Characterizing the fluid dynamics of the inverted frustoconical shaking bioreactor

The authors conducted a three‐dimensional computational fluid dynamics (CFD) simulation to calculate the flow field in the inverted frustoconical shaking bioreactor with 5 L working volume (IFSB‐5L). The CFD models were established for the IFSB‐5L at different operating conditions (different shaking speeds and filling volumes) and validated by comparison of the liquid height distribution in the agitated IFSB‐5L. The “out of phase” operating conditions were characterized by analyzing the flow field in the IFSB‐5L at different filling volumes and shaking speeds. The values of volumetric power consumption (P/V_L) and volumetric mass transfer coefficient (k_La) were determined from simulated and experimental results, respectively. Finally, the operating condition effect on P/V_L and k_La was investigated. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:478–485, 2018     

Effect of pH,aeration and sucrose feeding on the invertase activity of intactS. cerevisiae cells grown in sugarcane blackstrap molasses

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O₂L^–1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L^–1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attainingS. cerevisiae cells suitable for invertase production were: temperature=30°C; pH=5.0; DO=3.3 mg O₂L^–1; (S)=0.5 g L^–1 and sucrose added into the fermenter according to the equations: (V–V_o)=t²/16 or (V–V_o)=(V_f–V_o)·(e^0.6t–1)/10.This work was supported by FAPESP     

The relationship between nutrient feed rate and specific growth rate in fed batch cultures

Summary Equations are described which relate nutrient feed rate to specific microbial growth rate in fed batch culture. Fed batch cultures are classified into three types: 1) those allowing constant specific microbial growth rate, 2) those in which the rate of change of flow rate is constant and 3) those in which the nutrient flow rate is constant. The basic properties of these three types are described.Symbols F medium flow rate, L³ T^–1 - F _o medium flow rate at zero time, L³ T^–1 - g rate of change of flow rate with time, L³ T^–2 - K _v volume constant, being the total cell weight at zero time divided by the product of the yield coefficient and growth-limiting substrate concentration in the feed, L³ - s _r growth limiting substrate concentration in the feed, ML^–3 - V volume of liquid in the growth vessel, L³ - V _f volume of medium fed to the growth vessel, L³ - V _o volume of liquid in the growth vessel at zero time, L³ - X total weight of cells, M - x concentration of cells, ML^–3 - X _g total weight of cells grown, M - X _o total weight of cells at zero time, M - Y yield coefficient, weight of cells grown per unit weight of growth-limiting substrate - specific microbial growth rate, T^–1     

Acclimation of photosynthetic capacity to irradiance in tree canopies in relation to leaf nitrogen concentration and leaf mass per unit area

