A quenched-flow apparatus which allows the measurement of the kinetics of a reaction in one stroke

A chemical quenched-flow apparatus is described which measures, in a unique stroke, enough data points (8–11) for establishing the kinetics curve of a reaction. Only very small volumes of reaction solutions (2 × 500 μl) are required. The time intervals between which the kinetic data may be measured range from 5 to 37 ms and from 120 to 450 ms with the corresponding mixing times of 0.6 and 5 ms, respectively. This apparatus was used to investigate the pre-steady-state domain of the aminoacylation reaction of tRNA^Val by valyl-tRNA synthetase from yeast.     

Construction of a microprocessor-controlled pulsed quench-flow apparatus for the study of fast chemical and biochemical reactions

The construction and operation of a microprocessor-controlled quenched-flow machine are described. Two sets of syringes are moved by high-torque stepping motors to achieve any desired mixing scheme. The dead time of the instrument is pulsed quench-flow operation is of the order of 10 ms. The fastest possible reaction time is 12 ms with the reaction chamber used here; this might be extended downward with other reaction chambers. The reactant volume required is low; 1 ml of solution for each reactant is sufficient for more than 40 kinetic measurements. The machine is tested by alkaline hydrolysis of 2,4-dinitrophenylacetate and used in the investigation of the reaction mechanism of the EcoRI restriction endonuclease and of GTPase activity of ribosomes.     

Two- and three-phase mixing in a concentric draft tube gas-lift fermentor

Two- and three-phase mixing studies were carried out in a 44-L concentric draft tube gas-lift fermentor. It was proposed to use the fermentor for the production of solvents using immobilized bacteria. Bubble size, gas holdup, liquid velocities, circulation, and mixing times were determined for various superficial gas velocities in distilled water, starch, carboxymethyl cellulose, and ethanol solutions. The observed trends for two phase mixing were similar to other studies but the results were found to be more sensitive to liquid properties. This was possibly due to the large value of downcomer to riser area used in this study. Mixing in three phases highlighted the difficulty in predicting the effect of adding solids to the gas-liquid system. Results showed that the gas-lift fermentor was ideally suited to dealing with three phases but more work is necessary before accurate models can be developed to account for the effect of solids.     

A Computer-Controlled Spraying-Freezing Apparatus for Millisecond Time-Resolution Electron Cryomicroscopy

Apparatus is described for the kinetic investigation of biological reactions by electron cryomicroscopy with time resolution on the order of milliseconds. This involves layering a grid with one reactant and then spraying on a second reactant immediately before freezing. Two-stage mixing can be achieved by mixing two solutions, holding them in a delay line for a preset interval, and then spraying the aged solution onto a grid carrying a third reactant. The individual steps of these procedures are under software control and can be adjusted independently. Spray-freezing is widely applicable since solutions of small molecules, proteins, and protein assemblies can be delivered as aerosols. Thus the method can be used to study both the effects of small molecules on macromolecules and for monitoring protein–protein interactions. It may also be useful in other situations, for instance in light microscopy.     

Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method.

Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water.     

A double-beam rapid-scanning stopped-flow spectrophotometer.

A double-beam rapid-wavelength-scanning stopped-flow spectrophotometer system based on the Norcon model 501 spectrometer was construced, which enables u.v.-or visible absorbance spectra to be recorded at the rate of 800/s after the rapid mixing (within 3ms) of two reactant solutions. Each spectrum spans about 200nm in 1ms. It is possible to record difference spectra during reactions with half-lives less than 10ms involving absorbance changes of less than 0.1 absorbance unit. Analogue circuitry is used to produce spectra of absorbance against wavelength. Up to 32 such spectra can be recorded at pre-selected times during a reaction and stored in an 8Kx8-bit-word hard-wired data-capture system to be subsequently displaned individually or simultaneously. Time-courses at different wavelengths can also be displayed. By averaging up to 216 spectra it is possible to record spectra under conditions of low signal-to-noise ratios...     

A simple,reliable rapid-mixing apparatus for continuous-flow studies

We have developed a simple and economical system for rapid mixing of solutions where a continuous-flow, chemically quenched mode of operation is appropriate. This has proved especially useful for studies of pyruvate dehydrogenase which produces a stable, enzyme-bound intermediate when the terminal acceptors are absent. It has also proved convenient for studies of the overall reaction in which fluorescence of pyridine nucleotide can be measured after quenching in alkaline solution.Syringes containing reactants are driven by a Harvard infusion pump, which provides sufficient speed and power (torque) to give good mixing with conventional commercially available mixing chambers and delay lines. Liquid chromatography fittings and valves are used for the switching operations necessary in the continuous-flow mode.     

