Optimum design of a series of continuous stirred tank reactors containing immobilised growing cells

The optimum design of a series of continuously operated stirred-tank reactors containing immobilised growing cells is described. Optimal design is defined as the minimal total holding time over the reactor series to achieve a certain degree of conversion. The analysis is made under the assumptions that there is a constant and equal concentration of immobilised biomass in all bioreactors along the series, no diffusion limitation takes place, all growth of the immobilised biomass will lead to an increase in suspended biomass, and that maintenance of the immobilised cells can be neglected. It is shown that the use of more than three bioreactors in series is likely to be de trop.     

Continuous biotransformation of glycopeptide antibiotic A40926 in a cascade of three airlift bioreactors using immobilized Actinoplanes teichomyceticus cells

Immobilized cells of Actinoplanes teichomyceticus ATCC 31121 were used to selectively cleave the acyl group of A40926 yielding the deacylated form of the molecule. The feasibility of this particular biotransformation in a series of three perfectly mixed airlift bioreactors with immobilized cells was examined. A continuously operated airlift cascade was designed using a model for a series of reactors with immobilized biocatalyst beads obeying Michaelis–Menten kinetics. In independent experimental runs the cascade bioreactor system was operated continuously for 56 days with an overall conversion of 99%. Model estimates for reactor volumes and relative conversions were found to be in a good agreement with the experimental results.     

Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process

The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg‐based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin‐Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25–30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers ≥8.6 log₁₀ FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn‐around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production. Biotechnol. Bioeng. 2010;106: 906–917. © 2010 Wiley Periodicals, Inc.     

Proteome analysis of the Escherichia coli heat shock response under steady-state conditions

In this study a proteomic approach was used to investigate the steady-state response of Escherichia coli to temperature up-shifts in a cascade of two continuously operated bioreactors. The first reactor served as cell source with optimal settings for microbial growth, while in the second chemostat the cells were exposed to elevated temperatures. By using this reactor configuration, which has not been reported to be used for the study of bacterial stress responses so far, it is possible to study temperature stress under well-defined, steady-state conditions. Specifically the effect on the cellular adaption to temperature stress using two-dimensional gel electrophoresis was examined and compared at the cultivation temperatures of 37°C and 47.5°C. As expected, the steady-state study with the double bioreactor configuration delivered a different protein spectrum compared to that obtained with standard batch experiments in shaking flasks and bioreactors. Setting a high cut-out spot-to-spot size ratio of 5, proteins involved in defence against oxygen stress, functional cell envelope proteins, chaperones and proteins involved in protein biosynthesis, the energy metabolism and the amino acid biosynthesis were found to be differently expressed at high cultivation temperatures. The results demonstrate the complexity of the stress response in a steady-state culture not reported elsewhere to date.     

Enhanced aerobic granulation with extracellular polymeric substances (EPS)-free pellets

Extracellular polymeric substances (EPSs) were secreted by cells after they agglomerated into a compact aggregate. This study shows that the EPS initially embedded in seed sludge before granulation may sterically slow subsequent microbe–microbe contact, thereby delaying aerobic granulation. Three identical bioreactors were used in this study using glucose as the sole carbon and energy source. Reactor 1 (R1) was seeded with EPS-free pellets and operated in sequencing batch reactor (SBR) mode. Reactor 2 (R2) was seeded with the original sludge flocs and operated in SBR mode. Reactor 3 (R3) was seeded with EPS-free pellets and operated in continuously stirred tank reactor (CSTR) mode. Granulation occurred in R1 earlier than in R2; the granules that formed in R1 were larger and more compact than those in R2 at the same cultivation time. The few mature granules in R3 suggest that aerobic granulation can occur in a CSTR when a reactor is seeded with EPS-free pellets.     