The observation of acclimation in leaf photosynthetic capacity to differences in growth irradiance has been widely used as support for a hypothesis that enables a simplification of some soil‐vegetation‐atmosphere transfer (SVAT) photosynthesis models. The acclimation hypothesis requires that relative leaf nitrogen concentration declines with relative irradiance from the top of a canopy to the bottom, in 1 : 1 proportion. In combination with a light transmission model it enables a simple estimate of the vertical profile in leaf nitrogen concentration (which is assumed to determine maximum carboxylation capacity), and in combination with estimates of the fraction of absorbed radiation it also leads to simple ‘big‐leaf’ analytical solutions for canopy photosynthesis. We tested how forests deviate from this condition in five tree canopies, including four broadleaf stands, and one needle‐leaf stand: a mixed‐species tropical rain forest, oak (Quercus petraea (Matt.) Liebl), birch (Betula pendula Roth), beech (Fagus sylvatica L.) and Sitka spruce (Picea sitchensis (Bong.) Carr). Each canopy was studied when fully developed (mid‐to‐late summer for temperate stands). Irradiance (Q, µmol m^?2 s^?1) was measured for 20 d using quantum sensors placed throughout the vertical canopy profile. Measurements were made to obtain parameters from leaves adjacent to the radiation sensors: maximum carboxylation and electron transfer capacity (V_a, J_a, µmol m^?2 s^?1), day respiration (R_da, µmol m^?2 s^?1), leaf nitrogen concentration (N_m, mg g^?1) and leaf mass per unit area (L_a, g m^?2). Relative to upper‐canopy values, V_a declined linearly in 1 : 1 proportion with N_a. Relative V_a also declined linearly with relative Q, but with a significant intercept at zero irradiance (P < 0·01). This intercept was strongly related to L_a of the lowest leaves in each canopy (P < 0·01, r² = 0·98, n= 5). For each canopy, daily lnQ was also linearly related with lnV_a(P < 0·05), and the intercept was correlated with the value for photosynthetic capacity per unit nitrogen (PUN: V_a/N_a, µmol g^?1 s^?1) of the lowest leaves in each canopy (P < 0·05). V_a was linearly related with L_a and N_a(P < 0·01), but the slope of the V_a : N_a relationship varied widely among sites. Hence, whilst there was a unique V_a : N_a ratio in each stand, acclimation in V_a to Q varied predictably with L_a of the lowest leaves in each canopy. The specific leaf area, L_m(cm² g^?1), of the canopy‐bottom foliage was also found to predict carboxylation capacity (expressed on a mass basis; V_m, µmol g^?1 s^?1) at all sites (P < 0·01). These results invalidate the hypothesis of full acclimation to irradiance, but suggest that L_a and L_m of the most light‐limited leaves in a canopy are widely applicable indicators of the distribution of photosynthetic capacity with height in forests.     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

Response surface methodology-based optimization of glucose acylation bio-catalyzed by immobilized lipase

This paper describes in detail the selection and optimization of immobilized lipases for enhanced regioselective acylation of glucose into glucose monolaurate (GlcML). Initially, nature of biocatalyst, immobilization approach, reaction media, glucose, and lauric acid concentration were screened out. Finally, lipases from Rhizopus arrhizus immobilized on dead mycelia were investigated under various reaction conditions (Temperature, shaking speed, enzyme dose, and water content) following a fully rotatable central composite design (FRCCD) to optimize the activity of lipases. The immobilized lipases-based biocatalysts in the presence of polar solvents (tertiary alcohols) and higher concentrations of substrates i.e. glucose and lauric acid (100 and 300?mmol?L^?1, respectively) offered conversion rate of 1.5 mmolmin^?1?L^?1. Moreover, optimization of reaction conditions revealed that 162.5 lipase units/100mL at 31.25?°C, 3% water content, and 105?RPM shaking speed enhanced the conversion rate by 0.5 mmolmin^?1?L^?1 rendering the reaction more economical. Hence, lipases-based immobilized biocatalysts may provide an intelligent and green choice for commercial scale synthesis of GlcML for food and pharmaceutical industries.     

Enhanced activity,enantioselectivity and stability of papain in asymmetric hydrolysis of d,l-p-hydroxyphenylglycine methyl ester with ionic liquid

Papain-mediated asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester (D,L-HPGME) was examined in the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF₄) and different solvents. The activity of the enzyme varied widely with change in BMIM·BF₄ concentration, with 12.5% (v/v) being the optimum BMIM·BF₄ concentration for the reaction. Papain displayed much higher hydrolytic activity and enantioselectivity in phosphate buffer solution of 12.5% (v/v) BMIM·BF₄ (pH 7.0) than in other media examined. Comparative studies on the kinetics and activation energy (E_a) of this reaction performed in different media showed a higher V_max, a lower K_m and a lower E_a for the reaction taking place in phosphate buffer solution of 12.5% (v/v) BMIM·BF₄ than in other media tested. The stability of papain at 45°C was considerably enhanced in BMIM·BF₄ as compared with aqueous buffer, 2-propanol and acetonitrile. A half-life time of 169 h was observed with BMIM·BF₄ in the presence of substrate, which was 9.2–16.8-fold higher than those with the other solvents. These results suggested that BMIM·BF₄ is an excellent reaction medium for this reaction.     