Photosynthetic studies with a 10-psec resolution streak camera

The fluorescence decay times for the species Anacystis nidulans and Chlorella pyrenoidosa are measured by means of a streak camera to be 75 ± 10 and 41 ± 5 psec, respectively. Concentrated solutions of chlorophyll a are shown to have fluorescence decay times as short as 10 psec. Fluorescence decay curves similar to that of the living cells can be generated by mixing cellular component molecules at comparable concentrations to that present in living cells.     

The rate of ATP synthesis as a function of ΔpH in normal and dithiothreitol-modified chloroplasts

The rate of ATP synthesis catalyzed by normal and by dithiothreitol-modified ATPases is investigated as a function of ΔpH in spinach chloroplasts at constant pH_out. The transmembrane ΔpH was generated by an acid-base transition and the reaction time was limited to 150 ms by using a rapidly mixing quenched-flow apparatus. The result was that the functional dependence of the rate on ΔpH is shifted to lower ΔpH values and that the shape of this curve is altered after dithiothreitol modification. The maximal rate (400 ATP / CF₁ per s) is the same under both conditions.     

Calibration of stopped-flow spectrophotometers using a two-step disulfide exchange reaction

A coupled two-step reaction of Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid)) with excess thioglycerol produces a progress curve composed of two superimposed exponentials. The ratio of the two pseudo-first-order rate constants equals 22.5 and does not vary appreciably with ehanges of either pH value or temperature. Because the ratio of the two amplitudes is defined by the ratio of the rate constants, the reaction can be used to estimate both the apparent zero time of mixing (or the dead time) and the detector linearity of a stopped-flow instrument with a single mixing experiment. The reaction is used as a standard of performance for both absorbance and fluorescence measurements with a stopped-flow spectrophotometer.     

Mixing in liquid-impelled loop reactors

The liquid-impelled loop reactor is a new column-type bioreactor. The design of this device is based on the principle of the air-lift loop reactor. In the external-loop configuration used in this work, descending perfluorochemical drops bring about circulation of the continuous aqueous phase. Mixing of this continuous phase is characterized per section of the rector. Axial-dispersion coefficients for the tube with two-phase flow are determined and correlated with the energy dissipation in the tube. Comparisons with similar systems such as bubble columns and air-lift loop reactors are made. Overall mixing parameters are derived and used for calculation of the number of circulatins needed to achieve a certain degree of mixing. The hydrodynamic model from previous work is tested for the reactor configurations of this work. It can be useful to calculate circulation times.     

A capillary-based microfluidic instrument suitable for immunoaffinity chromatography

The analysis of biological samples to produce clinical or research data often requires measurement of analytes from complex biological matrices and limited volumes. Miniaturized analytical systems capable of minimal sample consumption and reduced analysis times have been employed to meet this need. The small footprint of this technology offers the potential for portability and patient point-of-care testing. A prototype microfluidic system has been developed and is presented for potential rapid assessment of clinical samples. The system has been designed for immunoaffinity chromatography as a means of separating analytes of interest from biological matrices. The instrument is capable of sub-microliter sample injection and detection of labeled antigens by long wavelength laser-induced fluorescence (LIF). The laboratory-constructed device is assembled from an array of components including two syringe pumps, a nano-gradient mixing chip, a micro-injector, a diode laser, and a separation capillary column made from a polymer/silica (PEEKsil) tube. An in-house program written with LabVIEW software controls the syringe pumps to perform step gradient elution and collects the LIF signal as a chromatogram. Initial columns were packed with silica beads to evaluate the system. Optimization of the device has been achieved by measuring flow accuracy with respect to column length and particle size. Syringe size and pressure effects have also been used to characterize the capability of the pumps. Based on test results, a 200-microm x 25-mm column packed with 1-microm silica beads was chosen for use with a 500-microL syringe. The system was tested for mixer proportioning by pumping different compositions of buffer and fluorescent dye solutions in a stepwise fashion. A linear response was achieved for increasing concentrations of fluorescent dye by online mixing (R2=0.9998). The effectiveness of an acidic gradient was confirmed by monitoring pH post-column and measuring premixed solutions offline. Finally, assessment of detectability was achieved by injecting fluorescent dye solutions and measuring the signal from the LIF detector. The limit of detection for the system with these solutions was 10.0 pM or 10.0 amol on-column. As proof-of-principle, immunoaffinity chromatography was demonstrated with immobilized rabbit anti-goat IgG and a fluorescent dye-goat IgG conjugate as a model antigen.     