Application of a continuous bioreactor cascade to study the effect of linoleic acid on hybridoma cell physiology

The aim of the present study is to demonstrate the use of controlled bioreactors for toxicological studies. As a model system the effect of linoleic acid on hybridoma cells is studied in two well-controlled continuously operated bioreactors placed in series. In the first reactor the effect on rapid proliferating cells can be studied, while in the second reactor a special steady state is created, which allows studying the effect on apoptotic cells. Experiments are done at 0, 25, and 50 microM linoleic acid. At the end of the experiment with 50 microM linoleic acid, the concentration of linoleic acid is increased stepwise to determine the cytotoxic level. For rapid proliferating cells exposed to 25 and 50 microM stimulation of growth was observed. At 50 microM there was at the same time an increase in cell death through apoptosis. For stressed apoptotic cells linoleic acid caused partial growth inhibition at 25 and 50 microM and arrest of cell proliferation in the G(2)/M phase at 50 microM. For both, rapid proliferating cells and stressed apoptotic cells, complete growth inhibition occurred at 85 microM, with cells being arrested in the G(2)/M phase and dying mainly through necrosis. Cells in the bioreactor system appeared to be more sensitive towards linoleic acid than cells grown in multi-well plates. (IC(50) = 300 microM; IC(100) = 400 microM). Altogether the results of the present study reveal that the biostat experiments allow detailed analysis of the effect of a bioactive ingredient on cell physiology and behavior.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

A continuous process for the production of baculovirus using insect-cell cultures

Summary A continuous culture of insect cells (Spodoptera frugiperda) was used for continuous production of baculovirus (nuclear polyhedrosis virus fromAutographa californica). The system consisted of a cascade of two continuous stirred tank reactors (CSTRs). In CSTR I the insect cells were grown in suspension. This suspension was fed continuously to CSTR II where the virus infection occurred. For a period of about 25 days the average volumetric productivity was about 10⁷ polyhedra (virus particles occluded in protein capsules) and 10⁸ infectious NOVs (non-occluded virus particles) per cm³ effluent. This is equivalent to 25 polyhedra and 250 NOVs per infected cell, respectively. In one case, the percentage of infected cells was 65%, which is close to the theoretical value of 68%. After a run-time of 32 days a decrease of process productivity was observed, probably due to the so-called passage effect, a degeneration of the virus DNA.     

Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses

Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated. (c) 1996 John Wiley & Sons, Inc.     

Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection.

Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.     

Continuous production of baculovirus in a cascade of insect-cell reactors

Summary Insect cells (Spodoptera frugiperda) were cultured in a continuous stirred-tank reactor. The effluent was led to a cascade of another two reactors, each containing half the volume of the cell-growth reactor, where the cells were infected with Autographa californica nuclear polyhedrosis virus. For about 10 days production of 10⁷ polyhedra (virus particles embedded in a protein capsule) per cm³ was achieved. This short production time compared to previous experiments involving an analogous system with a single infection vessel of equal volume to the cell-growth vessel is ascribed to the accelerated occurence of the so-called passage effect (a decrease of infectious virus with time). From the results of a computer model it was concluded that this passage effect was accelerated by the change in residence time distribution as compared to the earlier experiments. time distribution as compared to the earlier experiments. Offprint requests to: F. L. J. van Lier     

Continuous ethanol production and cell growth in an immobilized-cell bioreactor employing Zymomonas mobilis

Ethanol-producing bioreactors employing cells of Zymomonas mobilis attached to glass-fiber pads were operated continuously for as long as 28 days. Ethanol production, which is related to bed-associated biomass levels, was found to occur in three distinct phases: an exponential phase, a linear phase, and a "steady-state" phase. After prolonged operation, a bacterial floc developed in the reactor. The maximum effluent ethanol concentration and the maximum volumetric productivity were 6.4% and 152 g L(-1) h(-1), respectively, and both were attained at a liquid residence time of from 10-15 min. Both maxima occurred after the development of the bacterial floc. The flocculant bacterium has been isolated and tentatively identified as a flocculant strain of Z. mobilis.     

