Jose C. Merchuk

Aqueous two-phase systems for protein separation: Studies on phase inversion

Phase equilibrium studies were done with the PEG 400-phosphate system, obtaining equilibrium binodal lines, tie lines and phase inversion points. A method of calculation of the critical point on the binodal curve is described. The influence of the presence of NaCl in solution was studied, and the comparative results are presented. It was found that in some range of concentration the shift produced in the binodal line can be important. The rate of phase separation can be used as an indication of which of the phases is continuous. Using this method the phase inversion point can be determined in the system for each tie line. A range of ambiguity was found, where the continuity of the phases is affected not only by the composition of the mixture, but also by the fluid dynamics. Within this range, gentle agitation produces a bottom-continuous suspension. while strong agitation produces a top-continuous suspension. Two inversion points exist therefore on each tie line, delimiting on the phase equilibrium plane a region where the phase continuity depends on fluid dynamics. The convergence of this region towards the critical point can be used to control of the consistency of the experimental data.

Hepatocyte behavior within three-dimensional porous alginate scaffolds

A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.

A model integrating fluid dynamics in photosynthesis and photoinhibition processes

Abstract A mathematical representation of the growth of a photosynthetic system in an alternating light/dark regime is proposed, integrating fluid dynamics and maintenance in the three-state model developed earlier by Eilers and Peeters (1998. Ecological Modeling , 42 , 199–215). The model was solved analytically and the constants were fitted to experimental data obtained in a thin film tubular reactor. The theoretical prediction that the introduction of light/dark cycle may enhance the growth, was confirmed by the experimental results, supporting the idea of utilizing ordered mixing to obtain more efficient growth. The model allows to predict collapse of cultures in photobioreactors either under light-deficit or light-excess conditions, as well as the influence of mixing on these critical phenomena. It has also been shown that chlorophyll fluorescence measurements can be used to estimate the growth at steady state. This paper presents an approach to model the kinetics of photosynthetic systems for photobioreactor design under conditions of simultaneous occurrence of photoinhibition in one region of the reactor, and photolimitation in another. The model takes into account the movement of the cells from one region to the other.

Interaction between CO2-mass transfer, light availability, and hydrodynamic stress in the growth of phaeodactylum tricornutum in a concentric tube airlift photobioreactor

The microalga Phaeodactylum tricornutum was grown in a concentric tube airlift photobioreactor. A maximum specific growth rate of 0. 023 h-1 was obtained using a superficial gas velocity around 0.055 m/s. Lower or higher gas flow rates limited the culture performance. To establish if the observed limitation was due to CO2 or to the photosynthetically active irradiance, characteristic times for mixing, mass transfer and CO2 consumption, and the photon flux absorbed by the culture were analyzed. The CO2-gradients in the culture were shown to be responsible for the limitation during the exponential growth phase, and both CO2 and light irradiance were limiting in the linear growth phase. The decrease in specific growth rate relative to the maximum was found to be related to the specific gas-liquid interfacial area, the length scale of the microeddies and the shear rate. Copyright 1998 John Wiley & Sons, Inc.

Light/dark cycles in the growth of the red microalga Porphyridium sp.

The effect of light/dark cycles on the growth of the red microalga Porphyridium sp. was studied in a tubular loop bioreactor with light/dark cycles of different frequencies and in two 35-L reactors: a bubble column reactor and an air-lift reactor. Photon flux densities were in the range of 50 to 300 μE m-2 s-1, and flow rates were 1 to 10 L min-1. Under conditions of low illumination and high flow rates, similar results were obtained for the bubble column and air-lift reactors. However, higher productivities-in terms of biomass and polysaccharide-were recorded in the air-lift reactor under high light intensity and low gas flow rates. The interactions of both photosynthesis and photoinhibition with the fluid dynamics in the bioreactors was taken as the main element that allowed us to interpret the differences in performance of the bubble column and the air-lift reactor. It is suggested that the cyclic distribution of dark periods in the air-lift reactor facilitates better recovery from the photoinhibition damage suffered by the cells. Copyright 1998 John Wiley & Sons, Inc.

