtained a correlation for gas holdup estimation taking into account the non-Newtonian behaviour of the fermentation broths and the dry weight of the solid phase, as well. The concordance between the experimental data and those calculated with the proposed correlation was good, with a maximum error of ±17%. Also, a dimensionless correlation for gas holdup involving superficial velocities of gas and liquid, cross sectional areas ratio, dispersion height to riser diameter ratio, as well as Froude and Morton numbers, was obtained.     

黄河中游绿水系数变化及其生态环境意义

绿水和蓝水的概念和理论对于半干旱、半湿润区水资源的评价和管理有重要意义。引入流域尺度上绿水系数指标,定义为某一流域内由降水到绿水的转换系数。研究发现,1950—2011年间,黄河中游河口镇至龙门区间(河龙区间)的绿水系数呈现增大的趋势。除了气温、降水的影响外,水土保持是一个重要因素。水土保持措施减少了降雨到径流(蓝水)的转化率,增大了降雨到绿水的转化率。绿水系数的增大意味着坡面径流减弱和河流径流的减弱,从而减少了坡面侵蚀和流域产沙。同时,绿水系数的增大意味着植被蒸腾作用的增强,说明植被对地表的保护作用增强,这也会导致坡面侵蚀的减弱。河龙区间产沙量与流域绿水系数之间呈显著的负相关关系,产沙量变化的53.7%可以用流域绿水系数的变化来解释。水土保持实施后,梯田和坝地使得生产性绿水系数增大,从而增加了粮食产量。梯田、坝地面积与绿水系数和粮食产量之间都存在着显著的正相关关系,粮食产量与绿水系数之间也存在显著的正相关关系。绿水系数具有流域生态环境质量变化的指示意义,在年降水可比的情况下,流域绿水系数的减小意味着集水区生态环境环境质量降低,流域绿水系数的增大意味着集水区生态环境质量提高。依照绿水系数的变化,可以将近60余年来河龙区间生态环境的变化过程划分为3个阶段。     

Performance and spatial community succession of an anaerobic baffled reactor treating acetone-butanol-ethanol fermentation wastewater

An anaerobic baffled reactor with four compartments (C1-C4) was successfully used for treatment of acetone-butanol-ethanol fermentation wastewater and methane production. The chemical oxygen demand (COD) removal efficiency was 88.2% with a CH₄ yield of 0.25 L/(g COD_removed) when organic loading rate (OLR) was 5.4 kg COD m⁻³ d⁻¹. C1 played the most important role in solvents (acetone, butanol and ethanol) and COD removal. Community structure of C2 was similar to that in C1 at stage 3 with higher OLR, but was similar to those in C3 and C4 at stages 1-2 with lower OLR. This community variation in C2 was consistent with its increased role in COD and solvent removal at stage 3. During community succession from C1 to C4 at stage 3, abundance of Firmicutes (especially OTUs ABRB07 and ABRB10) and Methanoculleus decreased, while Bacteroidetes and Methanocorpusculum became dominant. Thus, ABRB07 coupled with Methanoculleus and/or acetogen (ABRB10) may be key species for solvents degradation.     

Molecular simulation of Tyr105 phosphorylated pyruvate kinase M2 to understand its structure and dynamics

Microstructure of dibenzo-18-crown-6 (DB18C6) and DB18C6/Li⁺ complex in different solvents (water, methanol, chloroform, and nitrobenzene) have been analyzed using radial distribution function (RDF), coordination number (CN), and orientation profiles, in order to identify the role of solvents on complexation of DB18C6 with Li⁺, using molecular dynamics (MD) simulations. In contrast to aqueous solution of LiCl, no clear solvation pattern is found around Li⁺ in the presence of DB18C6. The effect of DB18C6 has been visualized in terms of reduction in peak height and shift in peak positions of g_Li-Ow. The appearance of damped oscillations in velocity autocorrelation function (VACF) of complexed Li⁺ described the high frequency motion to a “rattling” of the ion in the cage of DB18C6. The solvent-complex interaction is found to be higher for water and methanol due to hydrogen bond (HB) interactions with DB18C6. However, the stability of DB18C6/Li⁺ complex is found to be almost similar for each solvent due to weak complex-solvent interactions. Further, Li⁺ complex of DB18C6 at the liquid/liquid interface of two immiscible solvents confirm the high interfacial activity of DB18C6 and DB18C6/Li⁺ complex. The complexed Li⁺ shows higher affinity for water than organic solvents; still they remain at the interface rather than migrating toward water due to higher surface tension of water as compared to organic solvents. These simulation results shed light on the role of counter-ions and spatial orientation of species in pure and hybrid solvents in the complexation of DB18C6 with Li⁺. Graphical Abstract

