Dynamic modeling of immobilized cell reactor: application to ethanol fermentation

A mathematical model has been developed for the unsteady-state operation of an immobilized cell reactor. The substrate solution flows through a mixed-flow reactor in which cells immobilized in gel beads are retained. The substrate diffuses from the external surface of the gel beads to some internal location where reaction occurs. The product diffuses from the gel beads into liquid medium which flows out of the reactor. The model combines simultaneous diffusion and reaction, as well as cell growth, and it can predict how the rates of substrate consumption, product formation, and cell growth vary with time and with initial conditions. Ethanol fermentation was chosen as a representative reaction in the immobilized cell reactor, and numerical calculations were carried out. Excellent agreement was observed between model predictions and experimental data available in the literature.     

An immobilized cell reactor with simultaneous product separation. I. Reactor design and analysis

A four-phase reactor-separator (gas, liquid, solid, and immobilized catalyst) is proposed for fermentations characterized by a volatile product and nonvolatile substrate.In this reactor, the biological catalyst is immobilized onto a solid column packing and contacted by the liquid containing the substrate.A gas phase is also moved through the column to strip the volatile product into the gas phase. The Immobilized Cell Reactor-Separator (ICRS) consists of two basic gas-liquid flow sections: a cocurrent "enricher" followed by a countercurrent-"stripper".In this article, an equilibrium stage model of the reactor is developed to determine the feasibility and important operational variables of such a reactor-separator. The ICRS concept is applied to the ethanol from whey lactose fermentation using some preliminary immobilized cell reactor performance data. A mathematical model for a steady-state population based on an adsorbed monolayer of cells is also developed for the reactor. The ICRS model demonstrated that the ICRS should give a significant increase in reactor productivity as compared to an identically sized Immobilized Cell Reactor (ICR) with no separation. The gas-phase separation of the product also allows fermentation of high inlet substrate concentrations. The model is used to determine the effects of reactor parameters on ICRS performance including temperature, pressure, gas flow rates, inlet substrate concentration, and degree of microbial product inhibition.     

Immobilization by Polyurethane of Pseudomonas dacunhae Cells Containing l-Aspartate beta-Decarboxylase Activity and Application to l-Alanine Production

Whole cells of Pseudomonas dacunhae containing l-aspartate beta-decarboxylase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol; W. R. Grace & Co., Lexington, Mass.). The immobilized cell preparation was used to convert l-aspartic acid to l-alanine. Properties of the immobilized P. dacunhae cells containing aspartate beta-decarboxylase activity were investigated with batch reactors. Retention of enzyme activity was observed to be as much as 100% when cell lysis was allowed to occur before immobilization. The pH and temperature optima were determined to be 5.5 and 45 degrees C, respectively. Immobilized P. dacunhael-aspartate beta-decarboxylase activity was stabilized by the addition of 0.1 mM pyridoxal-5-phosphate and 0.1 mM alpha-ketoglutaric acid to a 1.7 M ammonium aspartate (pH 5.5) substrate solution. Under conditions of semicontinuous use in a batch reactor, a 2.5% loss in immobilized l-aspartate beta-decarboxylase activity was observed over a 31-day period.     

Immobilization of Escherichia coli Cells Containing Aspartase Activity with Polyurethane and Its Application for l-Aspartic Acid Production

Whole cells of Escherichia coli containing aspartase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol). The immobilized cell preparation was used to convert ammonium fumarate to l-aspartic acid. Properties of the immobilized E. coli cells containing aspartase were investigated with a batch reactor. A 1.67-fold increase in the l-aspartic acid production rate was observed at 37 degrees C as compared to 25 degrees C operating temperature. The pH optimum was broad, ranging from 8.5 to 9.2. Increasing the concentration of ammonium fumarate to 1.5 M from 1.0 M negatively affected the reaction rate. l-Aspartic acid was produced at an average rate of 2.18 x 10 mol/min per g (wet weight) of immobilized E. coli cells with a 37 degrees C substrate solution consisting of 1.0 M ammonium fumarate with 1 mM Mg (pH 9.0).     

