Process engineering aspects of the bioleaching of copper ores

The bioleaching of minerals is a complex process that is affected by a number of biological, mineralogical, electrochemical and engineering factors. This work presents and discusses the most significant process engineering aspects involved in the bacterial leaching of copper ores, i.e. bacterial population, type of mineral and particle size, nutrients and inhibitors, oxygen and carbon dioxide, temperature and pH, leaching kinetics and operation mode.It is concluded that more work is needed in this area in order to gain a deeper insight in the many factors that govern this process. This would allow to significantly improve its overall productivity.List of Symbols C _L kg/m³ dissolved oxygen concentration - C ^* kg/m³ equilibrium oxygen concentration - d, e, f, g % percentage of C, H, O and N in the cell - D m impeller diameter - K consistency index - K _S, K₁, K_c constants - k _La h^–1 volumetric oxygen transfer coefficient - M _b mol/kg biomass apparent molecular weight - N s^–1 rotation frequency - n behavior index - P kg/m³ ungassed agitation power, product concentration - P _g kW/m³ gassed agitation power - p % pulp density - Q m³/h air flow rate - S kg/m³ limiting substrate concentration - W kg/(m³ · h) mass transfer rate per unit volume - X cells/cm³ biomass concentration - Y _o g cells/g Fe oxygen cell yield - Y _x g cells/g Fe substrate cell yield - h^–1 specific growth rate - _m h^–1 maximum specific growth rate     

Optimisation of agitation and aeration in fermenters

The problem of optimising agitation and aeration in a given fermenter is addressed. The objective function is total electric power consumed for agitation, compression and refrigeration. The major constraint considered is to ensure that the dissolved oxygen concentration is above the critical value. It is shown that it is possible to analytically calculate the optimal pair (air flowrate, stirrer speed) and that, at least for the industrial antibiotics fermentation used as case-study, the optimum lies within a window for satisfactory operation, limited by other possible constraints to the problem. Savings achievable by optimal operation as compared with current industrial procedure were found to be around 10% at pilot plant scale (0.26 m³) and 20% at full scale (85 m³).List of Symbols A fermenter cross sectional area (m²) - C dissolved oxygen concentration (mole m^–3) - C ^* DO concentration in equilibrium with the gas (mole m^–3) - C _crit critical DO concentration (mole m^–3) - C _p specific heat of air at constant pressure (J kg^–1 K^–1) - C _sp dissolved oxygen set point (mole m^–3) - C _v specific heat of air at constant volume (J kg^–1 K^–1) - D agitator diameter (m) - f pressure correction of air flow-rate - (Fl _g)_F aeration number at flooding - (Fr _g)_F froude number at flooding - k coefficient in expression for mass transfer coefficient - K _La volumetric oxygen transfer coefficient (s^–1) - m power exponent in expression for mass transfer coefficient - n gas flow rate exponent in expression for mass transfer coefficient - n ^* number of impellers - N rotation speed (s^–1) - N _F rotation speed at flooding (s^–1) - N _p unaerated power number - N _pg aerated power number - OUR Oxygen Uptake Rate (mole m^–3 s^–1) - p ₀ atmospheric pressure (N m^–2) - p ₁ compressor exit pressure (N m^–2) - p ₂ pressure at the bottom of the fermenter (N m^–2) - p ₃ pressure at the top of the fermenter (N m^–2) - P _c compression power (W) - P _d power added by expansion (W) - P _ev power removed by evaporation (W) - P _g agitation power (W) - P _m power added by metabolism (W) - P _r power removed by refrigeration (W) - P _t total power (W) - Q air flow-rate at atmospheric conditions (m³ s^–1) - Q _f air flow-rate at average fermenter conditions (m³ s^–1) - s ₀ absolute humidity at atmospheric conditions - s ₃ absolute humidity at fermenter exit - T tank diameter (m) - V liquid volume (m³) - v _s gas superficial velocity (m s^–1) - _i parameter defined in the text - safety margin for dissolved oxygen (mole m^–3) - ratio of specific heats of air - _g agitation efficiency - _c compression efficiency - _r refrigeration efficiency - liquid density (kg m^–3) - _g air density (kg m^–3) - latent heat of vaporisation of water (J kg^–1) The authors are grateful to Elsa Silva, Carlos Lopes, Carlos Aguiar, Fernando Mendes, and Alexandre Cardoso, who helped with parts of this work, and to CIPAN for permission to publish these data.     

