Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized β-galactosidase from Aspergillus oryzae

This work studied the hydrolysis of lactose using β-galactosidase from Aspergillus oryzae immobilized with a combination of adsorption and glutaraldehyde cross-linking onto the ion exchange resin Duolite A568 as a carrier. A central composite design (CCD) was used to study the effects of lactose concentration and feed flow rate on the average hydrolysis reaction rate and lactose conversion in a fixed bed reactor operating continuously with an upflow at a temperature of 35 ± 1 °C. The optimal conditions for the average hydrolysis reaction rate and the lactose conversion included a lactose concentration of 50 g/L and a feed flow rate of 6 mL/min. The average reaction rate and conversion reached 2074 U and 65%, respectively. The immobilized enzyme activity was maintained during the 30 days of operation in a fixed bed reactor with a 0.3 mL/min feed flow rate of a 50 g/L lactose solution at room temperature. Feed flows ranging from 0.6 to 12 mL/min were used to determine the distribution of residence times and the kinetics of the fixed bed reactor. A non-ideal flow pattern with the formation of a bypass flow in the fixed bed reactor was identified. The conditions used for the kinetics study included a lactose solution concentration of 50 g/L at pH 4.5 and a temperature of 35 ± 1 °C. Kinetic models using a PFR and axial dispersion methods were used to describe the lactose hydrolysis in the fixed bed reactor, thus accounting for the competitive inhibition by galactose. To increase the lactose conversion, experiments were performed for two fixed bed reactors in series, operating in continuous duty with upflow, with the optimal conditions determined using the CCD for a fixed bed reactor. The total conversion for the two reactors in series was 82%.     

Cell growth and death rates as factors in the long-term performance, modeling, and design of immobilized cell reactors

The viable fraction of immobilized cells in a bioreactor may be critical in predicting long-term or steady-state reactor performance. The assumption of near 100% viable cells in a bioreactor may not be valid for portions of immobilized cell reactors (ICRs) characterized by conditions resulting in appreciable death rates. A mathematical model of an adsorbed cell type ICR is presented in which a steady-state viable cell fraction is predicted, based on the assumptions of no cell accumulation in the reactor and a random loss of cells from the reactor. Data on cell death rates, cell growth rates, and productivity rates as functions of temperature, substrate, and ethanol concentration for the lactose utilizing yeast K. fragillis were incorporated into this model. The steady-state reactor viable cell fraction as predicted by this model is a strong function of both temperature and ethanol concentration. For example, a stable 20% viable fraction of the immobilized cells is predicted in ICR locations experiencing continuous conditions of either 30 g/L ethanol at 40 degrees C, or 95 g/L ethanol at 25 degrees C. Steady-state ICR "plug flow" concentration profiles and column productivities are predicted at three operating temperatures, 20, 30, and 40 degrees C using two different models for ethanol inhibition of productivity. These profiles suggest that the reactor operating temperature should be low if higher outlet ethanol concentrations are desired. Three reactor design strategies are presented to maximize the viable cell fraction and improve long-term ethanol productivity in ICR's: (1) reducing outlet ethanol concentrations, (2) rotating segments of an ICR between high and low ethanol environments, and (3) simultaneous removal of the ethanol produced from the reactor as it is formed.     

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.     

Mammalian cell and protein distributions in ultrafiltration hollow fiber bioreactors

The heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high-molecular-weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell-side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 x 10(4) D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell-side convective flow and its influence on the inoculum and high-molecular-weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have been doubled.     

Continuous beer fermentation using immobilized yeast cell bioreactor systems

Traditional beer fermentation and maturation processes use open fermentation and lager tanks. Although these vessels had previously been considered indispensable, during the past decades they were in many breweries replaced by large production units (cylindroconical tanks). These have proved to be successful, both providing operating advantages and ensuring the quality of the final beer. Another promising contemporary technology, namely, continuous beer fermentation using immobilized brewing yeast, by contrast, has found only a limited number of industrial applications. Continuous fermentation systems based on immobilized cell technology, albeit initially successful, were condemned to failure for several reasons. These include engineering problems (excess biomass and problems with CO(2) removal, optimization of operating conditions, clogging and channeling of the reactor), unbalanced beer flavor (altered cell physiology, cell aging), and unrealized cost advantages (carrier price, complex and unstable operation). However, recent development in reactor design and understanding of immobilized cell physiology, together with application of novel carrier materials, could provide a new stimulus to both research and application of this promising technology.     