Operation of packed-bed immobilized cell reactor featuring active β-galactosidase inclusion body-containing recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

In this study, we developed a packed-bed immobilized cell reactor containing active β-gal (β-galactosidase) inclusion body (IB)-containing Escherichia coli (E. coli) cells in alginate beads. This packed-bed reactor was operated using a substrate feed solution 0.72 ∼ 38.4 mM ONPG (o-nitrophenyl-β-D-galactoside) prepared in Z buffer supplemented with chloroform and 0.1% SDS (sodium dodecyl sulfate). The production rate of ONP (o-nitrophenol) in the reactor containing cells that were incubated with α-MG (α-methyl D-glucospyranoside) or D-fucose after induction was superior to those prepared with cells that were not incubated with α-MG or D-fucose. The ONP production rate was increased proportionally with ONPG concentration in the substrate feed up to a concentration of 38.4 mM. However, as the ONPG concentration was increased in the substrate feed solution, galactose inhibition inside the alginate beads was increased. This most likely occurred due to problems with diffusion. In addition, partial breakage of alginate beads was observed during the later periods of operation. In this study, we demonstrated that active β-gal IB-containing E. coli cells were sustained in the immobilized cell reactor during operation. Particularly, these findings demonstrate the feasibility of using active IBs in an enzymatic reaction without the need for any purification step. In addition, we showed that these IB-containing cells could be directly used in an immobilized reactor.     

Photometabolic production of hydrogen from organic substrates by free and immobilized mixed cultures of Rhodospirillum rubrum and Klebsiella pneumoniae*

A culture of R. rubrum cells apparently contaminated with K. pneumoniae were immobilized by entrapment in agar. This system was used as model for hydrogen production by photometabolic means. Observed results indicated that the contaminant exerted a major influence on the observed results. This preparation, when immobilized and used in a specifically designed reactor with glucose substrate, showed operational half-lives of approximately 1000 hr. The feasibility of using this “mixed” culture for producing hydrogen from acid hydrolyzed cellulose and wood sawdust was also examined.     

Continuous hydrolysis of oil by immobilized lipase in a countercurrent reactor

Lipase from Pseudomonas fluorescens biotype I was immobilized by adsorption of anion exchange resin using glutaraldehyde to enhance the adsorption. The activity yield of the immobilized lipase was very low (below 1%) when lipase activity was measured using emulsion substrate. The activity yield was 10-70% when lipase activity was measured using non-emulsion substrate. Countercurrent reactors for hydrolysis of oil using non-emulsion substrate were studied. A fluidized bed reactor was found to be superior to a fixed bed one since in a fixed bed reactor the separation rate of the two layers was slow and the flow rate of the reactor had to be slower than the separation rate. A fluidized bed reactor system equipped with settling compartments and stirring compartments was devised. Continuous lipolysis at 60 degrees C and continuous separation of oily product and water soluble product were performed. After continuous operation for more than 3 months, 70% of the initial activity of the immobilized lipase was observed at the end of the reaction.     

Control of the degree of hydrolysis of a protein modification with immobilized protease by the pH-drop method

A mathematical model, DH = 1/k(10^?2ΔpH ? 1), between the pH-drop (ΔpH) and degree of hydrolysis (DH) of an enzymatic modification of casein was developed to assess the DH in a packed-bed column reactor by directly monitoring the pH value of the modified protein system. It was demonstrated that the linear DH range and the k value of the equation were dependent on the reactor type and the specificity of the proteolytic enzymes immobilized on chitin used in the present study, but no effect of the substrate casein concentration on the linear DH range was observed. Since DH and ΔpH values of the modified casein correlated with the flow rate in a packed-bed column reactor, it was suggested that the DH value, in a considerably wide range of casein modification with a certain immobilized protease in a column reactor, could be controlled by adjusting the flow rate of the substrate and monitored by a pH-meter. This relationship might be used as a basis for scale-up and long-term operation of enzymatic modification of proteins by immobilized protease in a column reactor.     