Backmixing and mass transfer in the design of immobilized-enzyme reactors

The effects of dispersion and mass transfer resistance on the degree of conversion in an immobilized-enzyme reactor have been considered theoretically. It is assumed that the immobilized enzymes obey a Michaelis–Menten relationship and backmixing can be characterized by a dispersion model. For two extreme cases (perfect mixing and piston flow), approximate equations are obtained, which can be readily used to evaluate the effect of mass transfer on degree of conversion. Numerical solutions are obtained for other intermediate cases. Design charts are given which set practical limits of enzyme reactor design.     

Mass transfer and mixing rates in fermentation vessels

The effects on mass-transfer and overall mixing rates of varying impeller geometry and operating speed have been studied for flat-bladed turbines in laboratory fermentors, in aerated aqueous solutions, and in unaerated and aerated suspensions (1.6% w/v) of paper pulp. In the absence of suspended solid, oxygen absorption rates could be correlated directly with power input. In the pulp suspension, oxygen absorption at a given power input was influenced by impeller geometry and operating speed. The data for the three-phase system can be correlated by a dimensionless equation relating oxygen-transfer rates and mixing times to the geometrical and operating parameters of the impellers.     

Rheology and Hydrodynamic Properties of Tolypocladium inflatum Fermentation Broth and Its Simulation

A physico-chemical, two phase simulated pseudoplastic fermentation (SPF) broth was investigated in which Solka Floc cellulose fibre was used to simulate the filamentous biomass, and a mixture of 0.1% (w/v) carboxymethyl cellulose (CMC) and 0.15 M aqueous sodium chloride was used to simulate the liquid fraction of the fermentation broth. An investigation of the rheological behaviour and hydrodynamic properties of the SPF broth was carried out, and compared to both a fungal Tolypocladium inflatum fermentation broth and a CMC solution in a 50 L stirred tank bioreactor equipped with conventional Rushton turbines. The experimental data confirmed the ability of the two phase SPF broth to mimic both the T. inflatum broth bulk rheology as well as the mixing and mass transfer behaviour. In contrast, using a homogeneous CMC solution with a similar bulk rheology to simulate the fermentation resulted in a significant underestimation of the mass transfer and mixing times. The presence of the solid phase and its microstructure in the SPF broth appear to play a significant role in gas holdup and bubble size, thus leading to the different behaviours. The SPF broth seems to be a more accurate simulation fluid that can be used to predict the bioreactor mixing and mass transfer performance in filamentous fermentations, in comparison with CMC solutions used in some previous studies.     

The pre-steady-state hydrolysis of ATP by porcine brain (Na+ + K+)-dependent ATPase.

The hydrolysis of [gamma-32P]ATP by porcine brain (Na+ + K+)-stimulated ATP phosphohydrolase (EC 3.6.1.3) has been studied at 28 degree C in a rapid mixing quenched-flow apparatus. An "early burst" in the release of Pi from ATP has been observed when the enzyme is mixed with ATP, Na+ and a relatively high concentration of K+ (10 mM) but the burst is less pronounced with 0.5 mM K+. This "early burst" of Pi release is suppressed when the enzyme is pre-mixed with 10 mM K+ or 20% (v/v) dimethylsulphoxide before mixing with ATP and Na+, and premixing of enzyme with Na+ antagonizes this effect of dimethylsulphoxide. The results have been analysed by a non-linear least squares regression treatment and are consistent with a mechanism involving three steps, one of which may be a relatively slow change in enzyme conformation following release of Pi from its covalent linkage with the enzyme, in addition to formation of the enzyme-substrate complex. Rate constants (and S.E.) for these steps have been calculated and the roles of phospho-enzyme and other intermediates in the reaction mechanism of the transport ATPase are dicussed.     

Studying Mixing in Non-Newtonian Blue Maize Flour Suspensions Using Color Analysis

Background

Non-Newtonian fluids occur in many relevant flow and mixing scenarios at the lab and industrial scale. The addition of acid or basic solutions to a non-Newtonian fluid is not an infrequent operation, particularly in Biotechnology applications where the pH of Non-Newtonian culture broths is usually regulated using this strategy.