Interactions of cell morphology and transport processes in the lovastatin fermentation

The cholesterol lowering drug, Lovastatin (Mevacor), acts as an inhibitor of HMGCoA reductase, and is produced from an Aspergillus terreus fermentation.Pilot scale studies were carried out in 800 liter fermenters to determine the effects of cell morphology on the oxygen transport properties of this fermentation. Specifically, parallel fermentations giving (i) filamentous mycelial cells, and (ii) discrete mycelial pellets, were quantitatively characterized in terms of broth viscosity, availability of dissolved oxygen, oxygen uptake rates and the oxygen transfer coefficient under identical operating conditions.The growth phase of the fermentation, was operated using a cascade control strategy which automatically changed the agitation speed with the goal of maintaining dissolved oxygen at 50% saturation. Subsequently stepwise changes were made in agitation speed and aeration rate to evaluate the response of the mass transfer parameters (DO, OUR, and k _L a). The results of these experiments indicate considerable potential advantages to the pellet morphology from the standpoint of oxygen transport processes.List of Symbols DO % sat. Dissolved oxygen concentration - k _L a h^–1 Gas-liquid mass transfer coefficient - OUR mmol/dm³h Oxygen uptake rate - P/V KW/m³ Agitator power per unit volume - V _s m/s Superficial air velocity - _app cP Apparent viscosity     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of light during growth on photoinhibition and recovery

Photoinhibition of photosynthesis was induced in intact kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson) leaves grown at two photon flux densities (PFDs) of 700 and 1300 mol·m^-2·s^-1 in a controlled environment, by exposing the leaves to PFD between 1000 and 2000 mol·m^-2·s^-1 at temperatures between 10 and 25°C; recovery from photoinhibition was followed at the same range of temperatures and at a PFD between 0 and 500 mol·m^-2·s^-1. In either case the time-courses of photoinhibition and recovery were followed by measuring chlorophyll fluorescence at 692 nm and 77K and by measuring the photon yield of photosynthetic O₂ evolution. The initial rate of photoinhibition was lower in the high-light-grown plants but the long-term extent of photoinhibition was not different from that in low-light-grown plants. The rate constants for recovery after photoinhibition for the plants grown at 700 and 1300 mol·m^-2·s^-1 or for those grown in shade were similar, indicating that differences between sun and shade leaves in their susceptibility to photoinhibition could not be accounted for by differences in capacity for recovery during photoinhibition. Recovery following photoinhibition was increasingly suppressed by an increasing PFD above 20 mol·m^-2·s^-1, indicating that recovery in photoinhibitory conditions would, in any case, be very slow. Differences in photosynthetic capacity and in the capacity for dissipation of non-radiative energy seemed more likely to contribute to differences in susceptibility to photoinhibition between sun and shade leaves of kiwifruit.Abbreviations and symbols F _o , F _m , F _v instantaneous, maximum, variable fluorescence - F _v /F _m fluorescence ratio - F _i =F _v at t=0 - F F _v at t= - K _D rate constant for photochemistry - k(F _p ) first-order rate constant for photoinhibition - k(F _r ) first-order rate constant for recovery - PFD photon flux density - PSII photosystem II - _i photon yield of O₂ evolution (incident light)     

High misses after odd flashes in oxygen evolution in thoroughly dark-adapted thylakoids from pea and Chenopodium album

