Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Shear rate and microturbulence effects on the synthesis of proteases by Jacaratia mexicana cells cultured in a bubble column, airlift, and stirred tank bioreactors

Cysteine proteases from Jacaratia mexicana, an endemic Mexican plant, could compete in industrial applications with papain. Currently the only way to obtain these proteases is by extracting them from the wild plant. An alternative source of these enzymes is by J. mexicana suspension culture. In this work, this culture was carried out in airlift, bubble column and stirred tank bioreactors, and the effects of shear rate and microturbulence on cell growth, protein accumulation and proteolytic activity were determined. The shear rates in the stirred tank, bubble column and airlift bioreactors were 274 1/s, 13 1/s and 36 1/s respectively, and microturbulences (symbolized by λ, in units of μm) were 46, 79, and 77 μm, respectively. Protein levels and proteolytic activity were linearly correlated with both shear rate and microturbulence. A higher shear rate and a more intensive microturbulence occurred in the stirred tank, producing higher protein accumulation and higher proteolytic activity compared with those of the other two bioreactor systems. Higher shear rate and microturbulence had an elicitor effect on protease synthesis, because microturbulence in stirred tank bioreactors was lower than the average length of J. mexicana cells. Furthermore, cells in the stirred tank were smaller and thinner than those grown in shake flask, bubble column and airlift bioreactors. In summary, proteases were produced by J. mexicana cell cultures in a stirred tank under conditions of high shear rate and intensive microturbulence, which are similar to those which occur in industrial stirred tanks. These results encourage continuation of the process development for large scale production of these proteases by this technology.     

Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: stimulatory effect of carbon dioxide and ethylene on alkaloid production

Airlift bioreactor operations have been studied for the growth-associated production of secondary metabolites from plant cell suspension cultures. The model system used in this work was Thalictrum rugosum producing berberine, an isoquinoline alkaloid. The airlift system was well suited for growth of Thalictrum cell suspension cultures unless the cell density was high. At high cell density, the airlift system with a draught tube was not adequate due to large aggregates clogging the recirculation paths. This was overcome by use of a cell scraper in the reactor. For berberine production, gas-stripping also played a significant role and it was discovered that CO(2) and ethylene were important for product formation. By supplying a mixture of CO(2) and ethylene into the airlift system, the specific berberine content was increased twofold. It is evident that continuous gas sparging was harmful for the production of berberine without supplementation with other gases.     

Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor

Aims:  This paper investigates a selection-based acclimation strategy for improving the performance and stability of aerobic granules at a high chloroanilines loading.
Methods and Results:  The experiments were conducted in a sequencing airlift bioreactor (SABR) to develop aerobic granules fed with chloroanilines (ClA). The evolution of aerobic granulation was monitored using image analysis and scanning electron microscopy, and PCR–DGGE analysis of microbial community was performed. The sludge granulation was apparently developed by decreased settling time and gradual increased ClA loading to 0·8 kg m⁻³ day⁻¹. A steady-state performance of the granular SABR was reached at last, as evidenced by biomass concentration of 6·3 g l⁻¹ and constant ClA removal efficiency of 99·9%. The mature granules had a mean size of 1·55 mm, minimal settling velocity of 68·4 m h⁻¹, specific ClA degradation rate of 0·181 g gVSS⁻¹ day⁻¹. Phylogenetic analysis of aerobic ClA-degrading granules confirmed the dominance of β - , γ -Proteobacteria and Flavobacteria.
Conclusions:  The chosen operating strategy involving step increase in ClA loading and enhancement of major selection pressures was successful in cultivating the aerobic ClA-degrading granules.
Significance and Impact of the Study:  This research could be helpful for improving the stability of aerobic granules via optimizing operating conditions and developing economic feasible full-scale granular bioreactor.     