DB18C6/Li⁺ complex in pure solvents (water, methanol, chloroform, and nitrobenzene) and water/nitrobenzene interface     

小兴安岭典型苔草和灌木沼泽N2O排放及影响因子

为了研究小兴安岭林区典型苔草和灌木沼泽N2O排放通量的季节动态、年际动态及其与环境因子的关系,并估算排放总量,2007和2008年在植物生长季采用静态箱-气相色谱法,对小兴安岭林区典型修氏苔草（Carex schmidtii）沼泽和油桦-修氏苔草（Betula ovalifolia-Carex schmidtii）灌木沼泽N2O排放进行了监测。结果表明,苔草和灌木沼泽2007年生长季N2O排放总量分别为0.14和0.29 kg/hm2;2008年分别为0.68和-0.10 kg/hm2。苔草和灌木沼泽N2O排放通量除灌木沼泽2008年变化规律性不明显外,均具有比较显著的季节变化,最大排放出现在夏季或夏、秋季节,其中2007年N2O排放平均通量为0.0037和0.0082 mg?m-2?h-1;2008年为0.016和-0.0025 mg?m-2?h-1。分析表明,苔草沼泽N2O排放年际差异不显著,灌木沼泽N2O排放年际差异显著;不同类型沼泽间N2O排放差异不显著;仅苔草沼泽2007年N2O排放通量与水位具有显著的负相关性（r=-0.52,P < 0.05,n=15）。     

Biotransformation in Double-Phase Systems: Physiological Responses of Pseudomonas putida DOT-T1E to a Double Phase Made of Aliphatic Alcohols and Biosynthesis of Substituted Catechols

Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP_ow (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of ≥2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested P. putida DOT-T1E tolerance to different aliphatic alcohols with a logP_ow value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP_ow around 1.5 are produced. P. putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow P. putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP_ow of aliphatic alcohols correlates with their toxic effects, as octanol (logP_ow = 2.9) has more negative effects in P. putida cells than 1-nonanol (logP_ow = 3.4) or 1-decanol (logP_ow = 4). A P. putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP_ow = 3.2) into 3-methylcatechol (logP_ow = 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.     

Model for the production of L-tryptophan from L-serine and indole by immobilized cells in a three-phase liquid-impelled loop reactor

Production of L-tryptophan from L-serine and indole catalyzed by Escherichia coli, immobilized in k-carrageenan gel beads, is technically feasible in the liquidimpelled loop reactor (LLR), using an organic solvent, e.g. n-dodecane.With L-serine in large excess intrinsic reaction kinetics is approximately first order with respect to indole, with a reaction constant of 8.5×10^–5 m³ kg _dw ^–1 s^–1.The overall process kinetics is jointly controlled by intrinsic kinetics and by intraparticle mass transfer resistance, which can be quantified using an effectiveness factor.Mass transfer of indole from the organic to the aqueous phase and from the aqueous to the gel phase are relatively fast and thus have negligible influence in the overall process kinetics, under the operational conditions tested. However, they may become important if the process is intensified by increasing the cell concentration in the gel and/or the gel hold-up in the reactor.A simple model which includes indole mass balances over the aqueous and organic phases, mass transfer and reaction kinetics, with parameters experimentally determined in independent experiments, was successful in simulating L-tryptophan production in the LLR.List of Symbols a, b, c coefficients of the equilibrium curve for indole between organic and aqueous phases - A, B, C, D, E, F auxiliary variables used in liquid-liquid mass transfer studies - a _x specific interfacial area referred to the volume of the aqueous phase (m^–1) - A _x interfacial area (m²) - a _Y specific interfacial area referred to the volume of the organic phase (m^–1) - A _Y interfacial area (m²) - C _b substrate concentration in the bulk of the aqueous phase (kg m^–3) - C _e substrate concentration in exit stream (kg m^–3) - C _E biocatalyst concentration referred to the aqueous phase (kg m^–3) - C _{E s} biocatalyst concentration referred to the volume of gel (kg m^–3) - C _s substrate concentration at the gel surface (kgm^–3) - d, e, f coefficients of the equilibrium curve for indole between aqueous and organic phases - dp particle diameter (m) - K ₂ kinetic constant (s^–1) - K ₁ kinetic constant K₂/K_M (kg^–1 m³ s^–1) - K _M Michaälis-Menten constant (kgm^–3) - K _X mass transfer coefficient referred to the aqueous phase (ms^–1) - K _Xa_X volumetric mass transfer coefficient based on the volume of the aqueous phase (s^–1) - k _Y mass transfer coefficient referred to the organic phase (ms^–1) - K _Ya_Y volumetric mass transfer coefficient based on the volume of the organic phase (s^–1) - N _X mass flux of indole from organic to aqueous Phase (kg m^–2s^–1) - N _Y mass flux of indole from aqueous to organic phase (kg m^–2s^–1) - Q _e volumetric flow rate in exit stream (m³s^–1) - Q _f volumetric flow rate in feed stream (m³s^–1) - _obs observed reaction rate (kg s^–1 m^–3) - intrinsic reaction rate (kg s^–1 m^–3) - Re Reynolds number - Sc Schmidt number - Sh Sherwood number - t time (s) - u superficial velocity (m s^–1) - V _max maximum reaction rate (kg s^–1m^–3) - V _S volume of the support (m³) - V _X volume of aqueous phase (m³) - V _Y volume of the organic phase (m³) - X indole concentration in the aqueous phase (kgm^–3) - Y indole concentration in the organic phase (kg m^–3 Greek Letters overall effectiveness factor - _e external effectiveness factor - _i internal effectiveness factor - Thiele module A fellowship awarded to one of us (D.M.R.)by INICT is gratefuly acknowledged.     