In Photosystem II (PS II), water is oxidized to molecular oxygen and plastoquinone is reduced to plastoquinol. The oxidation of water requires the accumulation of four oxidizing equivalents, through the so-called S-states of the oxygen evolving complex; the production of plastoquinol requires the accumulation of two reducing equivalents on a bound plastoquinone, Q_B. It has been generally believed that during the flash-induced transition of each of the S-states (S_n S_n+1, where n=0, 1, 2 and 3), a certain small but equal fraction of the PS II reaction centers are unable to function and, thus, miss being turned over. We used thoroughly dark-adapted thylakoids from peas (Pisum sativum) and Chenopodium album (susceptible and resistant to atrazine) starting with 100% of the oxygen evolving complex in the S₁ state. Thylakoids were illuminated with saturating flashes, providing a double hit parameter of about 0.07. Our experimental data on flashnumber dependent oscillations in the amount of oxygen per flash fit very well with a binary pattern of misses: 0, 0.2, 0, 0.4 during S₀ S₁, S₁ S₂, S₂ S₃ and S₃ S₀ transitions. Addition of 2 mM ferricyanide appears to shift this pattern by one flash. These results are consistent with the bicycle model recently proposed by V. P. Shinkarev and C. A. Wraight (Oxygen evolution in photosynthesis: From unicycle to bicycle, 1993, Proc Natl Acad Sci USA 90: 1834–1838), where misses are due to the presence of P⁺ or Q_A ^- among the various equilibrium states of PS II centers.Abbreviations miss parameter - double hit parameter - PS II Photosystem II - Q_A primary one-electron acceptor of PS II, a plastoquinone molecule - Q_B secondary plastoquinone two-electron acceptor of PS II - S-states (S_n, where n=0, 1, 2, 3 or 4) redox states of the oxygen evolving complex     

Averaging methods in the delayed-logistic equation

The delayed logistic equation is analyzed using the averaging method. Using the transformation of coordinates v=ln N/K it is shown that the first order term in perturbation theory yields N=K exp(r ^* cos t/2) when the delay time T exceeds some critical value T _c. The amplitude r^* is equal to (40/3 – 2)^1/2 and is an expansion parameter that is proportional to (T – T_c). Comparison of the exponential solution of N and numerical results for the ratio N _maximum/N _minimum provides a good fit for values of larger than the results using the N coordinate as the perturbed coordinate.     

Improved scale-up strategies of bioreactors

Effective scale-up is essential for successful bioprocessing. While it is desirable to keep as many operating parameters constant as possible during the scale-up, the number of constant parameters realizable is limited by the degrees of freedom in designing the large-scale operation. Scale-up of aerobic fermentations is often carried out on the basis of a constant oxygen transfer coefficient, k _L a, to ensure the same oxygen supply rate to support normal growth and metabolism of the desired high cell populations. In this paper, it is proposed to replace the scale-up criterion of constant k _L by a more direct and meaningful criterion of equal oxygen transfer rate at a predetermined value of dissolved oxygen concentration. This can be achieved by using different oxygen partial pressures in the influent gas streams for different scales of operation. One more degree of freedom, i.e., gas-phase oxygen partial pressure, is thus added to the process of scale-up. Accordingly, one more operating factor can be maintained constant during scale-up. It can be used to regulate the power consumption in large-scale fermentors for economical considerations or to describe the fluid mixing more precisely. Examples are given to show that the results of optimization achieved in the bench-scale study can be translated to the production-scale fermentor more successfully with only a small change in the gas-phase oxygen partial pressure employed in the bench-scale operation.List of Symbols a m²/m³ Specific gas/liquid interfacial area - C _L mole/m³ Dissolved oxygen concentration in bulk liquid phase - C ^* mole/m³ Equilibrium oxygen concentration at gas/liquid interface - D _i m Impeller diameter - D _T m Bioreactor diameter - H _L mole/m³ · atm Henry's-law constant - k _L m/s Liquid-phase mass transfer coefficient - N 1/s Impeller agitation speed - N _i Number of impellers - OTR mole/s · m³ Oxygen transfer rate per unit volume of the medium - P _g kW Power input in aerated fermentation - P _o kW Power input in non-gassed fermentation - p _g atm Gas-phase oxygen partial pressure - Q m³/s Volumetric gas flow rate - Re _i Impeller Reynolds number - T _Q Joule Torque applied to the mixer shaft - V m³ Liquid volume - v _s m/s Superficial gas velocity - kg/m · s Liquid viscosity - kg/m³ Liquid density     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Phosphate uptake inLemna gibba G1: energetics and kinetics

