Oxygen mass transfer and scale-up studies in baffled roller bioreactors

Oxygen mass transfer was studied in conventional, bead mill and baffled roller bioreactors. Using central composite rotational design, impacts of size, rotation speed and working volume on the oxygen mass transfer were evaluated. Baffled roller bioreactor outperformed its conventional and bead mill counterparts, with the highest k _L a obtained in these configurations being 0.58, 0.19, 0.41 min^?1, respectively. Performances of the bead mill and baffled roller bioreactor were only comparable when a high bead loading (40 %) was applied. Regardless of configuration increase in rotation speed and decrease in working volume improved the oxygen mass transfer rate. Increase in size led to enhanced mass transfer and higher k _L a in baffled roller bioreactor (0.49 min^?1 for 2.2 L and 1.31 min^?1 for 55 L bioreactors). Finally, the experimentally determined k _L a in the baffled roller bioreactors of different sizes fit reasonably well to an empirical correlation describing the k _L a in terms of dimensionless numbers.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

Nitrification in hybrid bioreactors treating simulated domestic wastewater

Aim

To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors).

Methods and Results

Three bench‐scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5‐day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2·5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia‐oxidizing bacteria, AOB; nitrite‐oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB–NOB activity were less apparent, resulting in incomplete nitrification.

Conclusions

Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification.

Significance and Impact of the Study

This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.     

Effects of immobilization by entrapment in alginate and scale-up on paclitaxel and baccatin III production in cell suspension cultures of Taxus baccata

Paclitaxel and baccatin III-producing cells of Taxus baccata were immobilized within Ca(2+)-alginate beads. Under established optimum conditions for the biosynthesis of both taxanes, the yields of paclitaxel and baccatin III in shake-flask cultures of free cells increased by factors of up to 3 and 2, respectively, in the corresponding cultures of immobilized cells. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities when both free and immobilized cells were grown in the same optimum conditions in three different bioreactor types (Stirred, Airlift, and Wave) running for 24 days in a batch mode and with the system optimized in each case, there was a considerable increase in the yields of paclitaxel and baccatin III. Among the reactors, the Stirred bioreactor was the most efficient in promoting immobilized cell production of paclitaxel, giving a content of 43.43 mg.L(-1) at 16 days of culture, equivalent to a rate of 2.71 mg.L(-1).day(-1). To our knowledge, the paclitaxel productivity obtained in this study is one of the highest reported so far by academic laboratories for Taxus species cultures in bioreactors.     

CFD simulation coupled with population balance equations for aerated stirred bioreactors

Mammalian cells have been widely used to produce therapeutic proteins in stirred bioreactors in suspension culture. Local hydrodynamics can have a great impact on cell proliferation and protein synthesis, but there are few reports on spatial heterogeneity of nutrients, gas bubbles, and mass transfer coefficients. We have employed computational fluid dynamics (CFD) coupled with population balance equations to study local hydrodynamics in a 20 L stirred bioreactor. The flow patterns, energy dissipation rates, gas volume fraction, gas bubble size distribution and local mass transfer coefficient have been displayed throughout the whole bioreactor. Their implications for mammalian cell culture have been discussed. This study provides an insight into rational design and optimum operation conditions in a stirred bioreactor for mammalian cell cultivation.     

Fast kinetics of fe oxidation in packed-bed reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe oxidized per liter per h (1,400 mmol of Fe oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

Application of bioreactor design principles to plant micropropagation

Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the context of propagating aggregates of plant tissue in liquid suspension bioreactors. Although micropropagated plants have a relatively low biological oxygen demand (BOD), the relatively large tissue size and localization of BOD in meristematic regions will typically result in oxygen mass transfer limitations in liquid culture. In contrast to the typical focus of bioreactor design on gas–liquid mass transfer, it is shown that media-solid mass transfer limitations limit oxygen available for aerobic plant tissue respiration. Approaches to improve oxygen availability through gas supplementation and bioreactor pressurization are discussed. The influence of media components on oxygen availability are also quantified for plant culture media. Experimental studies of polystyrene beads in suspension in a 30-l air-lift and stirred bioreactors are used to illustrate design principles for circulation and mixing. Potential limitations to the use of liquid suspension culture due to plant physiological requirements are acknowledged.     

Shaken helical track bioreactors: Providing oxygen to high-density cultures of mammalian cells at volumes up to 1000 L by surface aeration with air

A new scalable reactor was developed by applying a novel mixing principle that allows the large-scale cultivation of mammalian cells simply with surface aeration using air owing to increased liquid-gas transfer compared to standard stirred-tank bioreactors. In the cylindrical vessels (50 mL-1500 L) with a helical track attached to the inside wall, the liquid moved upward onto the track as the result of orbital shaking to increase the liquid-gas interface area significantly. This typically resulted in a 5-10-fold improvement in the volumetric mass transfer coefficient (k(L)a). In a 1500-L helical track vessel with a working volume of 1000 L, a k(L)a of 10h(-1) was obtained at a shaking speed of 39 rpm. Cultivations of CHO cells in a shaken 55-L helical track bioreactor resulted in improved cell growth profiles compared to control cultures in standard systems. These results demonstrated the possibility of using these new bioreactors at scales of 1000 L or more.     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

