Analysis of a continuous, aerobic, fixed-film bioreactor. I. Steady-state behavior

The analysis of a continuous, aerobic, fixed-film bioreactor is performed by simulating the behavior of penicillin production in a three-phase fluidized bed. Rigorous mathematical models are developed for a fluidized-bed fermentor in which bioparticles are fluidized by the liquid medium and air. The steady-state performance of the fluidized-bed reactor is appraised in terms of penicillin productivity and outlet concentration by considering the two extremes in contacting patterns, complete back-mix and plug flow, in the absence of a growing biofilm. The results show that the complete back-mix contacting pattern is preferred over that of plug flow due to the nature of the penicillin kinetic relationships. It is also shown that for the dual-nutrient (glucose and oxygen) penicillin reaction system the optimum biofilm thickness does not equal the penetration depth of a limiting nutrient, but depends upon the total reactor configuration.     

Anaerobic treatment of baker's yeast wastewater: I. Start-up and sodium molybdate addition

The anaerobic treatment of baker's yeast wastewater was studied using an anaerobic biological contact reactor (AnRBC) and a fixed-film reactor. The AnRBC had an active biomass developed within the reactor before this study commenced; however, the fixed-film reactor was started without attached biomass in a support structure. The gas production rates obtained for the AnRBC were between 0·55 and 0·61 litre methane per litre reactor per day. However, a gas production rate of only 0·46 litre methane per litre reactor per day was achieved after a four-month operating period for the fixed-film reactor. Higher chemical oxygen demand reduction was also found in the AnRBC. The results indicated that the presence of high sulfate concentration in baker's yeast wastewater affected teh start-up process. The reactor with fully developed active biomass was less susceptible to sulfate inhibition and showed improved anaerobic digestion. Results indicate that the reactor should be innoculated by feeding nutrient-balanced substrate before it was subjected to the digestion of baker's yeast wastewater. The fixed-film reactor was also fed with the substrate contianing sodium molybdate, an inhibitor of sulfate-reducing bacteria. The results indicated that both methanogenic and sulfate-reducing bacteria were inhibited.     

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.     

Development of a Polyaniline-coated Monolith Reactor for the Synthesis of Cephalexin Using Penicillin G Acylase Aggregates

A monolith reactor for the synthesis of cephalexin was developed using capillary columns. The micro channel in the monolith reactor was coated with polyaniline (PANI), and penicillin G acylase was aggregated with PANI using 0.5% of glutaraldehyde as a cross-linker. The developed monolith reactor exhibited many advantages over other enzyme reactors such as batch and continuous reactors. It showed fast enzyme reaction rates owing to the decrease in external mass transfer and internal diffusion limitations. The reactor can easily be scaled up by bundling together multiple monolith reactors, enabling a corresponding increase in feed rate. Furthermore, the monolith reactor showed good operational stability, with 95% of its original activity maintained after 48 h of continuous operation. The PANI coating on the surface of the capillary column increased the enzyme immobilization capacity and conversion was increased from 15.4% to 70.6% after PANI coating. The conversion ratio increased to approximately 70.6% with an increase in residence time and reactor length.     

Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation

In high cell density cultivation processes the productivity is frequently constrained by the bioreactor maximum oxygen transfer capacity. The productivity can often be increased by operating the process at low dissolved oxygen concentrations close to the limitation level. This may be accomplished with a closed-loop controller that regulates the dissolved oxygen concentration by manipulating the dominant carbon source feeding rate. In this work we study this control problem in a pilot 50l bioreactor with a high cell density recombinant P. pastoris cultivation in complex media. The study focuses on the design of accurate stable adaptive controllers, with guaranteed exponential convergence and its relation with the calibration of controller parameters. Two adaptive control strategies were tested in the pilot bioreactor: a model reference adaptive controller with a linear reference model and an integral feedback controller with adaptive gain. The latter alternative proved to be more robust to errors in the measurements of the off-gas composition. Concerning the instrumentation, algorithms were derived assuming that both the dissolved oxygen tension and off-gas composition are measured on-line, but also the case of only dissolved oxygen being measured is addressed. It was verified that the measurement of off-gas composition might not improve the controller performance due to measurement and process time delays.     