Immobilization of urease within a thin channel ultrafiltration cell

Urease [urea amidohydrolase, EC 3.5.1.5] has been immobilized within a thin channel ultrafiltration cell. Loss of enzymic activity as a result of concentration polarization and other causes was minimized. The flow characteristics of the reactor were fully characterized by analysis of the distribution of residence times (using F diagrams) and kinetic data were also obtained for the immobilized enzyme. These data show that under certain conditions the thin channel ultrafiltration reactor can be considered to be an ideally mixed vessel. After almost 8 days of continuous operation it was found that 15% of the original enzyme activity remained.     

Development of a hollow-fiber system for large-scale culture of mammalian cells

A flat-bed hollow-fiber cell culture system has been developed which maximizes the utilization of the large fiber surface while diminishing significantly the problems inherent in a cartridge-type reactor. The reactor core consists of a shallow bed of hollow fibers sandwiched between two stainless-steel microporous filter plates through which the media flow is directed normal to the plane of the fiber bed. Reactors with both 930 and 9300 cm² of fiber surface have been successfully constructed and operated. A variety of cells has been grown in these reactors including SV3T3 cells, baby hamster kidney cells, Vero cells, and rhesus money kidney cells, and cell products such as plasminogen activator and migration inhibition factor (MIF) were produced. This system offers an excellent prototype for scaleup design.     

Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis: Part II

Lignocellulose breakdown in biorefineries is facilitated by enzymes and physical forces. Enzymes degrade and solubilize accessible lignocellulosic polymers, primarily on fiber surfaces, and make fibers physically weaker. Meanwhile physical forces acting during mechanical agitation induce tearing and cause rupture and attrition of the fibers, leading to liquefaction, that is, a less viscous hydrolysate that can be further processed in industrial settings. This study aims at understanding how mechanical agitation during enzymatic saccharification can be used to promote fiber attrition. The effects of reaction conditions, such as substrate and enzyme concentration on fiber attrition rate and hydrolysis yield were investigated. To gain insight into the fiber attrition mechanism, enzymatic hydrolysis was compared to hydrolysis by use of hydrochloric acid. Results show that fiber attrition depends on several factors concerning reactor design and operation including drum diameter, rotational speed, mixing schedule, and concentrations of fibers and enzymes. Surprisingly, different fiber attrition patterns during enzymatic and acid hydrolysis were found for similar mixing schedules. Specifically, for tumbling mixing, slow continuous mixing appears to function better than faster, intermittent mixing even for the same total number of drum revolutions. The findings indicate that reactor design and operation as well as hydrolysis conditions are key to process optimization and that detailed insights are needed to obtain fast liquefaction without sacrificing saccharification yields.     

Detoxification of organophosphate nerve agents by immobilized dual functional biocatalysts in a cellulose hollow fiber bioreactor