Phosphate uptake was studied by determining [³²P]phosphate influx and by measurements of the electrical membrane potential in duckweed (Lemna gibba L.). Phosphate-induced membrane depolarization (E _m ) was controlled by the intracellular phosphate content, thus maximal E _m by 1 mM H₂PO ₄ ^- was up to 133 mV after 15d of phosphate starvation. The E _m was strongly dependent on the extracellular pH, with a sharp optimum at pH 5.7. It is suggested that phosphate uptake is energized by the electrochemical proton gradient, proceeding by a 2H⁺/H₂PO ₄ ^- contransport mechanism. This is supported also by the fusicoccin stimulation of phosphate influx. Kinetics of phosphate influx and of E _m , which represent mere plasmalemma transport, are best described by two Michaelis-Menten terms without any linear components.Abbreviations E _m electrical membrane potential difference - E _m phosphate-induced, maximal membrane depolarization - FW fresh weight     

The enzymatic system thiosulfate: Cytochrome c oxidoreductase from photolithoautotrophically grown Chromatium vinosum

Chromatium vinosum cells form a vesicular type intracytoplasmic membrane system during phototrophic growth on thiosulfate.—An enzyme protein transferring electrons from thiosulfate to cytochromes of type c was enriched from S-144. The colorless thiosulfate: cytochrome c oxidoreductase was characterized by a molecular weight of 36,000 (after dodecylsulfate treatment) and 35,000 (by gel filtration). Isoelectric focusing revealed a pI range of 4.4 to 4.7. Apparent K _m values for the cytochromes tested were in the M range. — The endogenous electron acceptor compound, isolated from the chromatophore fraction P-144, was found to be a membrane-bound cytochrome c-552. The homogeneous cytochrome protein had an average pI value of 4.65 and a molecular weight of 71,500 determined by gel filtration. By dodecylsulfate electrophoresis it was cleaved into two proteins representing particle weights of 45,000 and 20,000.Abbreviations HiPIP high potential nonheme iron protein - IEF isoelectric focusing - SDS dodecylsulfate, sodium salt - Temed N,N,N,N-tetramethylethylenediamine     

Characteristics of Chlorophyll Fluorescence and Membrane-Lipid Peroxidation During Senescence of Flag Leaf in Different Cultivars of Rice

With japonica rice 98-08, indica hybrids Shanyou 63, Gangyou 881, and X07S/Zihui 100, and sub-species hybrid Peiai 64S/9311 as materials, chlorophyll (Chl) content, Chl a fluorescence parameters, and membrane lipid peroxidation in flag leaf were measured at late developmental stages under natural conditions. F_v/F_m, q_P, _PS2, and electron transport rate gradually decreased while q_N increased conversely. Excessive photon energy led to the accumulation of active oxygen (O₂ ^–), H₂O, malonyldialdehyde, and products of membrane lipid peroxidation, and resulted in reduced Chl content and early ageing subsequent to the photooxidation during flag leaf senescence. There was obvious diversification of these parameters among rice cultivars. In comparison with japonica cv. 98-08 (tolerant to photooxidation), F_v/F_m decreased in indica cv. Shanyou 63 (susceptible to photooxidation) with greater accumulation of active oxygen and a sharp drop in Chl content, which resulted in yellowish early ageing, and affected the filling and setting of rice grains. The mechanism for premature ageing in indica rice was related to irradiance and temperature at filling stages. On a sunny day at above 25 °C, the reaction centre of photosystem 2 (PS2) exhibited a dynamic change on reversible inactivation. Under the intense irradiance at noon, PS2 function in indica rice exhibited obvious down-regulation and photoinhibition. Under intense irradiance with lowered temperatures, PS2 resulted in photo-damage and early ageing, related to the degradation of PS2-D1 protein and the inhibition of endogenous protection systems such as the xanthophyll cycle and enzymes scavenging active oxygen. Hence for high-yield breeding, based on a good plant-type and utilising heterosis and tolerance of photooxidation, the selection of japonica rice or a sterile line with the japonica genotype as female is a strategy worthy of consideration.     

Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions

Summary The effect of soybean oil on the volumetric oxygen transfer coefficient during the cultivation ofAerobacter aerogenes cells is presented. For our aeration-agitation conditions (0.278 vvm and 500 rpm), it has been demonstrated that the use 19% (v/v) of soybean oil enabled a 1.85-fold increase of thek _l a coefficient (calculated on a per liter aqueous phase basis). For smaller volumetric oil fractions,k _L a increased linearly with the oil loading. Because of the oxygen-vector properties of soybean oil, this oil is able to significantly increase thek _L a of a bioreactor.Nomenclature C^*, C saturation and actual dissolved oxygen concentrations respectively (g/m³) - K_La volumetric oxygen transfer coefficient (h^–1) - K_La_initial k _La measured before the oil addition (h^–1) - M_O2 molar mass of oxygen (dalton) - N oxygen transfer rate (g/m³. h) - P_O2. P_N2 partial pressures ofO ₂ andN ₂ in the gas (atm) - P_H2OT partial pressure of water in air at the temperatureT (atm) - P_T total pressure (atm) - Q₀ volumetric flow rate of outlet air before seeding (m³/h) - Sp spreading coefficient (dynes/cm) - T absolute temperature of outlet gas (K) - V_i volume of the liquidi in the fermentor (m³) - V_M molar volume at 273 K and 1 atm (m³/mole) - _ij interfacial tension betweeni andj componants (dynes/cm) - _v volumetric fraction of the oil (v/v) - G gas - O oil - W water - i inlet - o outlet     

Correlation of liquid dispersion and oxygen transfer in bubble column

Summary In steady state, attained by continuous aeration after oxygen saturation of water in a bubble column, vertical composition distribution of liquid and gas phases has been determined. It has been assumed that, as a result of absorption at the bottom of the column, desorption in the upper section and vertical dispersion of dissolved oxygen flux, a closed oxygen circulation is created. Determination of the axial dispersion coefficient from hydrodynamic and oxygen transfer data verifies the mathematical model proposed. The results allow conclusions to be drawn about supersaturation and desorption and other phenomena expected in biological systems.Abbreviations C[-] Dimensionless oxygen concentration Unit=0.21 bar oxygen partial pressure or dissolved oxygen level in equilibrium with latter - E[m²/s] Axial dispersion coefficient - F[m²] Horizontal cross-section area - k _L a[s^-1] Overall oxygen transfer coefficient - u; u ₂[m/s; cm/s] Superficial velocity: related to state of bubbles leaving the sparger - x; x _atm[-] Signal registered in the experiment; signal recorded in O₂ saturated water, or water vapor saturated air stream, at temperature identical to the experiment under atmospheric pressure - y[m] Water column height - [s^-1] Dimensionless oxygen flux Indices a asorption - d desorption - g gas - l liquid - k dispersion - m measured value/in the case of hydrodynamically measured E/ Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Studies on transfer processes in mixing vessels: suspending of solid particles in liquid by modified Rushton turbine agitators