Methodology and Findings

We conducted mixing experiments in agitated vessels using Non-Newtonian blue maize flour suspensions. Acid or basic pulses were injected to reveal mixing patterns and flow structures and to follow their time evolution. No foreign pH indicator was used as blue maize flours naturally contain anthocyanins that act as a native, wide spectrum, pH indicator. We describe a novel method to quantitate mixedness and mixing evolution through Dynamic Color Analysis (DCA) in this system. Color readings corresponding to different times and locations within the mixing vessel were taken with a digital camera (or a colorimeter) and translated to the CIELab scale of colors. We use distances in the Lab space, a 3D color space, between a particular mixing state and the final mixing point to characterize segregation/mixing in the system.

Conclusion and Relevance

Blue maize suspensions represent an adequate and flexible model to study mixing (and fluid mechanics in general) in Non-Newtonian suspensions using acid/base tracer injections. Simple strategies based on the evaluation of color distances in the CIELab space (or other scales such as HSB) can be adapted to characterize mixedness and mixing evolution in experiments using blue maize suspensions.     

Optical analysis of liquid mixing in a minibioreactor

A novel optical sensor was used to study mixing and mean circulation time in a model minibioreactor (12.5 mL stirred vessel, equipped with a paddle impeller). Rotational rates in the range of 10-1,000 rpm corresponding to Reynolds number between 14 and 1,350 were studied. Results suggest that depending on the impeller rotational speed, mixing times up to 214 +/- 87 s can be reproducibly achieved. The minibioreactor was operated in the transitional regime, and it was determined that the non-dimensional form for mixing time, NTheta(M) was linearly dependent on Reynolds number. A linear correlation between mean circulation time and the inverse of rotational speed was also determined. The mean circulation time dependence on rotational speed in the 12.5 mL stirred vessel is similar to those found in large-scale stirred vessels. These results suggest that mixing and circulation times found in large-scale reactors can be replicated in minibioreactors.     

Time-resolved analysis of macromolecular structures during reactions by stopped-flow electrooptics.

A stopped-flow field-jump instrument and its use for the analysis of macromolecular structure changes during reactions is described. The operation of the new instrument is simple and reliable, owing to a new type of cell construction with electrodes directly integrated in a quartz cuvette: major advantages are the relatively low demand on sample quantities and a high time resolution. The stopped flow is characterized by a dead time of approximately 0.5 ms. Electric field pulses with field strengths up to 20 kV/cm and rise times in the nanosecond range are applied at adjustable times after stop of the flow. The time resolution of the optical detection is up to the nanosecond time range. The instrument may be used for the combination of stopped flow with temperature-jump and field-jump experiments. A particularly useful new application is the analysis of macromolecular reactions by electrooptical measurements, because electrooptical data provide information about structures. This is demonstrated for the intercalation of ethidium into double-helical DNA. The transients, measured at 313 nm, where the signal is exclusively due to ethidium bound to the DNA, demonstrate a relatively high negative dichroism at 0.5 ms after mixing. The absolute value of this negative dichroism increases in the millisecond time range and approaches the equilibrium value within about a second. The dichroism decay time constants demonstrate a clear increase of the effective DNA length due to ethidium binding, already 0.5 ms after mixing; a further increase to the equilibrium value is found in the millisecond time range. The analysis of these data demonstrate the existence of up to three relaxation processes, depending on the conditions of the experiments. The dichroism amplitudes, together with the decay time constants, indicate that all the reaction states found in the present investigation are complexes with insertion of ethidium residues between basepairs. Moreover, the data clearly show the degree of intercalation in the intermediate states, which is very useful information for the quantitative assignment of the mechanism.     

Discrete particle simulations predicting mixing behavior of solid substrate particles in a rotating drum fermenter.

A soft-sphere discrete particle model was used to simulate mixing behavior of solid substrate particles in a slow rotating drum for solid-state fermentation. In this approach, forces acting on and subsequent motion of individual particles can be predicted. The (2D) simulations were qualitatively and quantitatively validated by mixing experiments using video and image analysis techniques. It was found that the simulations successfully predicted the mixing progress as a function of the degree of filling and size of the drum. It is shown that only relatively large, straight baffles perpendicular to the drum wall (67% of the drum radius) increase the mixing performance of the rotating drum. Considering the different aspects of mixing dealt with in this work, it is concluded that the soft sphere discrete particle model can serve as a valuable tool for investigating mixing of solid substrate particles. Finally, it is expected that this model may evolve into a potential tool for design and scale-up of mixed solid-state fermenters.