Scale-down studies on the hydrodynamics of two-liquid phase biocatalytic reactors

The maintenance of constant interfacial area per unit volume is a key parameter for the successful scale-up of two-liquid phase bioconversion processes. To date, however, there is little published information on the hydrodynamics of such systems and a suitable basis for scale-up has yet to be defined and verified. Here we report power input and hydrodynamic data for a whole-cell bioconversion process using resting cells of Rhodococcus R312 to catalyse the hydration of a poorly water-soluble substrate 1,3-dicyanobenzene (1,3-DCB). Experiments were performed in geometrically similar 3-L and 75-L reactors, each fitted with a three-stage Rushton turbine impeller system. The two-phase system used comprised of 20% v/v toluene dispersed in 0.1 M aqueous phosphate buffer containing up to 10 g(ww) x L(-1) of resuspended biocatalyst and 20 g x L(-1) 1,3-DCB. The power input to the 3-L reactor was first determined using an air-bearing technique for both single-phase and two-phase mixing. In both cases, the power number attained a constant value of 11 at Re>10,000, while the measured power inputs were in the range 0.15-3.25 kW x m(-3). Drop size distributions and Sauter mean drop diameters (d(32)) were subsequently measured on-line in both reactors, using an in-situ light-backscattering technique, for scale-up on the basis of either constant power input per unit volume or constant tip speed. At both scales d(32) decreased with increasing agitation rate, while the drop size distributions obtained were log-normal. All the measured d(32) values were in the range of 30-50 microm, with the lowest values being obtained in systems with biocatalyst present. In all cases, constant power input per unit volume was found to be the most suitable basis for scale-up. This gave virtually identical d(32) values and drop size distributions at both scales. A number of correlations were also identified that would allow reasonable prediction of d(32) values for various agitation rates at each scale. While the results obtained are for a particular phase system, the scale-down methodology presented here would allow the rapid evaluation of other bioconversion processes in the 3-L reactor with a 25-fold reduction in scale. In this way, potential problems that might be encountered at the larger scale, such as the carryover of antifoam from the fermentation stage, could be quickly and efficiently identified.     

Quantitative measurements of mixing intensity in shake-flasks and stirred tank reactors. Use of the Mixmeter, a mixing process analyzer

A new mixing probe has been developed which measures the motions of the fluid during mixing as pressure fluctuations and converts the measurements into a mixing signal (MS). The MS is the root mean square (RMS) pressure fluctuation in the 1-64Hz range as determined by a sensitive pressure sensor and a digital signal processor specifically designed for the purpose. The MS is a measure of the actual mixing flow of the fluid rather than a measurement of the input motions or energies into the reactor system (e.g. RPM, torque or power). In other studies, the MS has been measured as a function of mixing speed in numerous sized reactors from 10 to 1000l, and provides consistent and reproducible measurements. The MS increases monotonically as a function of mixing speed, with a change of slope corresponding to the transition from laminar to turbulent mixing regimes. Maps of MS as a function of location in the reactor are useful in understanding stirred tank reactor design and performance. Quantitative measurements of mixing are especially useful during process development as a tool to increase the success of scale-up during the transition from process development to manufacturing. Measurements at a fixed location in a given reactor are useful in understanding changes in mixing that occur during the course of a given process, and are useful in manufacturing situations where validated documentation of lot-to-lot consistency of mixing is required (e.g. pharmaceutical manufacturing). In addition, the probe has been used to measure mixing in vessels with vibrational mixers with similar results. The probe has been successfully used in feedback loops to control either mixing speed or vibrational mixing amplitude in order to maintain constant mixing of the fluid during processing. With this system it is possible to maintain constant mixing over a wide range of fluid volumes in a given reactor, and, for instance, to compensate for changes in viscosity throughout the course of the process. Adaptations of this system for the measurement of mixing in shake-flasks is described in this paper.     