The modified blade turbines are attractive alternatives to the standard Rushton turbine as they do not require any modification in the electrical engine motor and drive assemblies are simple to manufacture and have a reduced power consumption.The modified blades were obtained through increase in the blade height of the Rushton turbine simultaneously with perforation of the blade surface. The field surface of the modified blade is equal to the blade surface of the standard Rushton turbine.In this study the modified blade turbine with the surface fraction of the perforations equal to 0.353 is used.The complete suspension speed and the power dissipation in transition and turbulent regimes using standard and modified Rushton turbine agitators positioned singly or doubly on same shaft, in five solid-liquid systems were investigated.The solid particles used have the mean diameter between 15–1000 m.The modified blade turbine, noted as TP3, was found to be more efficient than the standard turbine in complete and homogeneous suspension.List of Symbols A distance between turbine and the vessel bottom (m) - c dimensionless constant (-) - d agitator diameter (m) - d _p surface-to-volume mean diameter of the particle (m) - D vessel diameter (m) - (H _L )₁ suspension height for one turbine immersed (m) - (H _L )₂ suspension height for two turbines immersed (m) - K consistency index (Pa s ⁿ ) - l _k eddy-size characteristic (m) - N flow behaviour index (-) - N _p number of blades of the mixing system (-) - N agitator speed (s^–1) - N _js agitator speed that just causes complete suspension (s^–1) - Ne P_L/_LN³d⁵ power number in liquid system (-) - (Ne) _g P_g/_spN³d⁵ power number in solid-liquid system (-) - P _L power consumption in liquid system (W) - P _s power consumption in solid-liquid system (W) - r coefficient of correlation (-) - R distance between turbines (m) - Re _spNd²/ _a Reynolds number (-) - S suspension parameter in Zwietering equation (2) (-) - S _C full surface of the blade (m²) - S _G surface of the perforations applied on the blade (m²) - S _G /S _C surface fraction of the perforations (-) - X particle concentration (g/l) - w baffle width (m) - _js specific power input per mass at the complete suspension state (W/kg) - _a apparent viscosity under mixing conditions (Pa s) - _L kinematic viscosity of the liquid (m²/s) - _L density of liquid (Kg/m³) - _s density of solid (Kg/m³) - _sp density of suspension (Kg/m³)     

Effects of dissolved oxygen concentration on lipase production by Rhizopus delemar

Summary The influence of the concentration of oxygen on lipase production by the fungus Rhizopus delemar was studied in different fermenters. The effect of oxygen limitation ( 47 mol/l) on lipase production by R. delemar is large as could be demonstrated in pellet and filamentous cultures. A model is proposed to describe the extent of oxygen limitation in pellet cultures. Model estimates indicate that oxygen is the limiting substrate in shake flask cultures and that an optimal inoculum size for oxygen-dependent processes can occur.Low oxygen concentrations greatly negatively affect the metabolism of R. delemar, which could be shown by cultivation in continuous cultures in filamentous growth form (D_optimal=0.086 h^-1). Continuous cultivations of R. delemar at constant, low-oxygen concentrations are a useful tool to scale down fermentation processes in cases where a transient or local oxygen limitation occurs.Symbols and Abbreviations CO Oxygen concentration in the gas phase at time = 0 (kg·m^-3) - C_{O 2i} Oxygen concentration at the pellet liquid interface (kg·m^-3) - C_{O 2i} Oxygen concentration in the bulk (kg·m^-3) - D Dilution rate (h^-1) - ID_{O 2} Diffusion coefficient for oxygen (m²·s^-1) - dw Dry weight of biomass (kg) - f Conversion factor (rs _{O 2} to oxygen consumption rate per m³) (-) - k Radial growth rate (m·s^-1) - K Constant - kla Volumetric mass transfer coefficient (s^-1) - klA Oxygen transfer rate (m^-3·s^-1) - kl Mass transfer coefficient (m·s^-1) - K _{O 2} Affinity constant for oxygen (mol·m^-3) - K _w Cotton plug resistance (m^-3·s^-1) - M Henry coefficient (-) - NV Number of pellets per volume (m^-3) - R Radius (m) - RO Radius of oxygen-deficient core (m) - RQ Respiration quotient (mol CO₂/mol O₂) - rs _{O 2} Specific oxygen consumption rate per dry weight biomass (kg O₂·s^-1[kg dw]^-1) - rX Biomass production rate (kg·m^-3·s^-1) - SG Soytone glucose medium (for shake flask experiments) - SG ₄ Soytone glucose medium (for tower fermenter and continuous culture experiments) - V Volume of medium (m^-3) - X Biomass (dry weight) concentration (kg·m^-3) - XR _o Biomass concentration within RO for a given X (kg·m^-3) - Y _{O 2} Biomass yield calculated on oxygen (kg dw/kg O₂) - Thiele modulus - Efficiency factor =1-(RO/R)³ (-) - Growth rate (m^-1·s^-1·kg^1/3) - Dry weight per volume of pellet (kg·m^-3)     