Fed-batch cultivation of Saccharomyces cerevisiae in a hyperbaric bioreactor

Fed-batch is the dominating mode of operation in high-cell-density cultures of Saccharomyces cerevisae in processes such as the production of baker's yeast and recombinant proteins, where the high oxygen demand of these cultures makes its supply an important and difficult task. The aim of this work was to study the use of hyperbaric air for oxygen mass transfer improvement on S. cerevisiae fed-batch cultivation. The effects of increased air pressure up to 1.5 MPa on cell behavior were investigated. The effects of oxygen and carbon dioxide were dissociated from the effects of total pressure by the use of pure oxygen and gas mixtures enriched with CO(2). Fed-batch experiments were performed in a stirred tank reactor with a 600 mL stainless steel vessel. An exponential feeding profile at dilution rates up to 0.1 h(-)(1) was used in order to ensure a subcritical flux of substrate and, consequently, to prevent ethanol formation due to glucose excess. The ethanol production observed at atmospheric pressure was reduced by the bioreactor pressurization up to 1.0 MPa. The maximum biomass yield, 0.5 g g(-)(1) (cell mass produced per mass of glucose consumed) was attained whenever pressure was increased gradually through time. This demonstrates the adaptive behavior of the cells to the hyperbaric conditions. This work proved that hyperbaric air up to 1.0 MPa (0.2 MPa of oxygen partial pressure) could be applied to S. cerevisiae cultivation under low glucose flux. Above that critical oxygen partial pressure value, i.e., for oxygen pressures of 0.32 and 0.5 MPa, a drastic cell growth inhibition and viability loss were observed. The increase of carbon dioxide partial pressure in the gas mixture up to 48 kPa slightly decreased the overall cell mass yield but had negligible effects on cell viability.     

Strategies for penicillin fermentation in tower-loop reactors

Since it has not been possible to produce penicillin in tower-loop reactors with highly viscous filamentous molds of Penicillium chrysogenum which are employed in stirred-tank reactors, a new strategy has been developed to avoid the formation of this morphology and to use the pellet form of the fungi. When employing definite impeller speeds in the subculture in connection with definite inoculum amounts and substrate concentrations in the main culture (bubble column), it is possible to generate a suspension of isolated small pellets, which shows a low broth viscosity up to a sediment content of 45% over the entire fermentation time. Volumetric mass-transfer coefficients k(L)as are by a factor of 4 to 5 higher in these pellet suspensions than in filamentous broths. It was easy to maintain the necessary oxygen supply for penicillin production in these pellet suspensions. Under these conditions the specific penicillin productivities were higher with regard to power input (up to 90%), biomass, and consumed substrate than in the stirred-tank reactors with highly viscous filamentous morphology of the fungi. Under nonoptimized operating conditions the absolute penicillin production in the tower loop was 35% lower than in the stirred-tank reactor due to lower possible biomass concentrations. The separation of the biomass, and therefore the penicillin recovery, is much simpler when employing pellets. It is shown how the particular mass transfer resistances at the gas/liquid and liquid/pellet interfaces and within the pellets change with the pellet diameter. There should be a particular pellet diameter at which penicillin productivity has its maximum. These investigations indicate that the use of tower-loop reactors can, in the future, be an alternative for more economical penicillin production methods.     

Methane production using whole and screened dairy manure in conventional and fixed-film reactors

The technical feasibility of adopting the fixed-film reactor concept for biogas production from screened dairy manure was investigated. The methane production capability of laboratory-scale 4-L anaerobic reactors (conventional and fixed-film) receiving screened dairy manure and operated at 35 degrees C was compared. Dairy manure filtrate with 4.4% total solids (TS) and 3.4% volatile solids (VS) (average value) was prepared from 1:1 manure-water slurry. The feed material was added intermittently at loading rates ranging from 2.34 to 25 and 2.25 to 785 g VS/L d, respectively, for the conventional and fixed-film reactors. Maximum methane production rate (L CH(4)/L d) for the conventional reactor was 0.63 L CH(4)/L d achieved at a 6-day hydraulic retention time (HRT). For the fixed-film reactor the maximum production rate was 3.53 L CH(4)/L d when operated at a loading rate of 262 g VS/L d (3 h HRT). The fixed-film reactor was capable of sustaining a loading of 785 g VS/L d (1 h HRT). The fixed-film reactor performed much better than the conventional reactors. These results indicate that a large reduction of required reactor volume is possible through application of a fixed-film concept combined with a liquid-solid separation pretreatment of dairy manure.     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.     

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.     

Trichloroethylene mineralization in a fixed-film bioreactor using a pure culture expressing constitutively toluene ortho -monooxygenase

An aerobic, single-pass, fixed-film bioreactor was designed for the continuous degradation and mineralization of gas-phase trichloroethylene (TCE). A pure culture of Burkholderia cepacia PR1(23)(TOM(23C)), a Tn5transposon mutant of B. cepacia G4 that constitutively expresses the TCE-degrading enzyme, toluene ortho-monooxygenase (TOM), was immobilized on sintered glass (SIRANtrade mark carriers) and activated carbon. The inert open-pore structures of the sintered glass and the strongly, TCE-absorbing activated carbon provide a large surface area for biofilm development (2-8 mg total cellular protein/mL carrier with glucose minimal medium that lacks chloride ions). At gas-phase TCE concentrations ranging from 0.04 to 2.42 mg/L of air and 0.1 L/min of air flow, initial maximum TCE degradation rates of 0.007-0.715 nmol/(min mg protein) (equivalent to 8.6-392.3 mg TCE/L of reactor/day) were obtained. Using chloride ion generation as the indicator of TCE mineralization, the bioreactor with activated carbon mineralized an average of 6.9-10.3 mg TCE/L of reactor/day at 0.242 mg/L TCE concentration with 0.1 L/min of air flow for 38-40 days. Although these rates of TCE degradation and mineralization are two- to 200-fold higher than reported values, TOM was inactivated in the sintered-glass bioreactor at a rate that increased with increasing TCE concentration (e.g., in approximately 2 days at 0.242 mg/L and <1 day at 2.42 mg/L), although the biofilter could be operated for longer periods at lower TCE concentrations. Using an oxygen probe and phenol as the substrate, the activity of TOM in the effluent cells of the bioreactor was monitored; the loss of TOM activity of the effluent cells corroborated the decrease in the TCE degradation and mineralization rates in the bioreactor. Repeated starving of the cells was found to restore TOM activity in the bioreactor with activated carbon and extended TCE mineralization by approximately 34%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 674-685, 1997.     

Development of a predictive description of an immobilized-cell, three-phase, fluidized-bed bioreactor

A Large bioreactor is an inhomogenous system with concentration gradients which depend on the fluid dynamics and the mass transfer of the reactor, the feeding strategy, the saturation constant, and the cell density. The responses of Escherichia coli cells to short-term oscillations of the carbon/energy substrate in glucose limited fed-batch cultivations were studied in a two-compartment reactor system consisting of a stirred tank reactor (STR) and an aerated plug flow reactor (PFR) as a recycle loop. Short-term glucose excess or starvation in the PFR was simulated by feeding of glucose to the PFR or to the STR alternatively. The cellular response to repeated short-term glucose excess was a transient increase of glucose consumption and acetate formation. But, there was no accumulation of acetate in the culture, because it was consumed in the STR part where the glucose concentration was growth limiting. However, acetate accumulated during the cultivation if the oxygen supply in the PFR was insufficient, causing higher acetate formation. The biomass yield was then negatively influenced, which was also the case if the PFR was used to simulate a glucose starvation zone. The results suggest that short-term heterogeneities influence the cellular physiology and growth, and can be of major importance for the process performance. (c) 1995 John Wiley & Sons, Inc.     

Continuous production of manganese peroxidase by Phanerochaete chrysosporium immobilized on polyurethane foam in a pulsed packed-bed bioreactor

The bottleneck of the application of manganese peroxidase (MnP) on an industrial scale in pulp biobleaching or in degradation of hazardous compounds is the lack of an efficient production system. Three main problems arise for the continuous production of MnP during secondary metabolism of Phanerochaete chrysosporium: enzyme production occurs only under specific physiological conditions corresponding to C or N limitation, high O(2) tension, and adequate Mn(+2) concentration; the enzyme that is produced is destabilized by extracellular proteases; and excessive growth of the mycelium blocks effective oxygen transfer. To overcome these drawbacks, continuous production of MnP was optimized by selecting a suitable bioreactor configuration and the environmental and operating conditions affecting both enzyme production and stability. The combination between a proper feed rate and the application of a pulsation in a packed-bed bioreactor permitted the maintenance of continuous secretion of MnP while limiting mycelial growth and avoiding bed clogging. Environmental factors as an Mn(+2) concentration of 5000 muM and high oxygen tension enhanced MnP production. The hydraulics of the bioreactor corresponding to a plug flow model with partial mixing and an operating hydraulic rentention time of 24 h were optimal to achieve stable operating conditions. This policy allowed long operation periods, obtaining higher productivities than the best reported in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 130-137, 1997.     

Performance of a membrane reactor for cellobiose hydrolysis

A continuous enzymatic hollow fiber reactor (HFR), obtained by immobilizing cellobiose active cells into the shell side of hollow-fiber modules, was studied. The HFR yield was monitored by glucose analysis resulting from hydrolysis of cellobiose. The residence time of substrate in the bioreactor to obtain convenient hydrolysis yields was calculated from tests carried out by varying the reactor dilution rate in the range 0.001-0.004 L/min. The glucose yield was measured for 300 h (continuous substrate flux). The yield decreased from 40 to 15%. This decrease was due to the loss of specific activity in the operating conditions and to the pressure drop increase from 0.2 to 1.7 atm. The pressure drop increase is in turn dependent on the cell loading (0.2-2.1 g dry cell) and the substrate flux.     

Enhanced mixing and mass transfer in a recirculation loop results in high cell densities in a roller bottle reactor

A recirculation loop added to a large-scale roller bottle reactor resulted in high cell densities as compared to standard roller bottles. Four different mammalian cell lines reached an average maximum density equal to 5.4 x 10(6) cells /mL (sigma = 0.263), which was between 2.13 and 2.95 times greater than the densities in roller bottles without recirculation using the same cell lines. The high densities were maintained over long durations (>25 days) while the reactor operated with continuous perfusion. The increased densities are attributed to enhanced liquid mixing and oxygen transfer that occur as a result of the recirculation loop. Models were developed that describe axial liquid flow and oxygen transfer in both the sample loop and the reactor growth chamber. Axial dispersion and oxygen transfer coefficients are presented for a variety of operating conditions. The increased oxygen transfer characteristics of the reactor allow for easy scale-up of roller bottle cultures by operating at larger volumes with greater liquid depths than conventional roller bottles permit. The surface-area-to-volume ratio in the tests performed was 0.206 versus 1.16 cm(-1) in a standard roller bottle.     

Enhanced production of poly(3-hydroxybutyrate) in a novel airlift reactor with in?situ cell retention using Azohydromonas australica

Economic production of biodegradable plastics is a challenge particularly because of high substrate and energy cost inputs for its production. Research efforts are being directed towards innovations to minimize both of the above costs to economize polyhydroxybutyrate (PHB) production. A novel airlift reactor (ALR) with outer aeration and internal settling was utilized in this investigation. Although it featured no power consumption for agitation, it facilitated increased oxygen transfer rate and better cell retention than stirred tank reactor (STR), thereby resulting in enhanced PHB productivity. ALR with in?situ cell retention demonstrated a significant improvement in biomass concentration and biopolymer accumulation. The total PHB production rate, specific biomass, and product yield in the ALR were observed to be 0.84?g/h, 0.43?g/g, and 0.32?g/g, respectively. The studies revealed that the volumetric oxygen mass transfer rate and mixing time for ALR were 0.016?s(-1) and 3.73?s, respectively, at 2.0?vvm as compared with corresponding values of 0.005?s(-1) and 4.95?s, respectively, in STR. This demonstrated that ALR has better oxygen mass transfer and mixing efficiency than STR. Hence, ALR with cell retention would serve as a better bioreactor design for economic biopolymer production than STR, particularly due to its lower cost of operation and simplicity along with its enhanced oxygen and heat transfer rates.     

Evaluation of feeding strategies in carbon-regulated secondary metabolite production through mathematical modeling

There is now growing evidence that the production of many secondary metabolic by microorganisms is subjected to carbon-catabolite regulation. Even though the exact mode of this regulation is not yet clear, an engineering analysis of the production process is still possible based upon a suitable hypothesis. By way of simulation of penicillin fermentation data obtained from the literature, a mechanistic model involving a substrate inhibition kinetics of product formation has been verified in this paper. Such a model has been found successful not only in predicting simple sugar-feeding strategy, but also a complicated computer guided strategy based upon controlling biomass growth rates in the tropo and idiophases. Using this model, for strategies for sugar feeding into penicillin fermentation have been investigated. These results show that similar penicillin productivities can be obtained using any of these strategies provided fermentations are carried out under optimal conditions corresponding to the strategy chosen. Effect of maximum oxygen transfer capacity of the fermentor under the conditions of fungal growth has been incorporated using an upper limit of biomass concentration on achievement of which the fermentations must be stopped due to serious oxygen limitations. Results of model simulations with such limits throw light upon the way in which different fermentors may behave with respect to product formation.     

Application of oxygen vectors to Claviceps purpurea cultivation

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.     

Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch cultures

The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis mediterranei in batch and fed-batch culture was investigated. Fed-batch culture using glucose as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with constant feeding rate, in bioreactor level, proved to be an alternative production system with a significant increase in both volumetric and specific antibiotic production. The maximal concentrations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained in fed-batch culture in bioreactor level under non-oxygen limitation. On the other hand, the rate of rifamycins production was increased from 6.58 to 12.13 mg/l x h for rifamycin B and from 9.47 to 31.83 mg/l x h for rifamycin SV on the bioprocess transfer and improvement from the conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.