Isoelectric precipitation of soy protein. II. Kinetics of protein aggregate growth and breakage

Protein aggregate growth and breakage in agitated suspensions are modeled. The model includes growth of particles by a turbulent collision mechanism and breakage by a hydrodynamic shear mechanism. In the model, breakage results in the splitting of the particles into several small fragments. The model parameters are a growth rate constant and a breakage rate constant. Aggregate size distributions were measured with a Coulter counter and the data interpreted using a population balance that governs the steady-state particle size distribution in a continuous stirred tank reactor. Effects of changes in the operating variables pH, concentration, mean residence time, ionic strength, and mixing power input on the model kinetic parameters are investigated.     

Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

Anaerobic degradation of solid material: importance of initiation centers for methanogenesis, mixing intensity, and 2D distributed model

Batch anaerobic codigestion of municipal household solid waste (MHSW) and digested manure in mesophilic conditions was carried out. The different waste-to-biomass ratios and intensity of mixing were studied theoretically and experimentally. The experiments showed that when organic loading was high, intensive mixing resulted in acidification and failure of the process, while low mixing intensity was crucial for successful digestion. However, when loading was low, mixing intensity had no significant effect on the process. We hypothesized that mixing was preventing establishment of methanogenic zones in the reactor space. The methanogenic zones are important to withstand inhibition due to development of acids formed during acidogenesis. The 2D distributed models of symmetrical cylinder reactor are presented based on the hypothesis of the necessity of a minimum size of methanogenic zones that can propagate and establish a good methanogenic environment. The model showed that at high organic loading rate spatial separation of the initial methanogenic centers from active acidogenic areas is the key factor for efficient conversion of solids to methane. The initial level of methanogenic biomass in the initiation centers is a critical factor for the survival of these centers. At low mixing, most of the initiation methanogenic centers survive and expand over the reactor volume. However, at vigorous mixing the initial methanogenic centers are reduced in size, averaged over the reactor volume, and finally dissipate. Using fluorescence in situ hybridization, large irregular cocci of microorganisms were observed in the case with minimal mixing, while in the case with high stirring mainly dead cells were found.     

Instability caused by high strength of cheese whey in a UASB reactor

The anaerobic digestion of cheese whey was studied in a UASB reactor. The profiles of the reactor, i.e., the distributions of the substrate concentration and pH under different operating conditions were developed. From the concentrations of substrates measured at various levels above the bottom of the reactor, two reaction stages, namely acidogenesis and methanogenesis, were distinguished. The instability caused by high influent concentration was interpreted as the accumulation of VFAs in the acidogenic stage beyond the assimilative capacity of the methanogenic stage. A range of stable operating conditions was predicted from the results of the profile measurements. The optimal influent concentration was found to be between 25 and 30 g COD/L at an HRT of 5 days for system stability. Other options fro stability control were discussed. (c) 1993 John Wiley & Sons, Inc.     

Oxytetracycline production by Streptomyces rimosus: gas and temperature patterns in a solid-state column reactor

During oxytetracycline production by Streptomyces rimosus TM-55 on sweet potato residue in a solid-state column reactor, the moisture content increased by between 2 and 5% (w/w) during incubation, from an initial content of 70 to 73%, and pH initially increased from 6.0 to 7.3, followed by a gradual decrease to 6.2. Appropriate aeration enhanced oxytetracycline production, while mixing only once daily decreased it. The temperatures in the centre and upper layers of each reactor were higher than elsewhere in static non-aerated cultures. The maximum CO₂ concentration ranged from 2.9 to 3.2% (v/v) and the minimum O₂ concentration was 11.0 to 17.2% (v/v) in static cultures. Under optimal conditions, each gram of dry substrate produced the equivalent potency of 12 mg oxytetracycline.The authors are with the Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 10617, Republic of China     

Effect of liquid-phase surface tension on hydrodynamics of a three-phase airlift reactor with an enlarged degassing zone