Comparison of stirred tank and airlift bioreactors in the production of polygalacturonases by Aspergillus oryzae

The production of endo and exo-polygalacturonase (PG) by Aspergillus oryzae IPT 301 was studied in a stirred tank bioreactor (STR) and an internal circulation airlift bioreactor. Using a factorial experimental design, a soluble culture medium was defined which allowed the production of exo- and endo-PG comparable to that obtained in a medium containing suspended wheat bran. The soluble medium was used in tests to compare the production of these enzymes in the STR and airlift bioreactor. In these tests, after 96 h, maximum enzymatic activity values achieved for exo- and endo-PG were 65.2 units (U) per mL and 91.3 U mL⁻¹, in the STR, with similar activity values of 60.6 U mL⁻¹ and 86.2 U mL⁻¹, respectively, being achieved in the airlift bioreactor. The airlift bioreactor also showed satisfactory results regarding the oxygen transfer rate in this process, indicating its potential to be used in an eventual larger scale production of exo- and endo-PG, with lower costs for both installation and operation.     

Influence of sparger on energy dissipation, shear rate, and mass transfer to sea water in a concentric-tube airlift bioreactor

Data on volumetric mass-transfer coefficient, K_La_L, in a 12 × 10⁻³ m³ airlift bioreactor are reported. Measurements were made in sea water. The superficial gas velocity ranged up to 0.21 m/s. Four cylindrical spargers (60–1000 μm pore size) were tested. In bubbly flow, the sparger pore size strongly influenced the K_La_L; the highest K_La_L values were obtained with the smallest pore size. In contrast, in the transition and heterogeneous flow regimes, the pore size had little influence on K_La_L. The best correlation of the mass transfer data was obtained when both gas holdup and liquid superficial velocity were taken as independent variables. Shear rates were estimated in the different zones of the reactor. The highest values were found in the bottom zone of the reactor and in the gas-liquid separator. The penetration and isotropic turbulence models were used to develop a semi-theoretical equation relating the volumetric mass-transfer coefficient to shear rate; hence providing a better understanding of how the operational variables may be manipulated to attain a moderate shear rate and an appropriate level of mass transfer, two extremely important parameters for the growth of sensible microorganisms as those used in marine biotechnology.     

The influence of geometry on hydrodynamic and mass transfer characteristics in an external airlift reactor for the cultivation of filamentous fungi

The influences of geometric configuration, mycelial broth rheology and superficial gas velocity (U_sg) were investigated with respect to the following hydrodynamic parameters: gas holdup (), oxygen transfer coefficient (K_La) and mixing time (t_m). Increases in U_sg and height of gas separator (H_t) caused an increase in and K_La, and a decrease in t_m. Consequently, a diameter ratio (D_d/D_r) of 0.71 and H_t 0.20 m were found to be the best geometry and operation parameters to achieve high aeration and mixing efficiency for the high viscous broth system in the cultivation of filamentous fungi. An external airlift reactor (EALR) was developed and designed for the cultivation of filamentous fungi. The EALR with two spargers excels in reliability and high aeration and mass transfer coefficiency, resulting in a fast mycelial growth and high biomass productivity in the cultivation of the fungus Rhizopus oryzae.     

A quantitative analysis of shear effects on cell suspension and cell culture of perilla frutescens in bioreactors

The short-time effects of shear on suspended cells of Perilla frutescens were quantitatively analyzed by exposing the cells to a well-defined flow field in a rotating drum reactor. It was found that both shear rate and shearing time significantly affected cell viability. The quantitative effects of shear on cell growth and the production of anthocyanin, a secondary metabolite, by the cell cultures were further investigated in a series of batch cultivations using a 5-L plant cell bioreactor with a marine impeller. The results indicated that there was an optimum range of shear rate; i.e., an average shear rate of 20 to 30 s(-1) or an impeller tip speed of 5 to 8 dm/s, which maximized all the values of the following parameters: the specific growth rate, the maximum cell concentration, the (specific) production and productivity of anthocyanin, and the cell and anthocyanin yields. (c) 1994 John Wiley & Sons, Inc.     

Effects of shear rate and suspending medium viscosity on elongation of red cells tank-treading in shear flow

Elongation measurements of red cells subjected to simple shear flow are usually performed using a single suspending medium (viscosity η(0) ) and varying the mean shear rate (y). Such data are often plotted versus the shear stress (tau = no(y) suggesting that the elongation scales with τ. In this work, normal blood samples were tested in a rheoscope varying both η(0) and(y.). The ranges of (y.) were chosen to restrict the elongation of the red cells to low values where the behavior is dominated by their intrinsic properties. It was found that the elongation scales with [formula: see text] with s decreasing from two at η(0) = 20 mPas to unity at η(0) = 70 mPas. Above η(0) = 70 mPas, the elongation is therefore essentially determined by the membrane elasticity alone. A side observation was a large variation of the elongation both intraindividually and interindividually.     