Reactor comparison and scale-up for the microaerobic production of 2,3-butanediol by Enterobacter aerogenes at constant oxygen transfer rate

Stirred tank (STR), bubble column (BCR) and airlift (ALR) bioreactors of 0.05 and 1.5 m³ total volume were compared for the production of 2,3-butanediol using Enterobacter aerogenes under microaerobic conditions. Batch fermentations were carried out at constant oxygen transfer rate (OTR=35 mmol/lh). At 0.05 m³ scale, the STR reactor achieved much higher biomass and product concentrations than the BCR and ALR reactors. At 1.5 m³ scale, however, exactly the same biomass and product concentrations could be obtained in both STR and ALR reactors. The 1.5 m³ ALR reactor performed also much better than its counterpart at small scale, achieving a productivity 2.4-fold as high as that of the 0.05 m³ BCL and ALR reactors. No differences in performances were observed between BCR and ALR. As compared to STR the tower reactors have a 12 time higher energetic efficiency (referred to product formation) and thus should be the choice for large scale production of 2,3-butanediol.The criterion of constant OTR or constant k _L a is not applicable for the scale-up of this oxygen-sensitive culture due to strong influence of reactor hydrodynamics under microaerobic conditions. The effects of mixing and circulation time on growth and metabolism of E. aerogenes were quantitatively studied in scaled-down experiments with continuous culture. For a successful scale-up of this microaerobic culture it is necessary to have an homogeneous oxygen supply over the entire reactor volume. Under conditions of inhomogeneous oxygen supply an optimum liquid circulation time exists which gives a maximum production of 2,3-butanediol.List of Symbols BD 2,3-butanediol - [mmol/l] saturation value of dissolved oxygen - D [h^–1] dilution rate - D [mm] reactor diameter - D _K [mm] top section diameter - D _R [mm] stirrer diameter - D _S [mm] draft tube diameter - EtOH ethanol - E _P [kg/kWh] energy efficiency refered to product formation - H [mm] height of reactor - HAc acetate - H _L [mm] height of liquid - k _L a [h^–1] volumetric oxygen transfer coefficient - N [rpm=min^–1] stirrer speed - OTR [mmol/lh] oxygen transfer rate - OUR [mmol/lh] oxygen uptake rate - p [Pa] pressure - P [kW] power input - P/V _L [kW/m³] specific power input - [mmHg] oxygen partial pressure (mmHg) or - [mmol/l] dissolved oxygen (mmol/l) - [mmol/gh] specific oxygen uptake rate - q _P [mmol/gh] specific productivity - R [Nm/kgK] gas constant, R = 287.06 - RQ respiration quotient - t _c [s] liquid circulation time - T [°C or K] temperature - TCA tricarboxylic acid - u _G [cm/s] mean superficial gas velocity - v _G [m/s] gas velocity at nozzels of gas distributor - V_G [l/h] aeration rate at inlet - V [m³ or l] total volume - V _L [m³ or l] liquid volume - V _N [l/mol] gas mole volume under normal conditions, V _N = 24.4116 - X [g/l] biomass concentration - CO₂ mole fraction in the effluent gas - O₂ mole fraction in the effluent gas - inlet (above the gas distributor) - ratio of oxygen consumed through TCA cycle to the total oxygen uptake rate - [g/l or kg/m³] density - [%] degree homogeneity - outlet of fermenter or top of the dispersion phase Dedicated to the 65th birthday of Professor Fritz Wagner.We thank Dr. C. Posten and T. Gabel for support with the computer control system UBICON. T.-G. Byun gratefully acknowledges financial support by DAAD.     

