Estimation of axial dispersion in horizontal rotating tubular bioreactor by means of a structured model

A horizontal rotating tubular bioreactor (HRTB) is designed as the combination of a "thin layer bioreactor" and a "biodisc" reactor. The investigation of mixing in HRTB was done by the temperature step method in a wide range of process conditions [residence time (t_z=360036000 s) and bioreactor rotation speed (n=0.0830.917 s^у)]. In all experiments heat losses were detected. A mathematical model based on "tank in series" concept was developed to describe the mixing in HRTB - a "spiral flow" model (SFM) which has incorporated heat losses. However, the simulations of SFM could be used for calculation of temperature response curves for the case when there is no heat losses. These corrected curves were used then to estimate Bodenstein number as a parameter of standard dispersion model (SDM). The obtained Bodenstein numbers were in the range 10-17. The simulations showed that SFM was more capable to describe the mixing in HRTB giving better fitting with experimental measurements than SDM, indicating that mixing pattern in HRTB is too complex to be described with this relatively simple, one-parameter model.     

Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

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.     

Primary production, light and vertical mixing in Potter Cove, a shallow bay in the maritime Antarctic

Phytoplankton photosynthesis was measured during spring-summer 1991-1992 in the inner and outer part of the shallow Potter Cove, King George Island. Strong winds characterise this area. Wind-induced turbulent mixing was quantified by means of the root-mean square expected vertical displacement depth of cells in the water column, Z_t. The light attenuation coefficient was used as a measure of the influence of the large amount of terrigenous particles usually present in the water column; 1% light penetration ranged between 30 and 9 m, and between 30 and 15 m for the inner and outer cove, respectively. Obvious differences between photosynthetic capacity [P*_max; averages 2.6 and 0.6 µg C (µg chlorophyll-a)^-1 h^-1] and photosynthetic efficiency {!*; 0.073 and 0.0018 µg C (µg chlorophyll-a)^-1 h^-1 [(µmol m^-2 s^-1)^-1]} values were obtained for both sites during low mixing conditions (Z_t from 10 to 20 m), while no differences were found for high mixing situations (Z_t>20 m). This suggests different photoacclimation of phytoplankton responses, induced by modifications of the light field, which in turn are controlled by physical forcing. Our results suggest that although in experimental work P*_max can be high, wind-induced mixing and low irradiance will prevent profuse phytoplankton development in the area.     

Tight control of growth and cell differentiation in photoautotrophically growing moss (<Emphasis Type="Italic">Physcomitrella</Emphasis><Emphasis Type="Italic">patens</Emphasis>) bioreactor cultures

The use of the moss Physcomitrella patens as a production system for heterologous proteins requires highly standardised culture conditions. For this purpose a semi-continuous photoautotrophic bioreactor culture of Physcomitrella was established. This culture grew stably for 7 weeks in a 5-l bioreactor with a dilution rate of 0.22/day. Enrichment of the air for aeration in a batch bioreactor culture with 2% (v/v) CO₂ resulted in an increase in the specific growth rate to 0.57/day. Changes in the pH of the semi-continuous bioreactor culture medium between pH 4.5 and pH 7.0 influenced protonema differentiation; however it did not negatively affect the growth rate compared to uncontrolled pH. The advantages of Physcomitrella as a system for the production of heterologous proteins in plants are discussed.     

Effect of continuous feeding of galactose on the production of recombinant glucose oxidase using Saccharomyces cerevisiae

A recombinant strain of Saccharomyces cerevisiae harboring GOD gene originated from Aspergillus niger was used for the production of extracellular glucose oxidase. The effect of continuous galactose feeding on the induction of GAL-10 promoter was examined in a 5 l bioreactor. The highest enzyme production level (164 U cm^х) was achieved at 96 h of cultivation. The production performance was compared with the results of fed-batch cultivations carried out in the same laboratory. Continuous feeding mode was found to be less productive due to excess ethanol formation and plasmid instability.     

Stochastic modelling of axial dispersion in external-loop airlift bioreactors

The paper presents a model of the motion of a particle subjected to several transport processes in connection with mixing in two phase flow. A residence time distribution technique coupled with a one-dimensional dispersion model was used to obtain the axial dispersion coefficient in the liquid phase, D_ax. The proposed model of D_ax for an external-loop airlift bioreactor is based on the stochastic analysis of the two-phase flow in a cocurrent bubble column and modified for the specific flow in the airlift reactor. The model takes into account the riser gas superficial velocity, the riser liquid superficial velocity, the Sauter bubble diameter, the riser gas hold-up, the downcomer-to-riser cross sectional area ratio. The proposed model can be applied with an average error of ᆨ.     

