Enzymatic Production of γ-Aminobutyric Acid in Soybeans Using High Hydrostatic Pressure and Precursor Feeding

The purpose of this study is to prepare Worcestershire sauce with higher levels of ethanol and aromatic components by a trickle bed bioreactor. Ethanol productivity in a trickle bed bioreactor was measured with changing circulation rates from 40 to 280 ml/min with a fixed aeration rate (200 ml/min) at 28°C. Compared with the lower circulation rate (40 ml/min), at 210 ml/min of circulation rate or higher, ethanol productivity increased 10.3-fold at 41 h of fermentation.     

Biofiltration of paint solvent mixtures in two reactor types: overloading by hydrophobic components

Steady-state performance characteristics of a trickle bed reactor (TBR) and a biofilter (BF) in loading experiments with increasing toluene/xylenes inlet concentrations while maintaining a constant loading rate of hydrophilic components (methyl ethyl and methyl isobutyl ketones, acetone, and n-butyl acetate) of 4 g m⁻³ h⁻¹ were evaluated and compared, along with the systems’ dynamic responses. At the same combined substrate loading of 55 g m⁻³ h⁻¹ for both reactors, the TBR achieved more than 1.5 times higher overall removal efficiency (RE_W) than the BF. Increasing the loading rate of aromatics resulted in a gradual decrease of their REs. The degradation rates of acetone and n-butyl acetate were also inhibited at higher loads of aromatics, thus revealing a competition in cell catabolism. A step-drop in loading of aromatics resulted in an immediate increase of RE_W with variations in the TBR, while the new steady-state value in the BF took 6–7 h to achieve. The TBR consistently showed a greater performance than BF in removing toluene and xylenes. Increasing the loading rate of aromatics resulted in a gradual decrease of their REs. The degradation rates of acetone and n-butyl acetate were also lower at higher OL_AROM, revealing a competition in the cell catabolism. The results obtained are consistent with the proposed hypothesis of greater toxic effects under low water content, i.e., in the biofilter, caused by aromatic hydrocarbons in the presence of polar ketones and esters, which may improve the hydrocarbon partitioning into the aqueous phase.     

Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized β-galactosidase from Aspergillus oryzae

This work studied the hydrolysis of lactose using β-galactosidase from Aspergillus oryzae immobilized with a combination of adsorption and glutaraldehyde cross-linking onto the ion exchange resin Duolite A568 as a carrier. A central composite design (CCD) was used to study the effects of lactose concentration and feed flow rate on the average hydrolysis reaction rate and lactose conversion in a fixed bed reactor operating continuously with an upflow at a temperature of 35 ± 1 °C. The optimal conditions for the average hydrolysis reaction rate and the lactose conversion included a lactose concentration of 50 g/L and a feed flow rate of 6 mL/min. The average reaction rate and conversion reached 2074 U and 65%, respectively. The immobilized enzyme activity was maintained during the 30 days of operation in a fixed bed reactor with a 0.3 mL/min feed flow rate of a 50 g/L lactose solution at room temperature. Feed flows ranging from 0.6 to 12 mL/min were used to determine the distribution of residence times and the kinetics of the fixed bed reactor. A non-ideal flow pattern with the formation of a bypass flow in the fixed bed reactor was identified. The conditions used for the kinetics study included a lactose solution concentration of 50 g/L at pH 4.5 and a temperature of 35 ± 1 °C. Kinetic models using a PFR and axial dispersion methods were used to describe the lactose hydrolysis in the fixed bed reactor, thus accounting for the competitive inhibition by galactose. To increase the lactose conversion, experiments were performed for two fixed bed reactors in series, operating in continuous duty with upflow, with the optimal conditions determined using the CCD for a fixed bed reactor. The total conversion for the two reactors in series was 82%.     