Effects of shear rate and suspending viscosity on deformation and frequency of red blood cells tank-treading in shear flows

The tank-treading rotation of red blood cells (RBCs) in shear flows has been studied extensively with experimental, analytical, and numerical methods. Even for this relatively simple system, complicated motion and deformation behaviors have been observed, and some of the underlying mechanisms are still not well understood. In this study, we attempt to advance our knowledge of the relationship among cell motion, deformation, and flow situations with a numerical model. Our simulation results agree well with experimental data, and confirm the experimental finding of the decrease in frequency/shear-rate ratio with shear rate and the increase of frequency with suspending viscosity. Moreover, based on the detailed information from our simulations, we are able to interpret the frequency dependency on shear rate and suspending viscosity using a simple two-fluid shear model. The information obtained in this study thus is useful for understanding experimental observations of RBCs in shear and other flow situations; the good agreement to experimental measurements also shows the potential usefulness of our model for providing reliable results for microscopic blood flows.     

Determination of the average shear rate in a stirred and aerated tank bioreactor

A method for evaluating the average shear rate () in a stirred and aerated tank bioreactor has been proposed for non-Newtonian fluids. The volumetric oxygen transfer coefficient (k _L a) was chosen as the appropriate characteristic parameter to evaluate the average shear rate (). The correlations for the average shear rate as a function of N and rheological properties of the fluid (K and n) were obtained for two airflow rate conditions (ϕ_air). The shear rate values estimated by the proposed methodology lay within the range of the values calculated by classical correlations. The proposed correlations were utilized to predict the during the Streptomyces clavuligerus cultivations carried out at 0.5 vvm and four different rotational impeller speeds. The results show that the values of the average shear rate () varied from 437 to 2,693 s⁻¹ by increasing with N and flow index (n) and decreasing with the fluid consistency index (K).     

Characterization of a pilot plant airlift tower loop reactor: III. Evaluation of local properties of the dispersed gas phase during yeast cultivation and in model media

The local properties of the dispersed gas phase (gasholdup, bubble diamater, and bubble velocity) were measured and evaluated at different positions in the riser and downcomer of a pilot plant reactor and, for comparison, in a laboratory reactor. These were described in Parts I and II of this series of articles during yeast cultivation and with model media. In the riser of the pilot plant reactor, the local gas holdup and bubble velocities varied only slightly in axial direction. The gas holdup increased considerably, while the bubble velocity increased only slightly with aeration rate. The bubble size diminished with increasing distance from the aerator in the riser, since the primary bubble size was larger than the equilibrium bubble size. In the downcomer, the mean bubble size was smaller than in the riser. The mean bubble size varied only slightly, the bubble velocity was accelerated, and the gas holdup decreased from top to bottom in the downcomer. In pilot plant at constant aeration rate, the properties of the dispersed phase were nearly constant during the batch cultivation, i.e., they depended only slightly on the cell concentration. In the laboratory reactor, the mean bubble sizes were much larger than in the pilot plant reactor. In the laboratory reactor, the bubble velocities in the riser and downcomer increased, and the mean gas holdup and bubble diameter in the downcomer remained constant as the aeration rate was increased.     

Rheology and Shear Stress of Centaurea calcitrapa Cell Suspension Cultures Grown in Bioreactor

Centaurea calcitrapa suspension cultures were grown either in Erlenmeyer flasks or in a mechanically stirred bioreactor. Its rheological behaviour, when fitted to the Oswald–de Waele model (power law), showed pseudoplastic characteristics in both cases. The flow behaviour index (n) decreased over the course of a growth cycle and the consistency index (K) increased, reached a value of 1.81 N sⁿ m⁻² run on 2 l bioreactor. Bioreactor cultivation of C. calcitrapa cells at different agitation rates (30, 60, 100 and 250 rpm), highlighted the influence of shear forces on cell viability loss (90–34%) and phenol accumulation (74–140 μg l⁻¹), due to increased stirring speeds. Analysis of these results suggests that this cell line is shear-sensitive. An empirical exponential correlation was defined between apparent viscosity and biomass concentration, under the studied conditions, giving the possibility to estimate the prevailing broth regime and to optimize bioreactor design. Revisions requested 10 October 2005; Revisions received 19 December 2005     