Transient sensitivity analysis of a cybernetic model of microbial growth on two substrates

When a microbe has a choice of two substrates for its growth in a fermentation medium, its preference varies with the substrates and with time. This "informed" choice is conveniently expressed by cybernetic models. For the growth of Klebsiella oxytoca in a medium of glucose and lactose, a one-parameter cybernetic model of growth has been employed in a batch bioreactor to analyse sensitivity of the fermentation to perturbations in the parameter, ! (0 h ! h 1). The sensitivity surfaces in the (!-time) space show interesting variations which are discussed. An important observation is that while growth is best promoted with !=1, i.e. sequential consumption of the substrates [8], low sensitivities require smaller values of !, i.e. simultaneous utilisation. Thus, in a realistic operation it may be necessary to compromise between high growth (with subsequent high productivity) and low sensitivity.     

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived “pure concentration” profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.     

Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae

We have performed a comparative analysis of the fermentation of the solutions of the mixtures of D-glucose and D-xylose with the yeasts Pachysolen tannophilus (ATCC 32691) and Candida shehatae (ATCC 34887), with the aim of producing bioethanol. All the experiments were performed in a batch bioreactor, with a constant aeration level, temperature of 30v°C, and a culture medium with an initial pH of 4.5. For both yeasts, the comparison was established on the basis of the following parameters: maximum specific growth rate, biomass productivity, specific rate of substrate consumption (q_s) and of ethanol production (q_E), and overall ethanol and xylitol yields. For the calculation of the specific rates of substrate consumption and ethanol production, differential and integral methods were applied to the kinetic data. From the experimental results, it is deduced that both Candida and Pachysolen sequentially consume the two substrates, first D-glucose and then D-xylose. In both yeasts, the specific substrate-consumption rate diminished over each culture. The values q_s and q_E proved higher in Candida, although the higher ethanol yield was of the same order for both yeasts, close to 0.4 kg kg^у.     

K L a Evaluation during the course of fermentation using data reconciliation techniques

The oxygen mass transfer coefficient often serves to compare the efficiency of bioreactors and their mixing devices as well as being an important scale-up factor. In submerged fermentation, four methods are available to estimate the overall oxygen mass transfer coefficient (K_La): the dynamic method, the stationary method based on a previous determination of the oxygen uptake rate (Q_O2X), the gaseous oxygen balance and the carbon dioxide balance. Each method provides a distinct estimation of the value of K_La. Data reconciliation was used to obtain a more probable value of K_La during the production of Saccharomyces cerevisiae, performed in 22.5-l fed-batch bioreactor. The estimate of K_La is obtained by minimising an objective function that includes measurement terms and oxygen conservation models, each being weighted according to their level of confidence. Weighting factors of measurement terms were taken as their respective inverse variance whereas weighting factors of oxygen conservation models were obtained using Monte Carlo simulations. Results show that more coherent and precise estimations of K_La are obtained.     

Purification of a periplasmic enzyme by a stirred adsorbent, and by expanded and packed beds

The recovery of a recombinant !-amylase expressed in the periplasm of E. coli using packed bed, expanded bed and batch adsorption is compared. Recovery of recombinant protein using a packed bed requires complete clarification using microfiltration, resulting in loss of yield, or high speed centrifugation which reduces efficiency for viscous periplasmic feedstreams at large scale. Expanded beds ease these problems, however, low levels of cell debris and dilution of the feedstream to 5% sucrose are required to prevent particle aggregation and blockage of adaptors. Batch adsorption provides an alternative option by allowing a rapid capture step (~20 min) which removes the target protein from the feedstream thereby allowing it to be purified by further purification stages, however, the mixing of adsorbent during batch adsorption poses problems.     

Clearance rates in the Arctic bivalves <Emphasis Type="Italic">Hiatella arctica</Emphasis> and <Emphasis Type="Italic">Mya </Emphasis>sp.