The effect of the addition of ethanol (10?g/l) to the liquid-phase on gas and solids holdup, circulation and mixing times and interstitial liquid velocity in a three-phase airlift reactor was investigated. The airlift reactor (60?l) is of the concentric draught-tube type with an enlarged degassing zone. Ca-alginate beads were used as solid-phase and airflow rate (from 1.9 to 90.2?l/min) and solids loading (0–30% (v/v)) were manipulated. Riser and downcomer gas holdup were found to increase with the addition of ethanol, leading to a decrease on the relative solids holdup. The presence of ethanol seems to have no influence on the circulation time. On the other hand, mixing time variation depends on the solids loading and airflow rate. Riser and downcomer interstitial liquid velocity are lower for ethanol solution than for water.     

Gas-phase influence on the mixing in a fluidized bed bio-reactor

Summary The liquid and solids mixing in fluidized bed bio-reactors containing particles with a density only slightly higher than water (1100 kg/m³) is generally consistent with the results found in previous studies for reactors with particles of higher density. The liquid mixing can be described by an axial dispersion model for a large variety of conditions while the solids follow the streamlines of the liquid. In the presence of a gas phase the degree of mixing of both the liquid and the solid phase increased. This effect became larger with increasing reactor diameter. In the extrapolation of laboratory data of three phase fluidized bed bio-reactors to pilot plant systems this effect should be taken into account. The liquid and solids mixing may have a substantial effect on overall conversion rates and on possible microbial stratification in the reactor.Nomenclature Bo Bodenstein number v L/D (-) - D _r diameter of the fluidized bed reactor (m) - D ₁ Dispersion coefficient of the liquid phase (m²/s) - D _g dispersion coefficient of the solid phase (m²/s) - E(in) normalized dye concentration function entering the ideally mixed tank reactor (-) - E(t) normalized dye concentration function as measured (-) - L length of the axial dispersed reactor (m) - t time after dye injection (s) - t _m time constant for microbial selection (s) - t _s solid mixing time constant (s) - t time interval in which a particle migrates within the bed (s) - v _t superficial gas velocity (m/s) - v _g superficial liquid velocity (m/s) - z migration distance of a particle in the bed (m) - ₁ in situ growth rate of a dominant organism (s^-1) - ₂ in situ growth rate of a recessive organism (s^-1) - average residence time in the axial dispersed reactor (s) - _t average residence time in the ideally mixed tank reactor (s)     

Characterization of a pilot plant airlift tower loop bioreactor. I: evaluation of the phase properties with model media

Investigations were carried out in a 9 m high, 4 m(3) volume, pilot plant airlift tower loop bioreactor with a draft tube. The reactor was characterized by measuring residence time distributions of the gas phase using pseudostochastic tracer signals and a mass spectrometer and by evaluating the mixing in the liquid phase with single-pulse tracer inputs. The local gas holdup and the bubble size (piercing length) were measured with two-channel electrical conductivity probes. The mean residence times and the intensities of the axial mixing in the riser and downcomer and the circulation times of the phases as well as the fraction of the recirculated gas phase were evaluated. The gas holdup in the riser is nearly uniform along the reactor. In the downcomer, it diminishes from top to bottom. The liquid phase dispersion coefficients, D(L), are smaller than those measured in the corresponding bubble columns. In the pilot plant with tap water the following relationship was found: D(Lr) = cw(SG) (n); with c = 203.4; n = 0.5;D(Lr)(cm(2) s(-1);) and W(SG)(cm s(-1)) where D(Lr) is the longitudinal dispersion coefficient in the riser and W(SG) is the superficial gas velocity. The gas phase dispersion coefficients in the riser of the pilot plant, D(Gr), are also enlarged with increasing superficial gas velocity, W(SG), however, no simple relationship exists. Parameter D(Gr) is the highest in the presence of antifoam agents, intermediate in tap water, and the smallest in ethanol solution.     