Comparison of photobioreactors for cultivation of the red microalga Porphyridium sp

The growth of the red microalga it Porphyridium sp was studied in three bench-scale bioreactors of 13 dm3 volume: a bubble column, an airlift reactor and a modified airlift reactor with helical flow promoters in the top of the downcomer. Most of the experiments reported were run with a photon flux density of 250 µE m−2 s−1, but other illuminances were studied as well. Superficial gas velocities were in the range of 5.4 × 10−4 to 82 × 10−4 m s−1 (0.033–0.5 vvm). Algal growth in the airlift reactor with helical flow promoters had lower gas requirements than in the other reactor configurations. This implies lower costs in air compression and in air and CO2 requirements. It was concluded that the advantages found are related to the particular fluid dynamic characteristics of the reactor. © 2000 Society of Chemical Industry

Bioreactor system design.

Design of a bioreactor system - overview. Part 1 Biological systems and media design: organism selection bacterial, yeast, and fungal cultures - effect of microorganism type and culture characteristics on bioreactor design and operation design, formulation, and optimization of media. Part 2 Bioreactor design: fundamentals of bioreactor design stirred tank bioreactors pneumatically agitated bioreactors membrane reactors immobilized microorganism bioreactors immobilized animal cell bioreactors plant cell bioreactors photobioreactors bioreactor operation modes bioreactor scale-up. Part 3 Bioreactor support systems: sterilization and containment bioreactor system supplies.

Why use air-lift bioreactors?

Abstract Air-lift bioreactors are a relatively new type of fermenter, offering several advantages for large-scale bioprocesses, for animal and plant cell culture in particular. This review presents these advantages as well as some of the limitations, all of which are determined by the fluid dynamics and mass transfer characteristics of air-lift reactors.

Studies of mixing in a concentric tube airlift bioreactor with different spargers

Abstract Gas hold-up, e, liquid velocity, JLr, axial dispersion coefficient, Ez, and mixing time, tm, have been measured in a concentric tube airlift bioreactor (12 × 10−3 m3 in volume) using sea water, as a function of the superficial gas velocity, JGr, (up to 0.21 m/s). Seven different spargers were tested. Four of them were cylindrical (pore size from 60 μm to 1 × 10−3 m) and three were porous plates (pore size from 30 to 120 μm). Three different flow regimes are observed in hold-up and liquid velocity over the range of JGr covered: uniform bubbly flow at low gas velocities, heterogeneous flow at high gas velocities and a transition flow between bubbly and heterogeneous. The spargers with smaller pore sizes produce more hold-up and slow down velocity, because more gas recirculates into the downcomer. The change from uniform bubbly flow to transition flow appears because of the start of coalescence. In heterogeneous flow, where the bubble size is set by the degree of coalescence, no influence of the difference between the spargers used could be detected. Gas hold-up in the riser, er, could be represented, over the whole range of JGr used, by E r =α J Gr J Lr β When the Bodenstein number for the gas-liquid mixture, BoLG, and the Froude number, Fr, are used to represent axial dispersion, three flow regimes appear which correspond to those observed when hold-up and liquid velocity are plotted vs JGr. The axial dispersion coefficient, Ez, could be represented by E z =K 5 d e J Gr E n 4 where de is the equivalent diameter of the reactor. This equation suggests that Ez is dominated by bubble slip, and fits 78% of the measured data with less than 20% error. It has also been applied satisfactorily to the results obtained by other authors who used different systems and liquid phases. Mixing time depends on sparger geometry and pore size only at low gas velocities. At high gas velocities, mixing time is practically independent of sparger and gas velocity.

Mathematical modelling and computer simulation of aqueous two-phase continuous protein extraction☆

An extended mathematical model has been developed to describe the continuous, steady state operation of an aqueous two-phase system for protein extraction. The basic model is based on steady state mass balances of the main components and phase equilibrium data. Phase equilibrium (binodial curve) was fitted by an equation that relates poly(ethylene glycol) (PEG) to phase forming salt (e.g. phosphate) and added NaCl. Experimental data on the separation of α-amylase from B. subtilis supernatant in a PEG4000/phosphate system was used. The data show the effect of NaCl was used to carry out the extraction of α-amylase into the PEG phase and back into the salt phase. The partition coefficient of the α-amylase was fitted to a sigmoidal Boltzman curve and gave a very good fit. Two simulations were carried out to show the effect of phase ration on purification and this was represented in an equilibrium system diagram of material balances. The model has been extended to account for phase separation kinetics and thus aspects of continuous processing. Modelling the rate of settling of the two phases using appropriate correlations has been presented and discussed.