Biochemical Engineering Journal (v.9, #2)

Bej Keywords (II).

Semi-continuous production of lauroyl kojic acid through lipase-catalyzed condensation in acetonitrile by Takashi Kobayashi; Shuji Adachi; Kazuhiro Nakanishi; Ryuichi Matsuno (85-89).
7-O-Lauroyl kojic acid was synthesized on some tens of grams scale through lipase-catalyzed condensation of kojic and lauric acids in acetonitrile using a semi-continuous system where unreacted substrates were reused after the product recovery. The acylation of kojic acid improved the solubility in hexane and soybean oil. The absorption spectra of lauroyl kojic acid chelated with ferric ion were observed in ethanol and soybean oil at various molar ratios. The color tone of the complex of lauroyl kojic acid and Fe3+ was similar to that of the complex of unmodified kojic acid and Fe3+ in ethanol.
Keywords: Bioconversion; CSTR; Kojic acid; Lauric acid; Lipase;

Production of l-glutamic acid (GA) was accomplished by using Brevibacterium flavum (DSM 20411) in 3.5 dm3 laboratory scale bioreactors at 30°C, initial pH 7.2, penicillin ratio 10 000 U g−1 DW, agitation rate 150–500 min−1, and air flow rate 0.1–0.2 vvm. Cell and GA concentrations and concentrations of other amino acids and organic acids were measured throughout batch fermentations. By-products varied in number and concentration depending on the amount of oxygen supplied to the broth. Very low oxygen transfer rate favoured pyruvic acid and lactic acid production; while at higher oxygen transfer levels α-ketoglutaric acid and succinic acid were favoured. The 0.1 vvm and 250 min−1 conditions were optimum for maximum GA production and selectivity.
Keywords: Glutamic acid fermentation; Brevibacterium flavum; Glutamic acid fermentation by-products; Oxygen transfer effects;

Membrane fouling during cross-flow microfiltration of Polyporus squamosus fermentation broth by Darko M. Krstić; Siniša L. Markov; Miodrag N. Tekić (103-109).
The application of microfiltration to biological systems is hindered by membrane fouling that results in a decrease in the filtrate flux and solute transmission with time. In this work, the effects of transmembrane pressure, cross-flow feed velocity, biomass structure and feed composition, on membrane fouling during cross-flow microfiltration of Polyporus squamosus fermentation broth, were investigated. The results of cross-flow trials with 0.2 μm aluminum oxide membrane showed the existence of the optimal operation transmembrane pressure and cross-flow velocity, in respect of membrane fouling, for examined range of operation conditions. It was noticed that the process of membrane fouling was moved from a predominantly surface layer phenomenon to internal membrane fouling as the cross-flow velocity was increased. Comminution of the fungal biomass prior to microfiltration to reduce particle size showed a beneficial effect on the transient flux. The steady-state flux and the transmission of solutes were not significantly affected by the biomass comminution. The observations of the filtrate flux and the transmission of solutes indicated a decrease in the rate of fouling with decreasing content of soluble components with large molecular weights in the feed.
Keywords: Microfiltration; Fermentation broth; Membrane fouling;

Recombinant fermentations carried out in large bioreactors are characterized by incomplete dispersion of fluid and a distribution of plasmid copy numbers among the cells. The effects of both features have been studied for the batch production of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by E. coli harboring the plasmid pBR Eco gap. GAPDH activity is maximized by a large average copy number (within metabolically permissible limits) of the cell population and intermediate optimal values of its variance and of dispersion in the broth (expressed by the Peclet number). The optimum conditions, however, should be chosen judiciously because, although GAPDH activities are high, the incremental benefits are lower than under non-optimal operation.
Keywords: Recombinant fermentation; Plasmid copy number distribution; Fluid dispersion; GAPDH activity;

Crude preparations of porcine pancreatic lipase (PPL) contain apart from the PPL a significant number of other hydrolases as impurities. The treatment of crude PPL preparations with humid supercritical carbon dioxide leads to a quality improvement of the preparations. Five hundred milligrams of PPL preparation were incubated in a high pressure reactor (140 ml) at 150 bar and 75°C for 24 h. Different amounts of water were put into the reactor prior incubation. A set of different assays based on hydrolytic activities was used after the treatment. It was found out that the treated preparations are significantly more active with the long chain triglyceride triolein (675% residual activity with 100 μl water added) and an artificial substrate (1,2-o-dilauryl-rac-glycero-3-glutaric acid-resorufin ester (DGGR)), while there is a loss of activity towards the short chain triglycerides, tributyrin and triacetin. Treatment with humid supercritical carbon dioxide activates the PPL that is active towards long chain triglycerides and denatures the other hydrolases that are active only towards short chain triglycerides. Repeated incubation in dry supercritical carbon dioxide causes no loss of activity towards short chain triglycerides indicating the important role of the water in the supercritical phase for the denaturing effect on the hydrolase impurities. The treated preparations have a high temperature stability (no activity loss after 72 h at 60°C).
Keywords: Enzyme technology; Enzyme activity; Lipase; Protein denaturation; Supercritical carbon dioxide; Triglycerides;