新型生物反应器结构研究进展

生物反应器是生物工程的核心设备,其结构的合理性直接决定反应器生物加工的效率。生物反应器的研究一直是生物工程的核心问题之一。随着青霉素的工业化生产,机械搅拌式生物反应器应运而生,此后,随着动植物细胞培养,高等真菌培养,藻类培养等生物过程的发展,人们相应开发了大量的生物反应器,其中以机械搅拌式生物反应器和气升式样生物反应器尤为突出,本文总结了近年来文献报道的新型生物反应器,主要阐述了机械搅拌式和气升式两类生物反应器结构的研究进展,对目前国内外报道的11种新型反应器典型结构进行了总结与分析。     

Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.     

Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

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.     

High-yield continuous production of nicotinic acid via nitrile hydratase-amidase cascade reactions using cascade CSMRs

High yields of nicotinic acid from 3-cyanopyridine bioconversion were obtained by exploiting the in situ nitrile hydratase-amidase enzymatic cascade system of Microbacterium imperiale CBS 498-74. Experiments were carried out in continuously stirred tank UF-membrane bioreactors (CSMRs) arranged in series. This reactor configuration enables both enzymes, involved in the cascade reaction, to work with optimized kinetics, without any purification, exploiting their differing temperature dependences. To this end, the first CSMR, optimized for the properties of the NHase, was operated (i) at low temperature (5°C), limiting inactivation of the more fragile enzyme, nitrile hydratase, (ii) with a high residence time (24 h) to overcome reaction rate limitation. The second CSMR, optimized for the properties of the AMase, was operated (i) at a higher temperature (50°C), (ii) with a lower residence time (6h), and (iii) with a lower substrate (3-cyanopyridine) concentration to control excess substrate inhibition. The appropriate choice of operational conditions enabled total conversion of 3-cyanpyridine (up to 200 mM) into nicotinic acid to be achieved at steady-state and for long periods. Higher substrate concentrations required two CSMRs optimized for the properties of the NHase arranged in series to drive the first reaction to completion.     

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.     

Optimization of L-(+)-lactic acid production by ring and disc plastic composite supports through repeated-batch biofilm fermentation.

Four customized bioreactors, three with plastic composite supports (PCS) and one with suspended cells (control), were operated as repeated-batch fermentors for 66 days at pH 5 and 37 degrees C. The working volume of each customized reactor was 600 ml, and each reactor's medium was changed every 2 to 5 days for 17 batches. The performance of PCS bioreactors in long-term biofilm repeated-batch fermentation was compared with that of suspended-cell bioreactors in this research. PCS could stimulate biofilm formation, supply nutrients to attached and free suspended cells, and reduce medium channelling for lactic acid production. Compared with conventional repeated-batch fermentation, PCS bioreactors shortened the lag time by threefold (control, 11 h; PCS, 3.5 h) and sixfold (control, 9 h; PCS, 1.5 h) at yeast extract concentrations of 0.4 and 0.8% (wt/vol), respectively. They also increased the lactic acid productivity of Lactobacillus casei subsp. rhamnosus (ATCC 11443) by 40 to 70% and shortened the total fermentation time by 28 to 61% at all yeast extract concentrations. The fastest productivity of the PCS bioreactors (4.26 g/liter/h) was at a starting glucose concentration of 10% (wt/vol), whereas that of the control (2.78 g/liter/h) was at 8% (wt/vol). PCS biofilm lactic acid fermentation can drastically improve the fermentation rate with reduced complex-nutrient addition.     

Comparison of aerobic and anaerobic degradation of municipal solid waste in bioreactor landfills

Two landfill bioreactors were operated under aerobic and anaerobic conditions in a thermo-insulated room at a constant temperature of 32 °C. Reactors were filled with 19.5 kg of shredded synthetic solid waste prepared according to the average municipal solid waste compositions determined in Istanbul and operated under wet-tomb management strategy by using leachate recirculation. Aerobic conditions in the reactor were developed by using an air compressor. The results of experiments indicated that aerobic reactor had higher organic, nitrogen, phosphorus and alkali metal removal efficiencies than the anaerobic one. Furthermore, stabilization time considerably decreased when using aerobic processes with leachate recirculation compared to the anaerobic system with the same recirculation scheme.