Simulation of a multicolumn recirculated packed bed reactor (MRPBR) for penicillin acylase

Enzyme reactors for the industrial hydrolysis of penicillin are analyzed in terms of biocatalyst stability to pH. A multicolumn system with packed beds placed in parallel and operating under recirculating conditions is proposed as an adequate reactor for this process. The system is studied both experimentally and with the aid of a simulation program.List of Symbols A transversal area (cm²) - C _A ammonia concentration in the reaction mixture (M) - C ₁ concentration of KH₂PO₄ in buffer (M) - C ₂ concentration of K₂HPO₄ in buffer (M) - d _p biocatalyst diameter (cm) - E enzyme or biocatalyst concentration (gcat l^–1) - K _APA APA non competitive inhibition constant (M) - K _IS excess substrate inhibition constant (M) - Km constant Michaelis-Menten (M) - K _PAA PAA competitive inhibition constant (M) - Q recirculation flow rate (cm³ min^–1) - Q _T recirculation flow rate per column (cm³ min^–1) - Re Reynolds number - S _E substrate concentration entering the neutralization tank (M) - S ₀ initial substrate concentration (M) - S _T substrate concentration in neutralization tank (M) - t time (min) - v _i initial reactor rate (mol min^–1 gcat^–1) - V _s superficial velocity (cm seg^–1) - V _T volume of neutralization tank (cm³) - X _E substrate conversion entering tank - X _T substrate conversion in neutralization tank - X conversion - Z reactor length (cm) - z axial position in reactor (cm) - z ^* non-dimensional axial position in reactor - biocatalyst's density (gcat cm^–3) - p pressure drop in the packed-bed reactor     

n-carbamoyl-d-p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V_max and K_m of the immobilized d-hydantoinase at 50°C were 6.28 mm min⁻¹ g⁻¹ biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol⁻¹. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.     

Transesterification activity of a novel lipase from Acinetobacter venetianus RAG-1

Transesterification activity and the industrial potential of a novel lipase prepared from Acinetobacter ventiatus RAG-1 were evaluated. Purified lipase samples were dialyzed against pH 9.0 buffer in a single optimization step prior to lyophilization. The enzyme and organic phase were pre-equilibrated (separately) to the same thermodynamic water activities (a _w) ranging from a _w 0.33 to 0.97. Production of 1-octyl butyrate by lipase-catalyzed transesterification of vinyl butyrate with 1-octanol in hexane was monitored by gas chromatography. Production of 1-octyl butyrate and initial rate of reaction depended on water activity. Product synthesis and rate of transesterification increased sharply with increase from a _w 0.33 to 0.55. Highest product concentration (218 mM) and rate of reaction (18.7 μmol h⁻¹ · 10 μg protein) were measured at a _w 0.86. Transesterification activity in hexane represented 32% of comparable hydrolytic activity in aqueous buffer.     

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ⁵3β hydroxy steroids into the active Δ⁴3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ⁵3β HSD we have analysed the initial velocity rates (V_o) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ⁵3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/V_o versus 1/S plot, the x intercept being positive. Using a 1/V_o versus 1/S² plot the V_max was determined to be 1·0 ± 0·08 μmol min^?1 mg^?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the V_max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min^?1 mg^?1, n = 3) whilst the S_0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD⁺ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.¹ Extending the analysis of NAD⁺ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V_max and K_m being 0·84 ± 0·04 μmol min^?1 mg^?1 CL (n = 3) and 27 ± 7 μmol 1^?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.     

Distinctive and Synergistic Signaling of Human Adenosine A2a and Dopamine D2L Receptors in CHO Cells

