Biochemical Engineering Journal (v.9, #1)

Bej Keywords (IV).

Predictive modeling and loose-loop control for perfusion bioreactors by Jason E. Dowd; K.Ezra Kwok; James M. Piret (1-9).
Perfusion bioreactors are widely used to produce recombinant proteins and monoclonal antibodies for therapeutic and diagnostic use. Better control of the cellular environment can lead to higher volumetric productivity, ensure product consistency and optimize medium utilization. The objective was to manipulate and control substrate concentrations in the perfusion bioprocess using predictive modeling and control. The goal of the predictive controller was to minimize future deviations from the set point concentration, by structuring the controller output. The appropriate structure for the future manipulated variable was specified using the selected model of glucose uptake rates (GUR). When there was a deviation from the set point value, the flow rates were adjusted to drive the process close to the set point value in a defined first order manner. The shape of the first order process response depended on the magnitude of the deviation from the set point value. With daily sampling and glucose measurement, a feed rate profile (eight flow rates per day) was specified to control the bioprocess. Despite the infrequent sampling, the predictive control protocols demonstrated glucose variation of less than 0.4 mM in transient conditions, and less than 0.2 mM in pseudo-steady-state conditions. The non-linear controller allowed for rapid changes in set point concentrations (6–9 h) or a reference trajectory to be followed. Set point changes and reference trajectories were simulated and tested with real process data. Modeling error and measurement bias were simulated to have the greatest potential effect during exponential growth. With good model estimation of the GUR, predictive control was able to maintain the process at the set point with a level of variability approaching that of the glucose assay.
Keywords: Glucose-based control; Perfusion; Bioreactor control; Predictive control;

Factors affecting chromium(VI) reduction by Thiobacillus ferrooxidans by M. QuiIntana; G. Curutchet; E. Donati (11-15).
The ability of Thiobacillus ferrooxidans cultures to reduce chromium(VI) to chromium(III) was evaluated under different conditions. In T. ferrooxidans cultures with sulphur as energy source, the capacity for chromium(VI) reduction was related to the generation of sulphur compounds (sulphite, thiosulphate and polythionates) with high reducing power. In contrast with other chromium(VI)-reducing microorganisms, T. ferrooxidans showed higher chromium(VI) reduction at low pH. The reduction of chromium(VI) also increased with the age of the culture. A T. ferrooxidans cells were capable of growing under anaerobic conditions with chromium(VI) as the terminal-electron acceptor.
Keywords: Thiobacillus ferrooxidans; Chromium(VI); Bioreduction; Reducing compounds; Sulphur; Anaerobic; Aerobic conditions;

A response surface approach has been used to study the production of extracellular lipase by Candida cylindracea. Medium constituents were optimised for the production of lipase by central composite design. A 25 design matrix was used in the experiments with only the carbon source being varied. The optimal yield of lipase was 17.30 U/ml using glucose as the carbon source and 47.25 U/ml using olive oil as the carbon source. All the other medium constituents namely yeast extract, malt extract, peptone and tween 80 were simultaneously optimised. Experiments conducted at the predicted optimum levels fitted with the predicted optima. Based on these experimental conditions, medium containing olive oil appears to a better medium for fermentative production of C. cylindracea lipase.
Keywords: Enzyme production; Fermentation; Modelling and optimisation;

A modified anaerobic baffled bioreactor (MABR) was studied under steady-state conditions for treating palm oil mill wastewater. Methane gas production was in the range of 0.32–0.42 l-CH4  (g-COD)−1 removal, which corresponded to the methane content of 67.3–71.2% within the range of examined hydraulic retention time (HRT) of 3–10 days. The removal ranges of COD and grease/oil were from 87.4 to 95.3% and from 44.1 to 91.3%, respectively. The total volatile fatty acid production was 1450 mg l−1 at HRT of 3 days and gradually decreased to 608 mg l−1 at HRT of 10 days. Based on the experimental data, a kinetic model was discussed. Through the model the biokinetic parameters were evaluated, which represented the behavior of reactor very well.
Keywords: Anaerobic bioreactor; Kinetic model; Steady-state; Hydraulic retention time;

A study of the cross-linking immobilization of β-galactosidase from Kluyveromyces lactis on graphite surfaces is reported here. The cross-linking reagent was glutaraldehyde. Thermal deactivation of the immobilized enzyme at the temperatures from 35 to 55°C was investigated, and the deactivation rate was found to follow the Arrhenius law with the activation energy of about 200 kJ mol−1 for the deactivation of the immobilized enzyme. The temperature-activity curves are similar for both the free and immobilized enzyme. However, the maximum activity of the immobilized enzyme was shifted up from 40°C to 50°C compared with that of the free enzyme. The pH for the maximum activity of the immobilized enzyme to occur has been found to increase by 1.1 to 7.7 U compared with the free enzyme. Lactose hydrolysis in a skim milk using the immobilized enzyme has also been investigated in a continuous enzymatic reactor. The related mechanisms of the hydrolysis process are discussed.
Keywords: β-Galactosidase; Graphite; Enzyme activity; Kluyveromyces lactis; Lactose hydrolysis; Immobilized enzyme; Glutaraldehyde; Thermal deactivation; Bioreactor;

A semi-batch affinity ligand ultrafiltration system used for the separation of trypsin from a simulated mixture containing trypsin (MW: 23,800) and chymotrypsin (MW: 27,000) has been studied using regenerated cellulose membrane (MWCO: 30,000) and soybean trypsin inhibitor (STI) as ligand. The concentrations of both trypsin and chymotrypsin are 0.02 mg/ml in the mixture while the ratio between STI and trypsin has been maintained at 50:1. Operating pressure of the ultrafiltration module was in the range of 199.4–395.5 kPa. Concentration of trypsin in the ultrafiltration module went down from its initial value to zero within 160 s whereas chymotrypsin took 300 s to go down to zero. Two deterministic mathematical models have been developed and have been compared with the experimental observations.
Keywords: Affinity ultrafiltration; Trypsin purification; Washing phase modelling;

The problem of finding the optimal regulatory structures was formulated as a mixed-integer linear programming problem for lysine synthetic pathway using (log)linear models obtained from nonlinear kinetic models developed previously by us. Optimal structures were obtained from a set of postulated regulatory structures. This method of metabolic design allowed the identification of the type and the strength of the necessary regulations, the activities of the corresponding enzymes and the intermediate metabolite levels in the lysine pathway. The calculated results indicate that >20% increase of the internal lysine flux can be obtained when only the inhibitory regulation was allowed, and eight optimal structures with one regulatory loop were adopted. When the regulation due to enzyme activation was allowed, the internal lysine flux can be increased by >70%. Changes of the participating precursor and cofactor concentrations may not improve the lysine flux significantly in this system.
Keywords: Metabolic regulation design; Lysine synthetic pathway; Model linearization;

Enhancement of anthocyanin production from grape (Vitis vinifera) callus in a viscous additive-supplemented medium by Eiji Nagamori; Kousuke Hiraoka; Hiroyuki Honda; Takeshi Kobayashi (59-65).
N-medium, which is the medium supplemented with carboxymethyl cellulose (CMC) as a viscous additive, was applied to the anthocyanin production from grape callus, Vitis vinifera cv. Bailey alicant A. Although the cell growth decreased with increasing CMC content in the medium, the anthocyanin content per packed cell volume (PCV) increased significantly. When N-B5-medium (0.8%), B5-medium containing 0.8% CMC, was used, approximately three times higher level of anthocyanin production was obtained than that from the control culture without CMC addition. In the repeated batch culture using N-B5-medium (0.8%), the productivity was found to continue at a high level during 30 batch cultures, while no anthocyanin productivity was obtained after 16th batch in the control culture. When the completely white callus after 16th batch culture was transferred to N-B5-medium (0.8%) and cultured repeatedly, anthocyanin productivity was completely recovered after 6th batch culture in N-B5-medium. In contrast, when calli obtained from N-B5-medium (0.8%) was cultured in conventional medium, the productivity was gradually decreased and became almost 0 after 8th repeated batches. Size distribution of calli obtained from the culture in N-B5-medium (0.8%) was also investigated. After repeated batch culture in N-B5-medium (0.8%), the size of calli was shifted to large size and the fraction of calli with less than 0.2 mm decreased remarkably. All calli in the fraction of more than 0.6 mm in size contained pigmented purple cells, while in the fraction of less than 0.2 mm in size the ratio of calli containing pigmented purple cells was less than 50%. From these results, it was confirmed that the enhanced and prolonged production of anthocyanin was due to the shift of size distribution to larger size and it was suggested that the shift of cell size was caused by lowering the damage to grape callus by hydrodynamic stress via increase of medium viscosity.
Keywords: Grape callus; Anthocyanin; Viscosity; N-medium; Shear stress;

Esterification by Rhizopus delemar lipase in organic solvent using sugar ester reverse micelles by Kazumitsu Naoe; Tomomi Ohsa; Mikio Kawagoe; Masanao Imai (67-72).
The esterification of oleic acid with octyl alcohol catalyzed by Rhizopus delemar lipase was investigated in reverse micellar system of sugar ester DK-F-110. High initial reaction rate was obtained by preparing the micellar organic phase with extremely low water content. The maximum rate was obtained at a W0  (=[H 2 O]/[amphiphile]) of 2.5. The highest reaction rate occurred at pH 6. With decreasing DK-F-110 concentration, the initial reaction rate was slightly decreased. The optimal temperature for lipase activity was around 313 K (40°C). Apparent activation energy of the esterification in the system was 43.7 kJ/mol. The lipase showed 40% of its esterification activity after 28 h incubation in the DK-F-110 micellar organic phase. The reaction kinetics in this system were recognized as ping-pong bi–bi mechanism. From a Michaelis–Menten analysis, apparent kinetic parameters were determined. The turnover number of the DK-F-110 system was larger than that of the sodium bis(2-ethylhexyl)sulfosuccinate (AOT) system.
Keywords: Reverse micelle; Esterification; Lipase; Non-ionic surfactant; Sugar ester;

Modeling of the gel-immobilized cell system requires accurate measurement of diffusion coefficients. Three methods of the quasi-steady-state (QSS) method, the time-lag (TL) method and a variant quasi-steady-state (VQSS) method were critically assessed and compared for the evaluation of diffusivities using the diffusion cell technique. Experimental data from our laboratory were used for the analysis of the influence of crucial theoretical assumptions not being fulfilled in each method. The results highlighted a risk in obtaining highly variable diffusion coefficients by not validating the QSS and the accuracy of the measurements. In the TL method, the estimation of diffusivities based on the plot intercept that was mostly used in the literature, results in a many fold lower value when compared to that based on the plot slope. The comparison with the QSS and VQSS methods confirmed similar diffusivity obtained by the TL method based on the plot slope. It thus suggested that the correct estimation of diffusivities by the TL method could be based on the plot slope only. Furthermore, the errors associated with the solute mass in the gel, the sample withdrawal and the non-negligible concentration changes in the chambers were also discussed. It is concluded that diffusion cell technique has to be employed cautiously for a correct evaluation of diffusivities.
Keywords: Diffusion cell; Alginate gel; Diffusion; Immobilized cells; The quasi-steady-state method; The time-lag method;