Filtration was studied in two Arctic clams, Hiatella arctica and Mya sp., collected in Young Sound, Northeast Greenland. Clearance rates were determined as a function of ambient temperature and algal cell concentration, using the clearance method and feeding with a unicellular flagellate. For both species, clearance rates increased with increasing temperature from <у up to 4-8°C. At higher temperatures, filtration ceased and the clams closed their valves. Clearance rates were also determined in temperate specimens of H. arctica collected on the west coast of Sweden. For these specimens, clearance rates increased with increasing temperature from 0 to 18-20°C. When weight-specific clearance rates were compared between the two populations and between species, there were no differences at 1°C. Clearance rates in Arctic H. arctica were maximal at algal cell concentrations corresponding to 2.5-8 µg chlorophyll a l^-1. Temperature compensation in Arctic bivalves is discussed and it is concluded that adaptations to constant low temperatures consist of a lower minimum temperature, for active filtration. Low clearance rates due to low temperatures did not seem to limit growth, under the prevailing conditions in Young Sound.     

Modelling mixing parameters in concentric-tube airlift bioreactors

The mixing behaviour of the liquid phase in concentric-tube airlift bioreactors of different scale (RIMP: V_L=0.070 m³; RIS-1: V_L=2.50 m³; RIS-2: V_L=5.20 m³) in terms of mixing time was investigated. This mixing parameter was determined from the output curves to an initial Dirac pulse, using the classical tracer response technique, and analyzed in relation to process and geometrical parameters, such as: gas superficial velocity, x_SGR; top clearance, h_S; bottom clearance, h_B, and ratio of the resistances at downcomer entrance, A_d/A_R. A correlation between the mixing time and the specified operating and geometrical parameters was developed, which was particularized for two flow regimes: bubbly and transition (x_SGRА.08 m/s) and churn turbulent flow (x_SGR> 0.08 m/s) respectively. The correlation was applied in bioreactors of different scale with a maximum error of ᆲ%.     

General relationship for volumetric oxygen transfer coefficient (k L a) prediction in tower bioreactors utilizing immobilized cells

A general relationship for prediction of the volumetric oxygen transfer coefficient (k_La) in a tower bioreactor utilizing immobilized Penicillium chrysogenum as function of air superficial velocity, suspension rheological parameters and liquid physical properties is proposed in this study. The relationship was applied to three different systems and a good agreement between the calculated values and the experimental data was obtained.     

Application of a two-dimensional disposable rocking bioreactor to bacterial cultivation for recombinant protein production

Disposable rocking bioreactors (RBs) are widely employed for cultivation of recombinant mammalian and insect cell lines, although the perception of inadequate mass transfer has prevented their application to bioprocesses based on microbial platforms. In this study, one-dimensional (1D) and two-dimensional (2D) RBs were assessed and compared with the conventional stirred tank reactor (STR) for recombinant therapeutic protein production in Escherichia coli. The comparison involved: (1) physical characterization of oxygen mass transfer efficiency and mixing intensity, (2) growth characteristics in batch cultivation, and (3) culture performance for the production of recombinant protein. Our results show that oxygen mass transfer was comparable between the 1D RB and STR at low working volume (WV), declining linearly with increasing WV, and was highest in the 2D RB for all tested WVs with the maximum mass transfer coefficient (k_La) at 3 L WV. Well mixing behavior was observed in all three systems for water and aqueous carboxymethylcellulose (CMC) solutions. Batch growth characteristics were similar in all bioreactor systems, although metabolite accumulation was significant in the 1D RB. Culture performance for the production of recombinant GST-hCD83ext (glutathione S-transferase-hCD83ext fusion protein) was similar in terms of soluble protein yield and inclusion body formation for all bioreactor systems.     

Feasibility of culturing C2C12 mouse myoblasts on glass microcarriers in a continuous stirred tank bioreactor

Commercial culturing of mammalian cell lines is increasing in importance as more biological products unique to mammals are being produced in genetically altered mammalian cells. Most mammalian cells are anchorage dependent, so they must be cultured on a support matrix. This limitation, along with the requirement of a low shear environment, severely effects the scale-up of bench-scale culture systems. The need to culture mammalian cells on a support matrix limits the increase in cell population to a factor of 10-20 before growth virtually stops due to contact inhibition. Commercial culturing systems for anchorage dependent cells are batch processes because of the combination of contact inhibition and support matrix requirements. Development of a continuous bioreactor system could allow both unlimited scale-up and continuous cell-mass production. To design a continuous reactor, a mathematical model to predict the reactor performance should be developed. This paper addresses the development of a mathematical model for predicting continuous bioreactor performance. It was found that anchorage dependent C2C12 mouse myoblast cells, a continuous cell line, followed Monod kinetics for glucose consumption and cell mass production in batch flask experiments, with w_max = 0.040 hr^у and K_m = 2.5 mM. Furthermore, it was found that these parameters could be used to predict the glucose consumption in a continuous bioreactor operated with constant feed of seeded microcarriers operated at two different residence times. The success of this model implies the possibility of developing a continuous cell harvesting and reinoculation system using a microcarrier bioreactor to produce cell mass.     