Relevance of rheological properties of gel beads for their mechanical stability in bioreactors

The mechanical stability of biocatalyst particles in bioreactors is of crucial importance for applications of immobilized-cell technology in bioconversions. The common methods for evaluation of the strength of polymer beads (mostly force-to-fracture or tensile tests) are, however, not yet proven to be relevant for the assessment of their mechanical stability in bioreactors. Therefore, we tested fracture properties of gel materials and investigated their relevance for abrasion in bioreactors. Abrasion of gel beads was assumed to be a continuous fracturing of the bead surface. At first, three rheological properties were considered: stress at fracture; strain at fracture; and the total fracture energy. If stress at fracture is the most important property, beads having a similar fracture energy, but a smaller stress at fracture, would abrade faster in a bioreactor than beads with a larger stress at fracture; if fracture energy the determining factor, beads that require less energy to fracture would abrade faster than those having a larger fracture energy for the same fracture stress. To determine this, beads of kappa-carrageenan and agar (at two different polymer concentrations) were tested for abrasion in four identical bubble columns under the same operating conditions. Agar beads were expected to abrade faster than those of carrageenan because agar had either a lower stress at fracture or a lower fracture energy. However, no correlation between fracture properties and abrasion rate was found in any of the combinations tested. Carrageenan beads abraded faster than those of agar in all combinations. Furthermore, both the stress and strain at fracture of agar and carrageenan beads decreased during the run and those of carrageenan decreased faster, suggesting that the gels are liable to fatigue in different ways. This hypothesis was confirmed by oscillating experiments in which gel samples were subjected to repeated compressions below their fracture levels. Their resistance to compression clearly decreased with the number of oscillations. Fatigue is probably related to the development of microcracks and microfracture propagation within the material. We concluded that: (a) the use of tests based on bead rupture do not provide relevant information on the mechanical stability of gel beads to abrasion; and (b) abrasion of polymer beads is likely to be related to fatigue of the gel materials. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 517-529, 1997.     

Fast Kinetics of Fe2+ Oxidation in Packed-Bed Reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe²⁺ oxidized per liter per h (1,400 mmol of Fe²⁺ oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

Quantification of power consumption and oxygen transfer characteristics of a stirred miniature bioreactor for predictive fermentation scale-up

Miniature parallel bioreactors are becoming increasingly important as tools to facilitate rapid bioprocess design. Once the most promising strain and culture conditions have been identified a suitable scale-up basis needs to be established in order that the cell growth rates and product yields achieved in small scale optimization studies are maintained at larger scales. Recently we have reported on the design of a miniature stirred bioreactor system capable of parallel operation [Gill et al. (2008); Biochem Eng J 39:164-176]. In order to enable the predictive scale-up of miniature bioreactor results the current study describes a more detailed investigation of the bioreactor mixing and oxygen mass transfer characteristics and the creation of predictive engineering correlations useful for scale-up studies. A Power number of 3.5 for the miniature turbine impeller was first established based on experimental ungassed power consumption measurements. The variation of the measured gassed to ungassed power ratio, P(g)/P(ug), was then shown to be adequately predicted by existing correlations proposed by Cui et al. [Cui et al. (1996); Chem Eng Sci 51:2631-2636] and Mockel et al. [Mockel et al. (1990); Acta Biotechnol 10:215-224]. A correlation relating the measured oxygen mass transfer coefficient, k(L)a, to the gassed power per unit volume and superficial gas velocity was also established for the miniature bioreactor. Based on these correlations a series of scale-up studies at matched k(L)a (0.06-0.11 s(-1)) and P(g)/V (657-2,960 W m(-3)) were performed for the batch growth of Escherichia coli TOP10 pQR239 using glycerol as a carbon source. Constant k(L)a was shown to be the most reliable basis for predictive scale-up of miniature bioreactor results to conventional laboratory scale. This gave good agreement in both cell growth and oxygen utilization kinetics over the range of k(L)a values investigated. The work described here thus gives further insight into the performance of the miniature bioreactor design and will aid its use as a tool for rapid fermentation process development.     

Application of multivariate analysis and mass transfer principles for refinement of a 3‐L bioreactor scale‐down model—when shake flasks mimic 15,000‐L bioreactors better