Modeling and simulation of a pressure-swing reactor for the conversion of poorly soluble substrate by immobilized enzyme: the case of d-hydantoinase reaction

An immobilized enzyme reactor system for converting poorly soluble substrate is proposed. In this stirred batch reactor, the solid substrate and immobilized enzyme suspensions are separated by a microporous filter. The advantage of separating the solid substrate from immobilized enzyme is that the fouling and breakage of the immobilized enzyme usually encountered in the stirred tank reactor can be prevented. Pressure swing can be applied to enhance the mass transfer between the two compartments. The hydrolytic reaction converting the poorly soluble substrate p-hydroxyphenylhydantoin (pHPH) into soluble N-carbamoyl-p-d-hydroxyphenylglycine (CpHPG) by immobilized d-hydantoinase is carried out in this reactor. The performance of this pressure-swing reactor is studied by simulation using a simple kinetic model. The pressure-swing operation increases the overall production rate significantly. The pressure swing also makes the reactor perform better for converting the solid substrate at higher concentration.     

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.     

Culture and nitrite uptake in immobilized Scenedesmus obliquus

Summary The green alga Scenedesmus obliquus was immobilized in Ca-alginate beads. The cell growth after immobilization was studied by cell counting. The nitrite uptake was not affected by immobilization, except that a longer lag phase was observed in immobilized cells than in free ones. That result could be due to a barrier effect of the matrix against nitrite diffusion inside the beads. The treatment of cells by glycerol prior to their immobilization in a batch reactor induced an increase of nitrite uptake by the cells. This effect disappeared after a few runs. The glycerol effect on specific rates seemed also to decrease when the number of immobilized cells increased. This decrease can be related to the decrease of light efficiency as well as substrate accessibility when a high cell concentration was used. Several alternating runs of Tris-HCl buffer containing nitrite growth medium depleted in combined nitrogen were tested. Cellular growth occurred inside the beads up to a maximum followed by a decrease of cell number in the beads.     

Hydrolysis of cellulose in a cellulase-bead fluidized bed reactor

Cellulase was immobilized in a collagen fibril matrix, and no leakage of cellulase from the collagen fibril matrix was observed. The immobilized cellulase was more stable than the native cellulase. The substrate cellulose was hydrolyzed quantitatively with immobilized cellulase. The final reaction product was identified as glucose. Immobilized cellulase was used in a fluidized bed reactor where the pressure drop of the fluidized bed reactor was low and constant. Cellulose was hydrolyzed to glucose by the cellulase-bead fluidized bed reactor. The minimum flow velocity (U_mf) was 0.5 cm/sec and the optimum flow velocity of the cellulose hydrolysis was 1 cm/sec.     

Growth and substrate consumption of Nitrobacter agilis cells immobilized in carrageenan: part 2. Model evaluation

A dynamic model that predicts substrate and biomass concentration profiles across gel beads and from that the overall substrate consumption rate by the gel beads containing growing cells was evaluated with immobilized Nitrobacter agilis cells in an airlift loop reactor with oxygen as the limiting substrate. The model predictions agreed well with the observed oxygen consumption rates at three different liquid phase oxygen concentrations. Image analysis showed that 90% of the immobilized cells after 42 days of cultivation was situated in the outer shells in a film of 140 mum, while the bead radius was about 1 mm. The maximum biomass concentration in the outmost film of 56 mum was 11 kg . m(-3) gel.     

Examination of substrate and product inhibition kinetics on the production of ethanol by suspended and immobilized cell reactors

Kinetic expressions for the fermentative production of relatively high concentrations [12% (w/v)] of ethanol have been examined. Several expressions which account for both substrate and product inhibition have been formulated, and have been applied to suspended cell and immobilized cell reactors. Experimental data have been used to validate the kinetic expressions used, and the impact of combined inhibition on optimal reactor configuration has been assessed. The process implications of combined substrate and product inhibition for suspended and immobilized cell systems have been discussed.     