The effects of mixing on bioprocesses. Concentration distributions and mechanical shear stress

The effects of mixing on batch alcohol fermentation of diluted solutions of starch hydrolysate is studied. The results of a limited number of samples simultaneously drawn at different locations in the reactor and after different reaction times have been used in a simple mathematical model to provide a picture of the concentration distributions within the reaction environment. The optimal mixing conditions for the fermentation are met at rotation speeds between 1.7 and 5.0 s^?1, while the broth homogeneity obviously increases indefinitely with increasing this parameter. This suggests the existence of a shear stress for the biomass, whose effect increases with the application time and seems to affect the process mainly at the end of the fermentation.     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: II. Operation of a laboratory-scale reactor

In Part I of this series,(1) we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory-scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow.     

Characterization of a pilot plant airlift tower loop bioreactor: II. Evaluation of global mixing properties of the gas phase during yeast cultivation

Saccharomyces cerevisiae was cultivated in a 4-m(3) pilot plant airlift tower loop reactor with a draft tube in batch and continuous operations and for comparison in a laboratory airlift tower loop reactor of 0.08 m(3) volume. The reactors were characterized during and after the cultivation by measuring the distributions of the residence times of the gas phase with pseudostochastic tracer signals and mass spectrometer and by evaluating the mixing in the liquid phase with a pulse-shaped volatile tracer signal and mass spectrometer as a detector. The mean residence times and the intensities of the axial mixing in the riser and downcomer, the circulation times of the gas phase, and the fraction of the recirculated gas phase were evaluated and compared.     

Factors controlling planktonic size spectral responses to autumnal circulation in a Mediterranean lake

1. Studies of planktonic size spectra have been common in recent years, but few concerning the effects of autumnal lake processes on those distributions have been reported. We carried out such a study for 93 days during early circulation in a small, mesotrophic, seepage lake with only benthivorous fish. Two distinct mixing periods occurred before full circulation in Las Madres lake (Spain). As a proxy of overall planktonic‐ and phytoplanktonic size distributions, the shape of a Pareto I power function was traced over time. The phytoplanktonic size spectrum was also used to test the hypothesis that phytoplankton might show a trophic cascade during early circulation. 2. Our results demonstrated that the overall size spectrum changed smoothly during the autumnal mixing, with sequential changes controlled by a combination of biotic and abiotic variables, albeit experiencing different temporal delays, always shorter than a week. A first combined, autogenic process, driven both by algal competition and a crustacean trophic effect, and a second physical‐forcing process, driven by the combined effect of convective cooling and decreasing irradiance on mixed layer dynamics, may be related to the overall planktonic spectrum in the first and second periods of mixing, respectively. 3. The phytoplanktonic size spectrum did not appear to be dictated by a trophic cascade in Las Madres lake, their dynamics being mostly controlled by physical forcing, along with some effect of non‐edible primary producers and cladocerans in the first and second periods of mixing, respectively. 4. Our results and others covering early circulation with longer sampling periods suggest that planktonic size spectral dynamics during lake circulation is context‐dependent (i.e. varying from one lake to another), thus preventing generalisation. However, when further studies with finer temporal resolution have been carried out, it is likely that clear‐cut patterns in the planktonic size spectrum will emerge, arising from the interplay of autogenic plankton dynamics, implying some resistance to community change because of external physical forcing, and the velocity of autumnal mixing.     

Investigations in commonness and rarity: a comparative analysis of co-occurring, congeneric Mexican trees

Population size distributions were examined for 12 species of trees co-occurring at Chamela Biological Station in Jalisco, Mexico. Species had been selected as congeneric pairs and trios similar in gross morphology and ecology in order better to identify correlates of relative abundance. Rarer species were found unanimously to have more irregular distributions of individuals among size classes than more common species when distributions were compared to a smooth, descending curve constructed from population mean stem diameters (an exponential distribution). Examination among species of patterns of deviation from these corresponding smooth distributions indicates that the most reasonably inferred cause for the observed pattern is consistent differences in degree of fluctuation in recruitment into adult size classes. These results thereby suggest a demographic difference between locally rarer and more common species that may be generally associated with observed differences in relative abundance and indicate a focus for management of rarity in forest trees.     