Optimization of protein extraction from Chlorella Vulgaris via novel sugaring‐out assisted liquid biphasic electric flotation system

Microalgae biomass has been consumed as animal feed, fish feed and in human diet due to its high nutritional value. In this experiment, microalgae specie of Chlorella Vulgaris FSP‐E was utilized for protein extraction via simple sugaring‐out assisted liquid biphasic electric flotation system. The external electric force provided to the two‐phase system assists in disruption of rigid microalgae cell wall and releases the contents of microalgae cell. This experiment manipulates various parameters to optimize the set‐up. The liquid biphasic electric flotation set‐up is compared with a control liquid biphasic flotation experiment without the electric field supply. The optimized separation efficiency of the liquid biphasic electric flotation system was 73.999 ± 0.739% and protein recovery of 69.665 ± 0.862% compared with liquid biphasic flotation, the separation efficiency was 61.584 ± 0.360% and protein recovery was 48.779 ± 0.480%. The separation efficiency and protein recovery for 5 × time scaled‐up system was observed at 52.871 ± 1.236% and 73.294 ± 0.701%. The integration of simultaneous cell‐disruption and protein extraction ensures high yield of protein from microalgae. This integrated method for protein extraction from microalgae demonstrated its potential and further research can lead this technology to commercialization.     

Highly pure 1,3‐propanediol: Separation and purification from crude glycerol‐based fermentation

A new separation and purification process was developed for recovering 1,3‐propanediol (1,3‐PD) from crude glycerol‐based fermentation broth with high purity. The downstream process integrated chitosan flocculation, activated carbon decolorization, fixed bed cation exchange resin adsorption, and vacuum distillation. Breakthrough curves were measured considering the effect of sample concentration, flow rate, temperature, and resin stack height. Yoon–Nelson model was proposed to fit the fixed bed adsorption. The characteristic column parameters were calculated. Optimal condition for adsorption was 1,3‐PD, 30.0 g/L; flow rate, 1.00 mL/min; stacking height, 30.0 cm; and temperature, 298 K. Ethanol‐water (75%, 1 mL/min) was used as eluent to separate 1,3‐PD and glycerol with 95.3% 1,3‐PD elution rate. After vacuum distillation, the overall purity and yield of 1,3‐PD were 99.2% and 80.8% in the purification process, respectively. This is a simple and efficient downstream strategy for 1,3‐PD purification.     

Ethanol fermentation by immobilized cells in a trickle bed reactor

A modified discontinuous packed bed reactor with CO₂ ventilation ports, resembling a trickle bed reactor was employed to overcome gas holdup and bed compaction problems which are commonly encountered in cell immobilized packed bed reactors for ethanol fermentation. The reactor consisting of yeast cells entrapped in alginate matrix was operated by varying the substrate concentration, bed volume and inlet flow rates. The number of recirculation cycles (passes) and total stages were dependent upon the liquid flow rate, though the total contact time for complete conversion remains the same for a particular initial substrate level. The total contact time was 1.5, 3 and 4.5 h for initial substrate concentrations of 0.555, 0.933 and 1.3 kmol/m³ respectively. The number of cycles and in turn stages increased with the increase in initial sugar level. A graphical method for predicting the number of stages required for complete conversion was proposed based on material balance equation and evaluated for the operating variables of the present study. The reactor was operated continuously for 30 days producing 1.05– 1.15 kmol/m³.     

Assessment of coronary microvascular resistance in the chronic infarcted pig heart

Pre‐clinical studies aimed at treating ischemic heart disease (i.e. stem cell‐ and growth factor therapy) often consider restoration of the impaired microvascular circulation as an important treatment goal. However, serial in vivo measurement hereof is often lacking. The purpose of this study was to evaluate the applicability of intracoronary pressure and flow velocity as a measure of microvascular resistance in a large animal model of chronic myocardial infarction (MI). Myocardial infarction was induced in Dalland Landrace pigs (n = 13; 68.9 ± 4.1 kg) by a 75‐min. balloon occlusion of the left circumflex artery (LCX). Intracoronary pressure and flow velocity parameters were measured simultaneously at rest and during adenosine‐induced hyperemia, using the Combowire (Volcano) before and 4 weeks after MI. Various pressure‐ and/or flow‐derived indices were evaluated. Hyperemic microvascular resistance (HMR) was significantly increased by 28% in the infarct‐related artery, based on a significantly decreased peak average peak flow velocity (pAPV) by 20% at 4 weeks post‐MI (P = 0.03). Capillary density in the infarct zone was decreased compared to the remote area (658 ± 207/mm² versus 1650 ± 304/mm², P = 0.017). In addition, arterioles in the infarct zone showed excessive thickening of the alpha smooth muscle actin (αSMA) positive cell layer compared to the remote area (33.55 ± 4.25 μm versus 14.64 ± 1.39 μm, P = 0.002). Intracoronary measurement of HMR successfully detected increased microvascular resistance that might be caused by the loss of capillaries and arteriolar remodelling in the chronic infarcted pig heart. Thus, HMR may serve as a novel outcome measure in pre‐clinical studies for serial assessment of microvascular circulation.     