Shear stress effects on cell growth and <Emphasis Type="SmallCaps">L</Emphasis>-DOPA production by suspension culture of <Emphasis Type="Italic">Stizolobium hassjoo</Emphasis> cells in an agitated bioreactor

Suspension cultures of Stizolobium hassjoo cells were cultivated in a 7l bioreactor. The growth rate and intracellular L-DOPA content of the cells using two different turbine impellers were compared. There were distinct differences in growth behavior and L-DOPA productivity in the range of 100 to 500 rpm for flat-blade turbine impeller. Disk turbine retarded significantly the cell growth but not so significantly for L-DOPA production in the range of 200 to 300 rpm. The shear force intensity of the two impellers at various rotational rates was compared with shear force index (SFI), and power input per unit mass and eddy length scale. There was good consistency among the three indexes for shear force intensity. Thus with SFI the shear force intensity of bioreactor can be indirectly estimated. A critical shear stress that may cause sublytic effect in cells was identified for flat-blade turbine operated at 400 rpm. The common effect between the shear stress and the proton elicitation in the bioreactor was elucidated with a hypothesis of signal transduction by second messenger, H⁺. Our results suggested that H⁺ transduced the signal to protoplast when S. hassjoo cells were stimulated by shear stress. This resulted in an increase of H⁺ which triggered a similar reaction to the pH control of culture broth and enhanced the L-DOPA production.     

Mixing characteristics and liquid circulation in a new multi-environment bioreactor

The theoretical and experimental aspects of the hydrodynamics and mixing in a new multi-environment bioreactor that uses the air-lift design were investigated. This study focused on the mixing characteristics, residence time distribution, liquid circulation between three zones of aerobic, microaerophilic and anoxic, and liquid displacement in the bioreactor at influent flow rates of 720–1,450 L/day and air flow rates of 15–45 L/min. The theoretical analysis of liquid displacement led to the estimation of the specific rate of liquid discharge from the bioreactor at any given influent flow rate, and the number of liquid circulations between various bioreactor zones before the discharge of a given quantity of wastewater. The ratio of mean residence time to the overall hydraulic retention time (t _m/HRT) decreased with the increase of air flow rate at any given influent flow rate, and approached unity at higher air flow rates. Mixing was characterized in terms of the axial dispersion coefficient and Bodenstein number, demonstrating a linear relationship with the superficial gas velocity. A correlation was developed between the Bodenstein number and the Froude number. The study of liquid circulation between the zones showed that less than 1.5 % of the circulating liquid escapes circulation at each cycle and flows towards the outer clarifier, while the percentage of escaped liquid decreases with increasing air flow rate at a given influent flow rate. The specific rate of liquid discharge from the bioreactor increased from 0.19 to 0.69 h^?1 with the increase of air and influent flow rates from 15 to 45 L/min and 500 to 1,450 L/day, respectively. Under the examined operating conditions, mixed liquor circulates between 364 and 1,698 times between the aerobic, microaerophilic and anoxic zones before 99 % of its original volume is replaced by the influent wastewater.     

Production of manganese peroxidase and laccase in laboratory-scale bioreactors by Phanerochaete chrysosporium

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) in laboratory-scale bioreactors was studied. One bioreactor was filled with cubes of polyurethane foam and the other with cubes of nylon sponge, in order to determine the more suitable carrier to produce high ligninolytic enzyme activities by this fungus. Both cultivations were carried out in batch. Manganese-dependent peroxidase activities about 600 U l^у were achieved in the bioreactor filled with cubes of nylon sponge, while up to 500 U l^у were detected in that filled with cubes of polyurethane foam. Furthermore, quite high levels of laccase appeared in both cultures: maximum activities of 114 U l^у and 62 U l^у were obtained on nylon and polyurethane supports, respectively.