Kinetics of the complexation of nickel(II) with 1,10-phenanthroline and its derivatives in acetonitrile-water mixed solvents

The kinetics of the complexation of Ni(II) with 1,10-phenanthroline(phen), 4,7-dimethyl-1,10-phenanthroline(dmphen), and 5-nitro-1,10-phenanthroline(NO₂phen) in acetonitrile-water mixed solvents of acetonitrile mole fraction x_AN = 0, 0.05, 0.1, 0.2 and 0.3 at 288, 293, 298 and 303 K have been studied by stopped-flow method at ionic strength of 1.0 (NaClO₄) and pH 7.4. The corresponding activation enthalpy, entropy, and free energy were determined from the observed rate constants. The complexation of Ni(II) with the three ligands has comparable observed rate constants; in pure water the observed rate constants are (×10³ dm³ mol⁻¹ s⁻¹) 2.31, 2.57, and 1.38 for phen, dmphen and NO₂phen, respectively. The corresponding activation parameters for the three ligands are, however, considerably different; in pure water the ΔH^‡/ΔS^‡ (kJ mol⁻¹/J K⁻¹ mol⁻¹) are 44.7/−30.2, 19.5/−114.1, and 32.2/−76.9 for phen, dmphen, and NO₂phen, respectively. The effects of solvent composition on the kinetics are also markedly different for the three ligands. The ΔH^‡ and ΔS^‡ showed a minimum at x_AN = 0.1 for phen; for dmphen and NO₂phen, however, maxima at x_AN = 0.2 were observed. Nevertheless, there is an effective enthalpy-entropy compensation for the ΔH^‡/ΔS^‡ of all the three ligands, demonstrating the significant effects of the changes in solvation and solvent structure on the complexation kinetics. As the rate-determining step of Ni(II) complexation is the dissociation of a water molecule from Ni(II), the solvent and ligand dependencies in the Ni(II) complexation kinetics are ascribed to the change in solvation status of the ligands and the altered solvent structures upon changing solvent composition.     

Stripping of nonionic surfactants from the coacervate phase of cloud point system for lipase separation by Winsor II microemulsion extraction with the direct addition of alcohols

Extractive microbial fermentation of lipase by Serratia marcescens ECU1010 in cloud point system was previously carried out in the cloud point system. The direct addition of different alcohols, including iso-butanol, 2-phenylethanol and 1-octanol, into the coacervate phase of the clear supernatant of the fermentation broth formed microemulsion, where the nonionic surfactants and lipase were unevenly partitioned between the different phases in the microemulsion system. The polarity of alcohols strongly affected the microemulsion type at room temperature condition. The results indicated that the Winsor II microemulsion, formed by the addition of iso-butanol or 2-phenylethanol as the organic solvent, favored the stripping of the nonionic surfactant into the O_m phase, whereas the lipase was left in the excess aqueous phase. However, the Winsor I microemulsion, formed by the addition of 1-octanol as the organic solvent, failed to separate the lipase from the nonionic surfactant in the coacervate phase of cloud point system, because the nonionic surfactant and lipase were partitioned into the W_m phase at the same time. Moreover, in the Winsor II microemulsion extraction with 2-phenylethanol as the organic solvent, in which case the protein–surfactant complexes were absent at the interface between the O_m phase and the excess aqueous phase, the high lipase recovery (above 80%) and good nonionic surfactant removal were achieved. The effect of nonionic surfactants on lipase activity was also presented.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.     

Measurement of Biologically Available Naphthalene in Gas and Aqueous Phases by Use of a Pseudomonas putida Biosensor

Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples. Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations. However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase. Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase. For this purpose, we reconstructed a bioluminescent Pseudomonas putida naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the sal promoter and the luxAB genes. Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase. Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively. The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase. In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase. This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 μM). Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices.