Biochemical Engineering Journal (v.7, #1)

Glucosamine measurement has been tested as the indirect method to estimate the biomass produced by Cunninghamella elegans during solid state cultivation (SSC). The independence of this cell constituent content from the age and the conditions of the culture have been verified. The influence of the medium composition, in particular the nature of the carbon source on glucosamine amount is presented. Glucosamine can be considered as a well-adapted biomass indicator, with the necessity to establish for each medium tested a prior correlation between biomass and glucosamine amount. This correlation should be defined in submerged conditions before applying the biomass estimating method in SSC.
Keywords: Glucosamine; Biomass estimation; Cunninghamella elegans; Solid state cultivation;

A mathematical model is developed to describe the performance of a three-phase airlift reactor utilizing a transverse magnetic field. The model is based on the complete mixing model for the bulk of liquid phase and on the Michaelis–Menten kinetics. The model equations are solved by the explicit finite difference method from transient to steady state conditions. The results of the numerical simulation indicate that the magnetic field increases the degree of bioconversion. The mathematical model is experimentally verified in a three-phase airlift reactor with P. chrysogenum immobilized on magnetic beads. The experimental results are well described by the developed model when the reactor operates in the stabilized regime. At relatively high magnetic field intensities a certain discrepancy in the model solution was observed when the model over estimates the product concentration.
Keywords: Mathematical modeling; Magnetic stabilization; Magneto airlift reactors; Penicillin production; P. chrysogenum;

On-line monitoring and controlling system for fermentation processes by Yen-Chun Liu; Feng-Sheng Wang; Wen-Chien Lee (17-25).
A personal computer-based on-line monitoring and controlling system was developed for the fermentation of microorganism. The on-line HPLC system for the analysis of glucose and ethanol in the fermentation broth was connected to the fermenter via an auto-sampling equipment, which could perform the pipetting, filtration and dilution of the sample and final injection onto the HPLC through automation based on a programmed procedure. The A/D and D/A interfaces were equipped in order to process the signals from electrodes and from the detector of HPLC, and to direct the feed pumps, the motor of stirrer and gas flow-rate controller. The software that supervised the control of the stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of glucose and ethanol concentration was programmed by using Microsoft Visual Basic under Microsoft Windows. The signal for chromatographic peaks from on-line HPLC was well captured and processed using an RC filter and a smoothing algorithm. This monitoring and control system was demonstrated to be effective in the ethanol fermentation of Zymomonas mobilis operated in both batch and fed-batch modes. In addition to substrate and product concentrations determined by on-line HPLC, the biomass concentration in Z. mobilis fermentation could also be on-line estimated by using the pH control and an implemented software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.
Keywords: Bioprocess monitoring; Control; Ethanol; Fermentation; Sensors; Chromatography;

The biotransformation of β-ionone by Aspergillus niger IFO 8541 entrapped in Ca-alginate beads was investigated in a two-phase liquid system, due to the low aqueous solubility of the precursor. Modelling of phase transfer processes of the substrate demonstrated that the solute was transferred from the organic droplets to the gas, giving a loss by stripping, and then from the gas to the aqueous solution where a chemical degradation occurred. The biological reaction took place after direct precursor transfer from the organic layer to the biocatalyst by surface adsorption. Studies on the biological process demonstrated the critical effect of the biomass content in the medium at the time at which β-ionone was added. Optimum conditions involved fed-batch feedings of both precursor and carbon source (sucrose) after the biomass concentration reached a value close to 6.8 g/l. The biotransformation process then took place at a constant rate of 0.046 mmol/l h with a reaction yield, defined with respect to β-ionone metabolised by the fungus, close to unity. Best results achieved in this study allowed to obtain 3.5 g/l biological compounds after 400 h reaction.
Keywords: Bioreactors; Biotransformations; Filamentous fungi; Immobilised cells; β-ionone; Two-phase liquid systems;

An aeration strategy was proposed for foam control in an airlift reactor with double wire mesh draft tubes. The airlift reactor was employed in the cultivation of Bacillus thuringiensis for thuringiensin production. The aeration strategy involved two situations. If the foam rose and touched the foam probe, the air flow rate was dropped to a low value for a certain period. However, if the DO value was already below 10% of the saturation when the air flow rate was dropped, the conventional foam control was employed. The production of thuringiensin based on the proposed strategy was up to 70% higher than that of using the conventional cultivation method with addition of antifoam agents for foam control.
Keywords: Foaming; Antifoam agent; Aeration; Airlift reactor; Bacillus thuringiensis;

Probabilistic neural networks (PNNs) were used in conjunction with the Gompertz model for bacterial growth to classify the lag, logarithmic, and stationary phases in a batch process. Using the fermentation time and the optical density of diluted cell suspensions, sampled from a culture of Bacillus subtilis, PNNs enabled a reliable determination of the growth phases. Based on a Bayesian decision strategy, the Gompertz based PNN used newly proposed definition of the lag and logarithmic phases to estimate the latent, logarithmic and stationary phases. This network topology has the potential for use with on-line turbidimeter for the automation and control of cultivation processes.
Keywords: Fermentation; Bioprocess monitoring; Probabilistic neural networks; Bayesian strategy; Growth kinetics; Modeling;

A mathematical model of the cell cycle of a hybridoma cell line by D.B.F Faraday; P Hayter; N.F Kirkby (49-68).
A one-dimensional age-based population balance model of the cell cycle is proposed for a mouse–mouse hybridoma cell line (mm321) producing immunoglobulin G antibody to paraquat. It includes the four conventional cell cycle phases, however, G1 is divided into two parts (G1a and G1b). Two additional phases have been added, a non-cycling state G1′, and a pre-death phase D. The duration of these additional phases is determined by cumulative glutamine content and ammonia concentration, respectively. It is assumed that glutamine is only consumed during G1 and antibody is only produced during G1b and S, the kinetics are assumed to be zero-order. Glucose is consumed throughout the cell cycle at a rate that is dependent upon its prevalent concentration. Ammonia and lactate are produced in direct proportion to glutamine and glucose consumption, respectively. Parameters in the model have been determined from experimental data or from fitting the model to post-synchronisation data. The model thus fitted has been used to successfully predict this cell lines behaviour in conventional batch culture at different initial glutamine concentrations, and in chemostat culture at steady-state and in response to a glutamine pulse. The model predicts viable cell, glutamine, glucose and lactate kinetics well, but there are some discrepancies in the prediction for ammonia and antibody. Overall, the results obtained support the assumptions made in the model relating to the regulation of cell cycle progression. It is concluded that this approach has the potential to be exploited with other cell lines and used in a model-based control scheme.
Keywords: Cell cycle; Modelling; Population balance; Hybridoma;

Perfluorocarbon (PFC) was used as an oxygen carrier in the cultures of insect cells and virus-infected insect cells. The cell suspensions were placed on a planar layer of PFC, which was re-oxygenated in an outer aeration unit and continuously recirculated, and were agitated by two sets of impeller blades, lower one of which was set in such a way that the ridge of the blade touched the PFC layer. The maximum cell density attained in the PFC-mediated aeration culture was higher than that in surface aeration culture. On viral infection, a recombinant protein yield was significantly high in the PFC-mediated aeration culture as compared with that in the surface aeration culture, though the production was largely decreased by setting apart the lower set of the blade from the PFC–medium interface. These results showed that the PFC-mediated aeration would be a useful technique for insect cell/baculovirus expression system. Overall mass-transfer coefficient K L for oxygen was examined in both the PFC-mediated aeration and surface aeration systems, by using a flask whose dimensions were identical to those of spinner flasks used for the cultures. The K L value in the PFC-mediated system was 2.60×10−3   cm s −1 , 1.6 times higher than that in the surface aeration system, when impeller blades were positioned at PFC–medium and medium–air interfaces, respectively. However, the K L values in both the PFC-mediated and surface aeration systems were decreased and their differences were brought so close, as the blade was set apart from the interfaces. DO behavior in the cultures was well explained by the model calculation using the determined K L values and oxygen-consumption rates of viable cells. This calculation further suggested that crucial DO, under which recombinant protein productions were unsuccessful, was 0.24–0.5 ppm (3–7%) in the insect cell/baculovirus expression system.
Keywords: Perfluorocarbon; Insect cell; Dissolved oxygen; Baculovirus;

The biosorption of reactive dyes (Reactive Blue 2 — RB2 and Reactive Yellow 2 — RY2) onto dried activated sludge was investigated. The dye binding capacity of biosorbent was shown as a function of initial pH, initial dye concentration and type of dye. The equilibrium data fitted very well to both the Freundlich and Langmuir adsorption models. The results showed that both the dyes uptake processes followed the second-order rate expression.
Keywords: Biosorption; Reactive dyes; Dried activated sludge;

Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases by Yoshitsune Shin-ya; Moo-Yeal Lee; Hirofumi Hinode; Toshio Kajiuchi (85-88).
Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide–maleic anhydride copolymer (PEO–MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis–Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.
Keywords: Enzyme technology; Enzyme activity; Kinetic parameters; N-acetylated chitosan; Modified enzyme; Enzymatic hydrolysis;