Determining specific biomass activity in anaerobic wastewater treatment processes

An experimental method for the measurement of specific gas production rate was developed and tested with biomass samples taken from anaerobic fluidized bed reactors, operating with a variety of carriers with molasses, condensate from cellulose production and brewery wastewater as feeds. The method is based on reactor sampling and offline gas volume measurement during a known time interval. Important factors are biomass and liquid sampling under oxygen-free conditions, using the liquid from the reactor as substrate, providing sufficient mixing and maintaining the physical integrity of the biomass. The method was developed in such a way that small samples (20 ml) were taken under anaerobic conditions (poising agent) for short-term (2–3 min.) gas rate measurements in a small fluidized bed (25 ml) batch reactor with U-tube. Biomass content was measured by an instrumental nitrogen method (Dumas), followed by weight determination of the carrier. The gas rates measured with the test system, and their dependence on substrate concentration, were in good agreement with those directly measured from the continuous fluidized bed reactor. Additions of molasses and acetate to the sample proved that the influence of concentration on the biomass activity can be obtained only by operating the continuous reactor at the concentration levels of interest. Comparison between the reactors showed large differences in the specific activity and the total reactor activity. It was found when comparing two reactors, that the values of the specific and the total activities permitted the calculation of the relative biomass quantities. In this way the influence of the carrier-type could be evaluated.     

Purification of exhaust air containing organic pollutants in a trickle-bed bioreactor

Summary This paper reports experiments on the purification of exhaust air containing organic pollutants by a new biological process using a trickle-bed reactor. Pollutant-specific microorganisms in high concentration were fixed to a suitable bed. The absorption and conversion of propionaldehyde as a model pollutant was measured by systematic variation of the gas and liquid flow rates in the reaction system. At a space velocity of 1000 h^–1, it was possible to achieve conversion rates of between 68 and 96%, depending on the trickling density. The degradation capacity of the biological trickle bed is over 500 g propionaldehyde/m³ of reactor per hour. By using a tube bundle (honeycomb tube), it was possible to ensure continuous operation of the reactor with reduced conversion and pressure loss.Dedicated to Professor Dr. Dr. D. Behrens on the occasion of 65th birthday     

Mixing and phase hold-ups variations due to gas production in anaerobic fluidized-bed digesters: influence on reactor performance

The influence of mixing and phase hold-ups on gas-producing fluidized-bed reactors was investigated and compared with an ideal flow reactor performance (CSTR). The liquid flow in the anaerobic fluidized bed reactor could be described by the classical axially dispersed plug flow model according to measurements of residence time distribution. Gas effervescence in the fluidized bed was responsible for bed contraction and for important gas hold-up, which reduced the contact time between the liquid and the bioparticles. These results were used to support the modeling of large-scale fluidized-bed reactors. The biological kinetics were determined on a 180-L reactor treating wine distillery wastewater where the overall total organic carbon uptake velocity could be described by a Monod model. The outlet concentration and the concentration profile in the reactor appeared to be greatly influenced by hydrodynamic limitations. The biogas effervescence modifies the mixing characteristics and the phase hold-ups. Bed contraction and gas hold-up data are reported and correlated with liquid and gas velocities. It is shown that the reactor performance can be affected by 10% to 15%, depending on the mode of operation and recycle ratio used. At high organic loading rates, reactor performance is particularly sensitive to gas effervescence effects. Copyright 1998 John Wiley & Sons, Inc.     

Adenosine transiently modulates stimulated dopamine release in the caudate–putamen via A1 receptors

Adenosine modulates dopamine in the brain via A₁ and A_2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast‐scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate–putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 μM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2 s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7%, similar to the 54 ± 6% decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 min. The A₁ receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A_2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A₁ receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain.