Batch in vitro hybridoma cell culture efficiency frequently is hindered by the depletion of the nutrient glutamine and the accumulation of toxic ammonia within the culture medium. This paper presents a novel approach for ammonia removal and glutamine replenishment in hybridoma cell culture through use of ex vivo immobilized glutamine synthetase (GS). Experiments involving soluble GS revealed that the ammonia conversion was sensitive to the concentration of reaction substrates. Glutamate and Mg2+ concentrations had the largest positive effects on initial ammonia conversion rate, whereas glutamate and ATP had the largest positive effects on total ammonia conversion. Ca2+ was found to be highly inhibitory to GS activity; 1.8 mM Ca2+ (as present in a standard culture medium) reduced GS conversion activity by 66%. Of three enzyme immobilization methods tested, covalent binding to Sepharose yielded the largest increase in GS thermostability at 37°C, but encapsulation in dialysis tubing provided the best overall conversion activity (100% of soluble GS activity). A hybridoma culture system incorporating encapsulated GS maintained the culture medium ammonia concentration below 4 mM and substantially prolonged hybridoma metabolic activity. Overall, immobilized GS shows promise as a means of extending the “useful life” of hybridoma cell cultures without the product diluting effect of medium perfusion.
Keywords: Hybridoma cultures; Glutamine synthetase; Immobilized enzymes; Bioreactors; Enzyme activity; Ammonia removal;

Economic analysis of immunoadsorption systems by Ben Hunt; Chris Goddard; Anton P.J. Middelberg; Brian K. O’Neill (135-145).
Immunoadsorption possesses an attractive combination of selectivity and yield, and in some cases may replace many conventional purification steps with a single step. The economic performance of immunoadsorption is dependent on apparatus configuration (e.g. Sepharose® bed, perfusive bed and membrane), operating conditions (e.g. bed depth, flow rate and concentration of adsorbate in the effluent at breakthrough) and antibody binding kinetics. The solution space for the lowest operating cost is potentially very large and is prohibitive to exhaustively investigate in the laboratory. A mathematical model of a generic immunoadsorption process is useful in identifying the most promising configurations, operating conditions and antibodies, without having to resort to experiments that explore every possibility. The model system was chosen to represent the purification of a valuable biochemical product (1000 mg) from mammalian cell culture media or milk where the protein is present at low concentration (2 mg l−1). The antigen–antibody kinetic rate constants for a bovine serum albumin–monoclonal antibody system are available in the literature and were representative for this study. A modified chromatographic model was developed to predict the dimensionless operating cost of the generic immunoadsorption system subject to different apparatus configurations, operating conditions and antibody kinetics. Optimal operating conditions for each of the three configurations were identified. Under these conditions, the operating costs for a perfusive bed and a membrane were comparable and 28% lower than for a packed bed. The antibody binding kinetics significantly affected the dimensionless operating cost of the system and, therefore, the model can help select the most suitable antibody from a panel of prospective antibodies.
Keywords: Affinity; Bioprocess design; Bioseparations; Chromatography; Modelling; Monoclonal antibodies;

Erythrocyte destruction during turbulent mixing by Ryszard Pohorecki; Jerzy Bałdyga; Adam Ryszczuk; Tomasz Motyl (147-154).
Destruction of erythrocyte cells in a high shear environment has been investigated during turbulent conditions. It has been observed that shear stress generated in viscous sub-range of turbulence is capable of destroying erythrocytes, although viscous sub-range has been so far considered generally safe for cells. The multifractal theory of turbulence has been proposed to explain erythrocyte cells destruction in this case. Two hypotheses of cell rupture, both based on a critical stress concept, were tested and kinetic constants for multifractal model as well as for Kolmogorov theory have been calculated.
Keywords: Kolmogorov theory; Multifractal theory; Turbulence; Hydrodynamic stress; Erythrocyte destruction;

Hydrodynamics and heat transfer were investigated in a 0.28 m diameter slurry bubble column for air–water–yeast cells system. Yeast cells of about 8 μm diameter were used and the effects of gas velocity and yeast concentrations were studied. Gas holdups exhibited a maximum value around a gas superficial velocity of 0.10 m/s when foam height was included. Without the foam layer, gas holdups increased with increasing gas velocity. Bubble population was measured by means of dynamic gas disengagement technique. Rise velocity of small bubbles decreased, while rise velocity of large-bubbles fraction increased with yeast concentration which was varied from 0–0.4% w/w. Local heat transfer coefficients were measured at different axial and radial locations inside the column. Heat transfer in the foam section was significantly lower than in the main slurry column. In the bulk and developing regions, the addition of yeast cells into water increased heat transfer in the center and decreased at the wall. This work is one of very few studies reported in the literature on the use of actual yeast cells in slurry bubble columns and opens up new opportunities on their potential use as bioreactors in the fermentation industries, wastewater biological treatment and other applications.
Keywords: Slurry bubble column; Yeast cells; Hydrodynamics; Bubbles fractions; Heat transfer;