Modeling of a Catalyzed Reaction Using Lipase Immobilized in a Poly(vinyl alcohol) Membrane

A mathematical model for the hydrolysis reaction of p‐nitro phenol laurate catalyzed by a lipase immobilized in a membrane was developed. In an earlier study this model reaction was found to show very different reaction rates when it was performed in aqueous micellar solution with free enzyme and with membrane immobilized enzyme. It was assumed that a local accumulation of substrate in the membrane is responsible for the observed rate enhancement. The conversion of p‐nitro phenol ester within the membrane was modeled by considering a combination of the convective flow through poly(vinyl alcohol) membrane pores, concentration polarization of substrate containing micelles at the membrane surface and the kinetics of the reaction with free enzymes. It was demonstrated that the model offered a comprehensive understanding of the interaction of the involved phenomena. The modeling results are in good agreement with the experimental data from 10 runs with different enzyme and substrate concentrations. The substrate concentration at the membrane surface increased by up to a factor of 3 compared to the feed concentration. This effect explains the observed rate enhancement. Moreover, the model was used to determine the unknown parameters, i.e., the intrinsic retention and the mass transfer coefficient, by fitting the model to the experimental data. The model may also be used to calculate the optimum operating conditions and design parameters of such a reactor.     

Continuous limonin degradation by immobilizedRhodococcus fascians cells in K-carrageenan

Limonin can be effectively degraded byRhodococcus fascians cells. These bacteria can be entraped in -carrageenan, and used in a continuous stirred tank reactor to degrade limonin in a continuous process. The effects of temperature limonin concentration, dilution rate, and aeration on the reactor behaviour have been tested, and the results correlated with changes in limonin conversion, substrate degradation rate, and free and immobilized biomass. Results showed that the immobilized cells were able to debitter limonin-containing media and the immobilized biomass was quite stable throughout the operational conditions tested. A population of free biomass was present in the reactor, the quantity of which was dependent on dilution rate. The immobilized bacteria increased its limonin-degrading capability when the substrate concentration was increased. The aeration was not strictly necessary for limonin degradation. Additionally, the immobilized cells were active and stable for more than 2 months of continuous operation, and were able to recover their limonin-degrading capability when used intermittently. Finally, none of the main components of a juice was noticeably altered during limonin degradation, so the reactor response was good enough to consider its application.     

固定化嗜热脂肪芽孢杆菌连续合成半乳糖寡糖的研究

利用固定了产β-半乳糖苷酶的嗜热脂肪芽孢杆菌,以乳糖为底物,在纤维床反应器中连续合成半乳糖寡糖(GOS),最高得率为50.7%。在连续反应体系中,研究了底物浓度、pH、反应温度和停留时间对半乳糖寡糖合成的影响,确定最佳反应条件为底物浓度450 g/L、反应温度55℃、pH7.0、停留时间100 min。在连续反应24h后,流加1.5%的D-半乳糖能提高合成GOS的能力,固定化细胞反应体系中连续稳定操作120 h。     

Effect of substrate concentration and nitrate inhibition on product release and heavy metal removal by a Citrobacter sp

A Citrobacter sp. accumulates heavy metals as cell-bound metal phosphates, utilizing phosphate released by the enzymatic cleavage of a phosphomonoester substrate. The effect of increased substrate (glycerol 2-phosphate, G2P) concentration on phosphate release and heavy metal accumulation was evaluated using a stirred tank reactor (STR) and a plug flow reactor (PFR). A significant improvement in metal removal was achieved with increased substrate concentration using immobilized Citrobacter cells in the PFR, which was not observed using free cells in the STR. Nitrate is an inhibitor of the Citrobacter phosphatase. This inhibition was concentration dependent and reversible. The rate of product release was restored by increasing the concentration of substrate (G2P). The ratio of rates of phosphate release under two different conditions (different nitrate and G2P concentrations) can be described by a equation developed from Michaelis-Menten kinetics. The concentration of substrate required for restoration of maximum velocity, V(max), in a batch and continuous-flow system can be predicted by substitution and calculation; this was confirmed by an experiment in model systems using cell suspensions and polyacrylamide gel immobilized cells in a flow-though column. For use in industrial situations it may be uneconomical or infeasible to supply additional substrate. Bioreactor activity was also restored by increasing the flow residence time, in accordance with a Michaelis-Menten-based model to describe removal of lanthanum from nitrate-supplemented flow in a PFR. (c) 1997 John Wiley & Sons, Inc. Biotechnol Biotechnol Bioeng 55:821-830, 1997.