     

Biofilm producing indigenous bacteria isolated from municipal sludge and their nutrient removal ability in moving bed biofilm reactor from the wastewater

In the present study, improved moving bed biofilm reactor (MBBR) was applied to enhance the nutrient removal ability of the municipal wastewater. A total of 18 indigenous bacterial isolates were screened from the sewage sludge sample and nitrate reductase, nitrite reductase and hydroxylamine oxidase was analyzed. The strains Pseudomonas aeruginosa NU1 and Acinetobacter calcoaceticus K12 produced 0.87 ± 0.05 U/mg and 0.52 ± 0.12 U/mg hydroxylamine oxidase, 1.023 ± 0.062 U/mg and 1.29 ± 0.07 U/mg nitrite reductase, and 0.789 ± 0.031 U/mg and 1.07 ± 0.13 U/mg nitrate reductase. Nitrogen and phosphate removal improved by the addition of nutrient sources and achieved > 80% removal rate. pH and temperature of the medium also affected nutrient removal and improved removal was achieved at optimum level (p < 0.05). MBBR was designed with R1 (aerobic), R2 and R3 (anoxic) reactors. MBBR reactors removed acceptable level phosphorus removal properties up to 7.2 ± 3.8%, 42.4 ± 4.6%, and 84.2 ± 13.1% in the R1, R2, R3 and R4 reactors, respectively. Denitrification rate showed linear relationship at increasing concentrations nitrogen content in the reactor and denitrification rate was 1.43 g NO₂-N /m²/day at 1.5 g NO₂-N /m²/day. Dehydrogenase activity was assayed in all reactors and maximum amount was detected in the aerobic biofilm reactor. Based on the present findings, MBBRs and the selected bacterial strains are useful for the degradation domestic wastewater with minimum working area.     

Nonparametric approaches for estimating regional lake nutrient thresholds

Nonparametric approaches including a classification and regression tree (CART), a nonparametric changepoint analysis (nCPA) and a Bayesian hierarchical modeling (BHM) method were developed to determine ecoregional nutrient response thresholds. A CART analysis revealed that hierarchical structure was important for predicting Chl a concentrations from total nitrogen (TN) and total phosphorus (TP). The nCPA and BHM methods confirmed the CART results for each node in the tree, and the 90% confidence interval for each threshold was calculated to quantify uncertainty. The CART, nCPA, and BHM methods suggested that the nutrient criteria differed significantly within certain nutrient ecoregions and that numerical nutrient criteria of 0.0150–0.222 mg/L TP and 0.300–1.766 mg/L TN may control Chl a concentrations in the various lake ecoregions. The results of this analysis suggest that the integration of CART, nCPA and BHM might be useful for determining nutrient thresholds.     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

Stop-flow studies of solute uptake in rat lungs

Stop-flow studies were used to characterize solute uptake inisolated rat lungs. These lungs were perfused at 8 or 34 ml/min for10-28 s with solutions containing¹²⁵I-albumin and two or more ofthe following diffusible indicators: [³H]mannitol,[¹⁴C]urea,³HOH,²⁰¹Tl⁺,or⁸⁶Rb⁺.After this loading period, flow was stopped for 10-300 s and thenresumed to flush out the perfusate that remained in the pulmonary vasculature during the stop interval. Concentrations of²⁰¹Tl⁺and⁸⁶Rb⁺in the venous outflow decreased after the stop interval, indicating uptake from exchange vessels during the stop interval. The amount ofthese K⁺ analogs lost from thecirculation during the stop interval was greater when the intervalswere longer. However, losses of²⁰¹Tl⁺at 90 s approached those at 300 s. Because extraction continued afterthe vasculature had been flushed, vascular levels had presumably fallento negligible levels during the stop interval. By 90 s of stop flow thevascular volume that was cleared of²⁰¹Tl⁺averaged 0.657 ± 0.034 (SE) ml in the experiments perfused at 8 ml/min and 0.629 ± 0.108 ml in those perfused at 34 ml/min. Increases in perfusate K⁺decreased the cleared volumes of²⁰¹Tl⁺and⁸⁶Rb⁺.Uptake of[³H]mannitol,[¹⁴C]urea, and³HOH during the stop intervals wasobserved only when the lungs were loaded at high flow for shortintervals. Decreases in²⁰¹Tl⁺and⁸⁶Rb⁺concentrations in the pulmonary outflow can be used to identify thefraction of the collected samples that were within exchange vessels ofthe lung during the stop interval and may help determine thedistribution of solute and water exchange along the pulmonary vasculature.