Adenosine A_2a receptor (A_2aR) colocalizes with dopamine D₂ receptor (D₂R) in the basal ganglia and modulates D₂R-mediated dopaminergic activities. A_2aR and D₂R couple to stimulatory and inhibitory G proteins, respectively. Their opposing roles in regulating neuronal activities, such as locomotion and alcohol consumption, are mediated by their opposite actions on adenylate cyclase, which often serves as “co-incidence detector” of various activators. On the other hand, the neural actions of A_2aR and D₂R are also, at least partially, independent of each other, as indicated by studies using D₂R and A_2aR knock-out mice. Here we co-expressed human A_2aR and human D_2LR in CHO cells and examined their signaling characteristics. Human A_2aR desensitized rapidly upon agonist stimulation. A_2aR activity (80%) was diminished after 2 hr of pretreatment with its agonist CGS21680. In contrast, human D_2LR activity was sustained even after 2 hr and 18 hr pretreatment with its agonist quinpirole. Long-term (18 hr) stimulation of human D_2LR also increased basal cAMP levels in CHO cells, whereas long-term (18 hr) activation of human A_2aR did not affect basal cAMP levels. Furthermore, long-term (18 hr) activation of D_2LR dramatically sensitized A_2aR-induced stimulation of adenylate cyclase in a pertussis toxin-sensitive way. Forskolin-induced cAMP accumulation was significantly increased after short-term (2 hr) human D_2LR stimulation and further elevated after long-term (18 hr) D_2LR activation. However, neither short-term (2 hr) nor long-term (18 hr) stimulation of A_2aR affected the inhibitory effects of D_2LR on adenylate cyclase. Co-stimulation of A_2aR and D_2LR could not induce desensitization or sensitization of D_2LR either. In summary, signaling t hrough A_2aR and D_2LR is distinctive and synergistic, supporting their unique and yet integrative roles in regulating neuronal functions when both receptors are present.     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

Whole-cell biocatalysis: Evaluation of new hydrophobic ionic liquids for efficient asymmetric reduction of prochiral ketones

A biphasic process design is often applied in whole-cell biocatalysis if substrate and product have low water solubility, are unstable in water or toxic for the biocatalyst. Some water immiscible ionic liquids (ILs) with adequate distribution coefficients have already been applied successfully as second liquid phase, which acts as a substrate reservoir and in situ extractant for the product. In this work, 12 new ILs were evaluated with respect to their applicability in biphasic asymmetric reductions of prochiral ketones in comparison to 9 already published ILs. The ILs under study are composed of seven different cations and three different anions. Recombinant Escherichia coli was used as whole-cell biocatalyst overexpressing the genes of a Lactobacillus brevis alcohol dehydrogenase (LB-ADH) and a Candida boidinii formate dehydrogenase (CB-FDH) for cofactor regeneration. Best results were achieved if ionic liquids with [PF6]- and [NTF]-anions were applied, whereas [FAP]-ILs showed minor qualification, e.g., the use of [HMPL][NTF] as second liquid phase for asymmetric synthesis of (R)-2-octanol resulted in a space–time-yield of 180 g L⁻¹ d⁻¹, a chemical yield of 95% and an enantiomeric excess of 99.7% in a simple batch process.     

Effect of periphyton on growth performance of grey mullet, Mugil cephalus (Linn.), in inland saline groundwater ponds

The present study attempts to assess the potential of artificial substrates to enhance fish production in inland saline groundwater ponds through periphyton production. Grey mullet, Mugil cephalus, was cultured for 100 days in ponds with substrate (treatment ponds) and without substrate (control ponds). To enhance the surface area, bamboo poles were used as substrate. The periphyton population, pigment concentration and hydrobiological characteristics of pond water were monitored. The studies revealed little difference in most of the water quality parameters observed in the two treatments. However, turbidity (27.0 ± 0.1–35.0 ± 0.1 Nephalo Turbidity Unit (NTU)), chlorophyll ‘a’ (6.6 ± 0.6–7.6 ± 0.6 μg L^?1), plankton population (phytoplankton 8.4 × 10³–9.4 ×10³ numbers L^?1; zooplankton 4.0 × 10³–5.1 × 10³ numbers L^?1) and NH₄–N (2.0 ± 0.2–2.3 ± 0.1 mg L^?1) were high in the treatment with no additional substrate; however, in the treatment with substrate the total Kjeldahl nitrogen (9.8 ± 0.8–10.8 ± 0.7 mg L^?1) and o‐PO₄ (0.1 ± 0.01–0.1 mg L^?1) remained significantly (P < 0.05) higher. Highest periphyton biomass in terms of dry matter (DM) (0.8 ± 0.01–1.4 ±0.01 mg cm^?2), ash free DM (0.4 ± 0.0–0.6 ± 0.01 mg cm^?2), chlorophyll ‘a’ (3.1 ± 0.2–8.1 ± 0.8 μg cm^?2) and pheophytin ‘a’ (1.9 ± 0.4–3.9 ± 0.5 μg cm^?2) was observed at 50 cm depth in ponds provided with additional substrate. Fifteen plankton genera showing periphytic affinity colonized the bamboo substrates. Fish growth (mean fish weight 524.3 ± 8.7 g and SGR 2.5 ± 0.1) was significantly (P < 0.05) higher in ponds provided with additional substrate compared with control ponds (387.2 ± 6.0). Length–weight relationship (LWR) (W = cLⁿ) also showed that the exponential value (‘n’) of length was high in substrate‐supported ponds (n = 2.36) in comparison with controls (n = 1.09). These studies suggest that a periphyton‐supported aquaculture system can be used successfully for the culture of herbivorous brackishwater fish species like M. cephalus in inland saline groundwaters and thus could contribute to the development of sound and sustainable aquaculture technology.     