Characterization of manufacturing processes is key to understanding the effects of process parameters on process performance and product quality. These studies are generally conducted using small‐scale model systems. Because of the importance of the results derived from these studies, the small‐scale model should be predictive of large scale. Typically, small‐scale bioreactors, which are considered superior to shake flasks in simulating large‐scale bioreactors, are used as the scale‐down models for characterizing mammalian cell culture processes. In this article, we describe a case study where a cell culture unit operation in bioreactors using one‐sided pH control and their satellites (small‐scale runs conducted using the same post‐inoculation cultures and nutrient feeds) in 3‐L bioreactors and shake flasks indicated that shake flasks mimicked the large‐scale performance better than 3‐L bioreactors. We detail here how multivariate analysis was used to make the pertinent assessment and to generate the hypothesis for refining the existing 3‐L scale‐down model. Relevant statistical techniques such as principal component analysis, partial least square, orthogonal partial least square, and discriminant analysis were used to identify the outliers and to determine the discriminatory variables responsible for performance differences at different scales. The resulting analysis, in combination with mass transfer principles, led to the hypothesis that observed similarities between 15,000‐L and shake flask runs, and differences between 15,000‐L and 3‐L runs, were due to pCO₂ and pH values. This hypothesis was confirmed by changing the aeration strategy at 3‐L scale. By reducing the initial sparge rate in 3‐L bioreactor, process performance and product quality data moved closer to that of large scale. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1370–1380, 2015     

Solid-state fermentation in rotating drum bioreactors: operating variables affect performance through their effects on transport phenomena

Aspergillus oryzae ACM 4996 was grown on an artificial gel-based substrate and on steamed wheat bran during solid-state fermentations in 18.7 L rotating drum bioreactors. For gel fermentations fungal growth decreased as rotational speed increased, presumably due to increased shear. For wheat bran fermentations fungal growth improved under agitated compared to static culture conditions, due to superior heat and mass transfer. We conclude that the effects of operational variables on the performance of SSF bioreactors are mediated by their effects on transport phenomena such as mixing, shear, heat transfer, and mass transfer within the substrate bed. In addition, the substrate characteristics affect the need for and the rates of these transport processes. Different transport phenomena may be rate limiting with different substrates. This work improves understanding of the effects of bioreactor operation on SSF performance.     

Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD)

A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s(-1). Oxygen transfer coefficients of >0.2 s(-1) can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L(-1) in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, mu, and biomass yield, Y(XS).     

Correlation between mass transfer coefficient k<Subscript>L</Subscript>a and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

Background

Among disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen k_La and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal k_La correlation.

Results

A Respiration Activity Monitoring System (RAMOS) was used to measure k_La values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for k_La. In this way, a scale- and volume-independent k_La correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.

Conclusion

The resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/? 30%. To our knowledge, this is the first k_La correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.

     

Bead-to-bead transfer of chinese hamster ovary cells using macroporous microcarriers

Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 5 1 bioreactor. Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads. Successful bead-to-bead transfer was achieved in various split ratios. The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring. Repeated transfers were performed and at least four transfers in spinner flasks were achieved.Two variations of bead-to-bead transfer were performed in the 5 1 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks orin situ transfer by adding fresh beads to the bioreactor. As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads. Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100–4500 cells/bead.The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.     

Bioremediation of phenol-contaminated water and soil using magnetic polymer beads

In order to easily separate pollutant-absorbing polymer beads from contaminated soil or water, novel polymer beads containing magnetic particles were developed. The polymer beads containing 4.67% (w/w) magnetic particles exhibited an almost identical partitioning coefficient for phenol compared to that of the pure polymer. A 1.5 L phenol solution of 2000 mg/L added to a bioreactor was reduced to 481 mg/L phenol within 3 h by adding 100 g of these magnetic beads, and the phenol was completely degraded by microorganisms in 16 h. The magnetized beads were then readily removed from the bioreactor by a magnet with 10,000 G, and subsequently detached for re-use. 500 g of soil contaminated with 4 mg-phenol/g-soil was also contacted with 100 g beads, and greater than 97% removal of phenol from the soil was achieved within 1 day. The phenol-absorbing beads were easily separated from the soil by the magnet and transferred into a fermentor. The phenol was released from the beads and was degraded by the microorganism in 10 h. Modifying polymers to possess magnetic properties has greatly improved the ease of handling of these sequestering materials when decontaminating soil and water sources, in conjunction with contaminant release in partitioning bioreactors.     

Aerobic dechlorination of low-chlorinated biphenyls by bacterial biofilms in packed-bed batch bioreactors

Cells of an aerobic three-membered bacterial co-culture, designated as ECO3, capable of cometabolizing and aerobically dechlorinating low-chlorinated biphenyls in the presence of biphenyl, were immobilized on Manville silica beads, on frosted-glass beads and on polyurethane foam cubes in packed-bed bioreactors continuously fed with a biphenyl-saturated air stream. The ECO3 biofilm reactors were found to be capable of extensively mineralizing several pure dichlorobiphenyls (75 mg/l) and Aroclor 1221 (75 mg/l) in batch mode. Immobilized ECO3 cells could aerobically degrade and dechlorinate the dichlorobiphenyls tested more extensively than suspended ECO3 cells. Among the three biofilm reactors, the glass bead bioreactor and the polyurethane bioreactor exhibited the highest capability of mineralizing both dichlorobiphenyls and Aroclor 1221; the polychlorinated biphenyl availability in the bioreactors, more than the biomass availability, both depending on the nature of the support employed, significantly governed the efficiency of the treatment. These results are of interest for the possible development of a bioreactor system for continuous treatment of polychlorinated-biphenyl-contaminated wastewaters.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.