A whole-cell technology for detoxification of organophosphates based on genetically engineered Escherichia coli cell expressing both cellulose-binding domain (CBD) and organophosphorus hydrolase (OPH) onto cell surface was reported recently (Wang et al., 2002). This study reports the application of these biocatalysts when immobilized in a cellulose hollow fiber bioreactor (HFB) for the biodetoxification of a model organophosphate, paraoxon, in a continuous flow mode. In 24 h, 0.79 mg wet cell/cm2 fiber surface were immobilized onto cellulose fibers specifically and strongly through the cellulose binding domain, forming a monolayer demonstrated by Scanning Electronic Micrograph, and essentially no cell was washed away by washing buffer. The immobilized biocatalyst had a high performance of detoxifying paraoxon solution of 5,220 mumol/h x L reactor or 990 mumol/h x m2 reactor. The immobilized biocatalysts maintained a stable degradation capacity for 15 uses over a period of 48 days with only 10% decline in degradation efficiency under operating and storage conditions. In addition, the bioreactor was easily regenerated by washing with 1% sodium dodecyl sulfate (SDS), with 86.7% immobilization capacity and 93.9% degradation efficiency recovery. This is the first report using the HFB in a non-traditional way, immobilizing whole-cell biocatalysts by specific adhesion thus rendering the catalysis operation the advantages of low pressure drop, low shear force, and low energy requirement. The successful application of this genetically engineered dual functional E. coli strain in a model bioreactor shows its promise in large-scale detoxification of organophosphate nerve agents in bulk liquid phase.     

Packed bed bioreactor with porous ceramic beads for animal cell culture

A packed bed bioreactor was investigated as means for the cultivation of mammalian cells. The packed bed is comprised of porous ceramic particles with pores sufficiently large for cell immobilization as well as for intraparticle convective flow. In this way, the transport of limiting nutrients such as oxygen can be significantly enhanced, allowing maintenance of cell viability and productivity in an environment protective of adverse shear effects. The extent of intraparticle convective medium flow was experimentally quantified relative to the reactor operating conditions, and was found to be the dominant mechanism of nutrient transport to cells immobilized in the particle interior. An approximate linear relationship was obtained between overall reactor productivity and the extent of intraparticle convection. As the latter can be controlled at the single-particle level through total flow rate control, this relationship is a useful scale-up tool for the design of bioreactors. The high cell densities and the high volumetric productivities achieved by using small lab-scale reactors underline the potential of this simple bioreactor configuration for large-scale cell culture applications. (c) 1993 John Wiley & Sons, Inc.     

Comparison of different bioreactor systems for the production of high titer retroviral vectors

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: I. Modeling and simulation

For the application of immobilized enzymes, fixed bed reactors are used almost exclusively. Fixed bed reactors have specific disadvantages, especially for processes with a deactivating catalyst. Therefore, we have studied a novel reactor type with continuous transport of the immobilized biocatalyst. Flow of biocatalyst is countercurrent to the substrate solution. Because of a stagewise reactor design, back-mixing of biocatalyst is very limited and transport is nearly plug flow. The reactor operates at a constant flow rate and conversion, due to constant holdup of catalytic activity. The reactor performance is compared with a configuration of fixed bed reactors. For reactions in the first-order regime, enzyme requirements in this new reactor are slightly less than for fixed bed processes. The multistage fluidized bed appears to be an attractive reactor design to use biocatalyst to a low residual activity. However, nonuniformity of the particles might affect plug flow transport of the biocatalyst. A laboratory scale reactor and experiments are described in Part II(1) of this series. Hydrodynamic design aspects of a multistage fluidized bed are discussed in more detail in Part III.(2).     

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.     

Assessing solid digestate from anaerobic digestion as feedstock for ethanol production

Ethanol production using solid digestate (AD fiber) from a completely stirred tank reactor (CSTR) anaerobic digester was assessed comparing to an energy crop of switchgrass, and an agricultural residue of corn stover. A complete random design was fulfilled to optimize the reaction conditions of dilute alkali pretreatment. The most effective dilute alkali pretreatment conditions for raw CSTR AD fiber were 2% sodium hydroxide, 130 °C, and 3 h. Under these pretreatment conditions, the cellulose concentration of the AD fiber was increased from 34% to 48%. Enzymatic hydrolysis of 10% (dry basis) pretreated AD fiber produced 49.8 g/L glucose, while utilizing 62.6% of the raw cellulose in the AD fiber. The ethanol fermentation on the hydrolysate had an 80.3% ethanol yield. The cellulose utilization efficiencies determined that the CSTR AD fiber was a suitable biorefining feedstock compared to switchgrass and corn stover.     