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Summary The V_max and K_M of various forms of lipase from Pseudomonas cepacia (powder, adsorbed onto Celite or covalently linked to polyethylene glycol) were determined in organic solvents preequilibrated to water activities (a _w) from <0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that K_M for the nucleophile increased with increasing a _w for all three lipase forms. V_max increased with increasing a _w for polyethylene glycol-lipase, whereas there was an optimum at intermediate a _w values (0.11 – 0.38) for lipase powder and Celite-immobilized lipase.     

Comparison of tomato root distributions by minirhizotron and destructive sampling

Calibration of minirhizotron data against root length density (RLD) was carried out in a field trial where three drip irrigation depths: surface (R0) and subsurface, 0.20 m (RI) and 0.40 m depth (RII) and two processing tomato cultivars: `Brigade' (CI) and `H3044' (CII) were imposed. For each treatment three minirhizotron tubes were located at 10, 37.5 and 75 cm of the way from one plant row to the next. Roots intersecting the minirizotrons walls were expressed as root length intensity (L _a) and number of roots per unit of minirhizotron wall area (N _ra). Root length density (RLD) was calculated from core samples taken for each minirhizotron tube at two locations: near the top of the minirhizotron (BI) and 15 cm apart from it, facing the minirhizotron wall opposite the plant row (BII). Minirhizotron data were regressed against RLD obtained at BI and BII and with their respective means. The results show that for all the situations studied, better correlations were obtained when RLD was regressed with L _a than with N _ra. Also was evident that the relationship between L _a and RLD was strongly influenced by the location of soil coring. RLD was correlated with L _a trough linear and cubic equations, having the last ones higher determination coefficients. For instance at 10 cm from the plant row when values from the top layer (0–40 cm) were analysed separately, L _a was significantly regressed with RLD measured at BII and described by the equations: RLD = 0.5448 + 0.0071 L _a (R ² = 0.51) and RLD = 0.4823 + 0.0074L _a + 8×10^–5 L _a ² – 5×10^–7 L _a ³ (R ² = 0.61). Under the 40 cm depth the highest coefficients of determination for the linear and cubic equations were respectively 0.47 and 0.88, found when L _a was regressed with RLD measured at BI. For minirhizotrons located at 75 cm from the plant row and for location BI it was possible to analyse jointly data from all depths with coefficients of determination of 0.45 and 0.59 for the linear and cubic equations respectively.     