     

High‐Throughput Chiral LC–MS/MS Method Using Overlapping Injection Mode for the Determination of Pantoprazole Enantiomers in Human Plasma with Application to Pharmacokinetic Study

A sensitive and high‐throughput chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of R‐pantoprazole and S‐pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid–liquid extraction in 96‐well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ‐RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 μL/min. The enantiomers were quantified on a triple‐quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole‐d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r² > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R‐pantoprazole and from 92.5% to 96.5% for S‐pantoprazole and the IS‐normalized matrix factor was 0.98 to 1.07 for R‐pantoprazole and S‐pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569–575, 2016. © 2016 Wiley Periodicals, Inc.     

Streamflow reduction induces early parental care in Salaria fluviatilis (Asso, 1801) males

This study investigated the effect of hydrological stress (streamflow reduction) on the reproductive and nesting behaviour of freshwater blenny (Salaria fluviatilis) males in a Mediterranean‐type stream in NE Spain (a tributary of the Ebro River). The investigation included two study periods: (i) before stream flow reduction (sampling in 2004) and (ii) afterwards (study year 2011). Nesting males in 2004 (N = 31) and 2011 (N = 11) were measured, weighed and photographed in the field. The size of the total egg cluster (male reproductive success) was measured and photographed for each nest found (N = 137 in 2004 and N = 28 in 2011). Nesting area was measured to determine nest density for each sampling period. The degree of secondary sexual traits (SSTs) development was measured later through the photographs. The age of males was assigned according to the length‐intervals established by Vinyoles and De Sostoa (2007) for this species in the same study area. After flow reduction in 2011, the flooded area of the river bed was reduced by more than 80%. A great proportion of small males (1 year old) with developed SSTs (cephalic crest and anal glands) were found to defend a nest. This is the first time that parental care is found for the male of the freshwater blenny at a young age. Total cluster size (mean ± SE) is halved under the low flow conditions (from 45.4 ± 2.8 cm² to 22.9 ± 2.7 cm²), but an increase in the number of partial clusters per nest was found. This study highlights the sexual plasticity of freshwater blenny males depending on environmental conditions and the vulnerability of this endangered species to the hydrological changes of anthropogenic origin in Spain.     

A biological active artificial saliva formulation containing flower mucilage from Ceylon Spinach (Basella alba Linn.)

Ceylon Spinach (Basella albe) is an edible perennial vine found in tropical Asia and Africa, known as vegetables containing mucilage. Its mucilage from flowers was extracted by microwaving and precipitated with 95% ethanol. Five artificial saliva formulations composing of mucilage from Ceylon Spinach, calcium chloride (CaCl₂), potassium chloride (KCl) and sodium fluoride (NF) were developed. The best formulation No.5 containing 0.61% of the mucilage with the non-Newtonian pseudoplastic flow (8.9 ± 0.2 cP) and the wetting time (12.50 ± 2.24 min) similar to the normal human saliva was selected. This artificial saliva formulation exhibited biological activities including an antioxidative activity by DPPH free radical scavenging with the SC₅₀ of 14.26 ± 2.00 mg/ml (0.05 folds of ascorbic acid), and the adhesion inhibition of S. mutans on hydroxyapatite beads at 17.01 ± 7.75%, while the natural human saliva exhibited an increase bacterial adhesion of 33.10 ± 9.70%. The safety of this formulation which gave no cytotoxicity on normal human gingival fibroblasts at 99.20 ± 21.09% cell viability was also demonstrated. The results from this study have indicated high biological activity and safety of the developed formulation containing mucilage from Ceylon Spinach which is potential to be used as artificial saliva for xerostomia patients.     

Effects of encouraged water drinking on thermoregulatory responses after 20 days of head-down bed rest in humans

We tested the hypothesis that encouraged water drinking according to urine output for 20 days could ameliorate impaired thermoregulatory function under microgravity conditions. Twelve healthy men, aged 24 ± 1.5 years (mean ± SE), underwent −6° head-down bed rest (HDBR) for 20 days. During bed rest, subjects were encouraged to drink the same amount of water as the 24-h urine output volume of the previous day. A heat exposure test consisting of water immersion up to the knees at 42°C for 45 min after a 10 min rest (baseline) in the sitting position was performed 2 days before the 20-day HDBR (PRE), and 2 days after the 20-day HDBR (POST). Core temperature (tympanic), skin temperature, skin blood flow and sweat rate were recorded continuously. We found that the −6° HDBR did not increase the threshold temperature for onset of sweating under the encouraged water drinking regime. We conclude that encouraged water drinking could prevent impaired thermoregulatory responses after HDBR.