A model investigation of the velocity and pressure spectra in vascular murmurs

A physical model consisting of an axisymmetrical jet in a rigid plexiglass pipe was used to study the flow and pressure fluctuations downstream from an aortic stenosis. The fluctuating velocity components, u and v, at several locations in the steady liquid jet were measured using a laser Doppler anemometer system. Simultaneous wall pressure fluctuations were monitored by an array of nine miniature pressure transducers wall mounted in the axial direction. This paper presents the detailed measurements of mean velocity profiles, turbulent intensity distributions and RMS pressure fluctuations. The energy spectra obtained for the pressure fluctuations and the u and v velocity components are compared. Contrary to earlier works, we found that the differences between peak frequencies of the pressure spectra and the characteristic frequencies of the velocity spectra vary with positions downstream from the nozzle. These differences are discussed in light of pseudosound generation by the eddy structures in the stenotic flow field.     

Characterization of stirrers for screening studies of enzymatic biomass hydrolyses on a milliliter scale

The evaluation of mixing quality is an important factor for improving the geometry of stirred-tank reactors and impellers used in bioprocess engineering applications, such as the enzymatic hydrolysis of plant materials. Homogeneity depends on different factors, including the stirrer type and the reactor type (e.g., ratio of diameter/height, ratio of impeller tip diameter/reactor diameter) with or without baffles. This study compares two impellers for enzymatic hydrolysis of suspensions of biomass particles on a milliliter scale. Both impellers were derived from industrially relevant geometries, such as blade and grid stirrers, although the geometry of the second stirrer was slightly modified to an asymmetric shape. The stirrers were investigated with different stirrer–reactor configurations. This was done experimentally and with the aid of computational fluid dynamics. The flow field, mixing numbers, power characteristics and initial conversion rates of sugars were considered to compare the two stirrers. The simulated mixing numbers and power characteristics in baffled and unbaffled milliliter-scale reactors were found to be in good agreement with the measured mixing times and power consumption. The mixing numbers required to reach homogeneity were much higher for the symmetric impeller and remained at least twice as high as the mixing numbers required when using the asymmetric impeller. The highest initial sugar releases from milled corn stover suspensions were achieved with the asymmetric impeller shape. Regardless of the differences in the flow fields or mixing times, diverging enzymatic sugar releases could be confirmed for Newtonian media only.     

Spectral fluctuation of a single fluorophore conjugated to a protein molecule

We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous buffer to methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to microenvironmental changes of fluorophores. The time dependence of the fluorescence spectra of fluorophores attached to S1 molecules solely showed a fluctuation with time over a time scale of seconds. Because the fluorescence spectra of the same fluorophores directly conjugated to a glass surface remained constant, the spectral fluctuation observed for the fluorophores attached to S1 is most likely due to slow spontaneous conformational changes in the S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.     

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...     

Diesel particulate emissions from used cooking oil biodiesel

Two different biodiesel fuels, obtained from waste cooking oils with different previous uses, were tested in a DI diesel commercial engine either pure or in 30% and 70% v/v blends with a reference diesel fuel. Tests were performed under a set of engine operating conditions corresponding to typical road conditions. Although the engine efficiency was not significantly affected, an increase in fuel consumption with the biodiesel concentration was observed. This increase was proportional to the decrease in the heating value. The main objective of the work was to study the effect of biodiesel blends on particulate emissions, measured in terms of mass, optical effect (smoke opacity) and size distributions. A sharp decrease was observed in both smoke and particulate matter emissions as the biodiesel concentration was increased. The mean particle size was also reduced with the biodiesel concentration, but no significant increases were found in the range of the smallest particles. No important differences in emissions were found between the two tested biodiesel fuels.