CH4 production from H2 and CO2 byMethanobacterium thermoautotrophicum cells fixed on hollow fibers

Summary Methane was produced from H₂ and CO₂ byMethanobacterium thermoautotrophicum cells fixed on the surface of hollow fibers. The mineral solution permeated through the inside of fibers was consumed by the cells, while the gaseous substrate flowing outside the fibers was directly metabolized to methane. Methane production was proportional to hollow fiber length i.e., contact area between cell layer and gas phase. In repeated batch cultures, the production rates of methane and cell mass were 33.1 L/L reactor/day and 1.75 g cells/L reactor/day, respectively with 90% conversion rate.     

Modelling and design of a high productivity ethanol process using Zymomonas mobilis — I

Growth kinetics and ethanol production of Zymomonas mobilis in a bioreactor with cell recycle were modelled. High specific growth rates can be used to control excessive biomass accumulation in the system. Predicted peak productivity with a cell concentration of 80 g l⁻¹, a dilution rate of 6.5 h⁻¹, and a feed glucose concentration of 120 g l⁻¹ is 350 g l⁻¹ h⁻¹. The design of a special recycle reactor using a filter which should permit the operating conditions required for the validation of the model is proposed.     

Conversion of cellulose into fungal cell mass in solid state culture

Summary To satisfy the demand for simple production technology (simple and cheap reactor, cheap recovery and finishing), solid state cultivations were carried out with pretreated straw in a simple fixed bed reactor under nonsterile conditions.The results of these investigations were compared with those evaluated in a stirred tank reactor. The same cell mass fractions were obtained in both reactors. However, about double the cultivation time is necessary for a solid state cultivation as compared to a submerse cultivation.Symbols N₂ nitrogen content of dry biomass (%) - P productivity on cell protein (%/h) - T temperature (°C) - t_F cultivation time (h) - X fungal cell mass fraction (%)     

Reduction of nitrate and nitrite in a cyclically operated continuous biological reactor

Biological reduction of nitrate and nitrite was studied with a continuously operated cyclic reactor. The medium was fed to the reactor during the first phase of the cycle, and the effluent was drawn from the reactor during the third phase of the cycle; reaction occurred throughout the cycle. The process was described mathematically based on kinetic expressions revealed in an independent study. The model equations were subjected to detailed analysis with numerical codes based on the bifurcation theory for forced systems. The analysis has shown that in the operating parameter space there are extensive regions where the system can reach up to three different periodic states. The results of this analysis are shown in the form of two-dimensional operating diagrams. Numerical results have also shown that under certain operating conditions nitrate can be completely eliminated, while nitrite remains practically untreated. An experimental unit was designed, constructed, and used in experiments with a strain of Pseudomonas denitrificans [American Type Culture Collection (ATCC) 13867] under different operating conditions. The experimental results confirmed the theoretical predictions both qualitatively and quantitatively. Conditions under which complete reduction of both nitrate and nitrite is achieved, were found and experimentally verified. The results of this study suggest a methodology for analysis and design of cyclically operated bioreactors employed in denitrification of wastewaters. (c) 1995 John Wiley & Sons, Inc.     

Hollow-fiber enzyme reactor operating under nonisothermal conditions

A hollow-fiber enzyme reactor, operating under isothermal and nonisothermal conditions, was built employing a polypropylene hollow fiber onto which beta-galactosidase was immobilized. Hexamethylenediamine and glutaraldehyde were used as spacer and coupling agent, respectively. Glucose production was studied as a function of temperature, substrate concentration, and size of the transmembrane temperature gradient. The actual average temperature differences across the polypropylene fiber, to which reference was done to evaluate the effect of the nonisothermal conditions, were calculated by means of a mathematical approach, which made it possible to know, using computer simulation, the radial and axial temperature profiles inside the bioreactor and across the membrane. Percent activity increases, proportional to the size of the temperature gradients, were found when the enzyme activities under nonisothermal conditions were compared to those measured under comparable isothermal conditions. Percent reductions of the production times, proportional to the applied temperature gradients, were also calculated. The advantage of employing nonisothermal bioreactors in biotechnological industrial process was discussed.