Two N 5, N 10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme

It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H₂-forming N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F₄₂₀-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH₄)₂SO₄, 2 M Na₂HPO₄, 1.5 M K₂HPO₄, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V _max rather than the K _m of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH₄)₂SO₄ the V _max was 4000 U/mg (k _cat=2400 s^-1) and the K _m for N ⁵,N ¹⁰-methylenetetrahydromethanopterin and for coenzyme F₄₂₀ were 80 M and 20 M, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K₂HPO₄ at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F₄₂₀-dependent N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.Abbreviations H₄MPT tetrahydromethanopterin - F₄₂₀ coenzyme F₄₂₀ - CH₂=H₄MPT N ⁵,N ¹⁰-methylenetrahydromethanopterin - CHH₄MPT⁺ N ⁵,N ¹⁰-methenyltetrahydromethanopterin - methylene-H₄MPT dehydrogenase N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase - Mops N-morpholinopropane sulfonic acid - Tricine N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

Synthesis and enzymology of modifiedN-benzyloxycarbonyl-L-cysteinylglycyl-3,3-dimethylaminopropylamide disulphides as alternative substrates for trypanothione reductase fromTrypanosoma crud: Part 3

Summary Kinetic data for alternative substrates of recombinant trypanothione reductase fromTrypanosoma cruzi were measured for a series ofN-substituted-L-cysteinylglycyl-3-dimethylaminopropylamides, in which the cysteineN-substituent was either a variant of the benzyloxycarbonyl group or was L-phenylalanine or L-tryptophan. Replacing the benzylic ether oxygen atom by CH₂. or NH had relatively minor effects on k_cat, but raised the value of K_m, 4.5- and 10-fold, respectively. Similarly, relative to the carbobenzoxy group, anN-L-phenylalanyl orN-L-tryptophanyl replacement on the cysteine hardly altered k_cat, but increased K_m, values by 16.6 and 7.4 fold, respectively. These observations were consistent with the K_m, values referring primarily to binding for this series of nonspecific substrates.Abbreviations DCC N,N-dicyclohexylcarbodiimide - dmapa dimethylaminopropylamine - DMF dimethylformamide - GR glutathione reductase - GSSG glutathione disulphide - GSH reduced glutathione - T[S]₂ trypanothione disulphide - Hbt hydroxybenzotriazole - TFA trifluoroacetic acid - TLC thin layer chromatography - T[SH]₂ reduced trypanothione as dithiol - TR trypanothione reductase - Z.cys.gly.dmapa N-benzyloxycarbonyl-Lcysteinylglycyl-3-dimethylpropylamide     

Heat-stress stimulation of oxygen uptake by Photosystem I involves the reduction of superoxide radicals by specific electron donors

A Photosystem I submembrane fraction isolated from spinach was used to study the mechanism of heat-stress stimulation of oxygen uptake by the photosystem. Various artificial electron donors were shown to generate electron transport reactions with various degrees of thermally induced stimulation. A strong stimulation was observed with durohydroquinone as electron donor with a maximal effect at 50 °C. The degree of stimulation obtained was independent from the redox potential of the electron donors and from their oxidation site because the enzyme superoxide dismutase fully inhibited the stimulation. Instead, it is proposed that thermal stress causes the release of membrane bound superoxide dismutase from the thylakoids thus allowing the reduced form of electron donors with specific properties to reduce O₂ ^– radicals to H₂O₂ besides the usual disproportionation of O₂ ^– into O₂ and H₂O₂.Abbreviations: PS photosystem - DCIP 2,6-dichlorophenolindophenol - MV methylviologen - TMPD N,N,N,N-tetramethylphenylenediamine - SOD superoxide dismutase - Chl chlorophyll - DQ duroquinone - DAD N,N,N,N-tetramethyl-1,4-benzenediamine - PMS 5-methylphenazium methyl sulfate - PC plastocyanin