Effect of organic solvents on oxygen mass transfer in multiphase systems: Application to bioreactors in environmental protection

The absorption of oxygen in aqueous–organic solvent emulsions was studied in a laboratory-scale bubble reactor at a constant gas flow rate. The organic and the gas phases were dispersed in the continuous aqueous phase. Volumetric mass transfer coefficients (k_La) of oxygen between air and water were measured experimentally using a dynamic method. It was assumed that the gas phase contacts preferentially the water phase. It was found that addition of silicone oils hinders oxygen mass transfer compared to air–water systems whereas the addition of decane, hexadecane and perfluorocarbon PFC40 has no significant influence. By and large, the results show that, for experimental conditions (organic liquid hold-up ≤10% and solubility ratio ≤10), the k_La values of oxygen determined in binary air–water systems can be used for multiphase (gas–liquid–liquid) reactor design with applications in environmental protection (water and air treatment processes).     

Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.     

Fish eggs as bioreactors: the production of bioactive luteinizing hormone in transgenic trout embryos

We demonstrated the production of goldfish luteinizing hormone (gfLH) by the use of 4-day-old rainbow trout embryos as novel bioreactors. This expression system has several advantages target proteins can be rapidly expressed at low cost, and recombinant proteins can be synthesized at low temperatures and can undergo complex post-translational modifications (PTMs). An expression vector containing gfLH cDNA was microinjected into fertilized trout eggs. After 4 days of incubation at 10°C, transgenic embryos were harvested and glycosylated recombinant gfLH was recovered, which stimulated testosterone production in testicular fragments from the goldfish. This is the first report on the successful production of bioactive recombinant gonadotropin originated from cyprinid. Further, these results demonstrate that trout-embryo bioreactors are a potentially powerful tool for the production of functional recombinant proteins.     

Single-cell-bioreactors as end of miniaturization approaches in biotechnology: progresses with characterised bioreactors and a glance into the future

Incidents with single cells and their genesis have not been the major focus of science up to now. This fact is supported by the difficulties one faces when wanting to monitor and cultivate small populations of cells in a defined compartment under controlled conditions, in vitro. Several approaches of up- and down-scaling have often led to poorly understood results which might be better elucidated by understanding the cellular genesis as a function of its microenvironment. This review of the approaches of scale-up and scale-down processes illustrates technical possibilities and shows up their limitations with regard to obtainable data for the characterisation of cellular genesis and impact of the cellular microenvironment. For example, stem cell research advances underline the lack of information about the impact of the microenvironment on cellular development. Finally, a proposal of future research efforts is given on how to overcome this lack of data via a novel bioreactor setup.     

Computational fluid dynamics (CFD) analysis of airlift bioreactor: effect of draft tube configurations on hydrodynamics,cell suspension,and shear rate

Bioprocess and Biosystems Engineering - The biomass productivity of microalgae cells mainly depends on the hydrodynamics of airlift bioreactor (ABR). Thus, the hydrodynamics of concentric tube ABR...     

Cellular content of ribonucleic acid and protein in Saccharomyces cerevisiae as a function of exponential growth rate: calculation of the apparent peptide chain elongation rate.

The average cellular content of ribonucleic acid and protein was determined in cultures of Saccharomyces cerevisiae growing exponentially at different rates in a variety of media. Estimations of the proportion of total cellular ribonucleic acid that is made up of ribosomal ribonucleic acid were used to calculate the average number of ribosomes per cell at the different growth rates. The fraction of ribosomes actively engaged in translation was estimated by sucrose gradient centrifugation of ribosomes and polysomes. These data were used in a calculation of the apparent time taken for the addition of an amino acid to the growing polypeptide chain; this value was found to vary linearly with growth rate over a fivefold range of doubling times.     

Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. (c) 1993 John Wiley & Sons, Inc.