Xylitol Production from D‐Xylose at Different Oxygen Transfer Coefficients in a Batch Bioreactor

Conversion of D‐xylose to xylitol by Candida boidinii NRRL Y‐17213 was studied under anaerobic and oxygen limited conditions by varying the oxygen transfer coefficient k_La. Shake flask experiments were used to provide the preliminary information required to perform experiments in a bioreactor. The yeast did not grow under fully anaerobic conditions, but anaerobic formations of xylitol, ethanol, ribitol, and glycerol were observed as well as D‐xylose assimilation of 11 %. In shake flasks, with an initial D‐xylose concentration of 50 g/L, an increase in k_La from 8 to 46 h^–1 resulted in a faster growth, higher rate of substrate uptake and lower yields of products. The highest xylitol productivity (0.052 g/L h) was attained at k_La = 8 h^–1. At k_La = 46 h^–1, 98.6 % of D‐xylose was consumed and mainly converted to biomass. Using 130 g/L D‐xylose, k_La was varied in the fermenter from 26 to 78 h^–1. The percentage of consumed D‐xylose increased from 31 % at k_La = 26 h^–1 to 93–94 % at all other aeration levels. Biomass yield increased with k_La, whereas ethanol, ribitol, and glycerol yields exhibited an opposite dependence on the oxygenation level. The most favorable oxygen transfer coefficient for xylitol formation, in the fermenter, was k_La = 47 h^–1 when its concentration (57.5 g/L) surpassed ethanol accumulation by 3.6‐fold, and the glycerol plus ribitol by 10‐fold. Concurrently, xylitol yield and productivity reached 0.45 g/g and 0.26 g/L h, respectively. The volumetric xylitol productivity was affected more by changes in the aeration than the corresponding yield.     

Characterizing the fluid dynamics in the flow fields of cylindrical orbitally shaken bioreactors with different geometry sizes

Orbitally shaken bioreactors (OSRs) are commonly used for the cultivation of mammalian cells in suspension. To aid the geometry designing and optimizing of OSRs, we conducted a three‐dimensional computational fluid dynamics (CFD) simulation to characterize the flow fields in a 10 L cylindrical OSR with different vessel diameters. The liquid wave shape captured by a camera experimentally validated the CFD models established for the cylindrical OSR. The geometry size effect on volumetric mass transfer coefficient (k_La) and hydromechanical stress was analyzed by varying the ratio of vessel diameter (d) to liquid height at static (h_L), d/h_L. The highest value of k_La about 30 h^?1 was observed in the cylindrical vessel with the d/h_L of 6.35. Moreover, the magnitudes of shear stress and energy dissipation rate in all the vessels tested were below their minimum values causing cells damage separately, which indicated that the hydromechanical‐stress environment in OSRs is suitable for cells cultivation in suspension. Finally, the CFD results suggested that the d/h_L higher than 8.80 should not be adopted for the 10 L cylindrical OSR at the shaking speed of 180 rpm because the “out of phase” state probably will happen there.     

Current–voltage–time records of ion translocating enzymes

Membrane currents, as non-linear functions of membrane voltage, V, and time, t, can be recorded quickly by triangular V protocols. From the differences, dI(V,t), of these relationships upon addition of a putative substrate of a charge-translocating membrane protein, the I(V,t) relationships of the transporter itself can be determined. These relationships likely comprise a steady-state component, I_a(V), of the active transporter, and a dynamic component, p_a(V,t), of its V- and time-dependent activity, p_a. Here, the steady-state component is modeled by a central reaction cycle, which senses a fraction _tr of the total V, whereas 1–_tr can be assigned to an inner and outer pore section with _i and _o, respectively (_i+_tr+_o = 1). For the enzymatic cycle, fast binding/debinding is assumed, plus V-sensitive and -insensitive reaction steps which may become rate limiting for charge translocation. At given substrate concentrations, I_a(V) is defined by eight independent system parameters, including a coefficient for the barrier shape of charge translocation. In ordinary cases, the behavior of p_a(V,t) can be described by two rate constants (for activation and inactivation) and their respective V-sensitivity coefficients. Here, the effects of the individual system parameters on I(V,t) from triangular V-clamp experiments are investigated systematically. The results are illustrated by panels of typical curve shapes for non-gated and gated transporters to enable a first classification of mechanisms. We demonstrate that all system parameters can be determined fairly well by fitting the model to experimental data of known origin. Applicability of the model to channels, pumps and cotransporters is discussed.