Biochemical Engineering Journal (v.5, #2)

Experiments have been carried out to study the reaction engineering behavior of the liquid membrane–encapsulated, sequential bienzymatic reaction system, n▪ 2n glucose ▪. A dynamic mathematical model, free from adjustable parameters, has been developed taking into account peri-emulsion mass transfer, intra-emulsion diffusion, membrane-related mass transfer limitations and substrate and product inhibitions. A finite difference-based, user-friendly software has been developed to solve the model equations. Experimental data satisfactorily correlate with the model. While it is understood that study of sequential bienzymatic reaction system immobilized in emulsion liquid is essential for their industrial exploitation, reaction engineering behavior of such a system in presence of both substrate and product inhibitions has not yet been reported in the literature. Therefore, the model predictions of the present investigations are expected to pave the way for scale-up and design of industrial bioreactors in this field.
Keywords: Bienzyme system; Sequential reactions; Immobilized enzymes; Liquid membrane;

Recombinant bacterial cells in a fermentation broth rarely contain the same number of plasmids, even though this simplification is often used. Recent work has however indicated limitations of the simplified approach. Based on these studies, the distribution of plasmid copy numbers per cell has been represented macroscopically here in a Gaussian form for the fraction of biomass as a function of the copy number. Applying this distribution and an experimentally validated kinetic model to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) synthesis by Escherichia coli containing the plasmid pBR Eco gap, it is seen that GAPDH production in a batch fermentation is maximized by a particular initial (non-zero) copy number variance and an optimal duration. To implement this distribution in a bioreactor, it is suggested that the profile may be discretized, inocula corresponding to the mean copy number of each fraction prepared, and then combined to obtain the seed culture.
Keywords: Recombinant fermentation; Plasmid copy number; Optimal distribution; GAPDH productivity;

Agitation induced mycelial fragmentation of Aspergillus oryzae and Penicillium chrysogenum by A. Amanullah; P. Jüsten; A. Davies; G.C. Paul; A.W. Nienow; C.R. Thomas (109-114).
Given the impact of mycelial morphology on fermentation performance, it is important to understand the factors that influence it, including agitation-induced fragmentation. The successful application of the energy dissipation/circulation function (EDC) to correlate fragmentation of Penicillium chrysogenum with agitation intensity and with different impeller types has already been demonstrated. The EDC function takes into account the specific energy dissipation rate in the impeller swept volume and the frequency of mycelial circulation through that volume. In order to explore whether the EDC function can be used more generally to correlate fragmentation of different filamentous species, the present study extended the concept to agitation-induced, off-line fragmentation of Aspergillus oryzae grown in chemostat culture. The work shows that at EDC values off-line greater than that in the chemostat, fragmentation with different impellers can be correlated with the EDC. For EDC values less than those used in the chemostat, fragmentation did not occur. The earlier results of Jüsten et al. with Penicillium chrysogenum are also reconsidered and found to behave similarly.
Keywords: Mycelial morphology; Agitation; Mycelial fragmentation; EDC; Aspergillus oryzae; Penicillium chrysogenum;

An external-loop airlift bioreactor, with a low ratio 2.9 of height-to-diameter of the riser and a ratio 6.6 of riser-to-downcomer diameter, was used to produce α-amylase from fermentation with dregs by Bacillus subtilis. The effects of gas flow rate and liquid volume on α-amylase production were investigated. After a 36-h fermentation time, an average of 432.3 U/ml α-amylase activity was obtained under the conditions of liquid volume 8.5 l and gas flow rate 1.2 vvm for the first 12 h of fermentation, 1.4 vvm from 12 to 27 h, and 1.2 vvm from 27 h to the end. The activity was higher than that obtained in shaking flasks (409.0 U/ml) and in a mechanically stirred tank bioreactor (397.2 U/ml) under optimized operating conditions. The fermentation cycle of the airlift bioreactor was shorter than the 48 h required for the shaking flasks and close to the 36 h of the mechanically stirred tank bioreactor. It was demonstrated that the external-loop airlift bioreactor could substitute for the traditional mechanically stirred tank bioreactor to produce α-amylase from fermentation by Bacillus subtilis with dregs.
Keywords: α-Amylase; Airlift bioreactor; Bacillus subtilis;

A study on the surface hydrophobicity of lipases by Yoshiaki Sugimura; Kimitoshi Fukunaga; Takahiro Matsuno; Katsumi Nakao; Masahiro Goto; Fumiyuki Nakashio (123-128).
Surface properties, including surface net and local hydrophobicities, of bovine serum albumin, γ-globulin, and six lipases of different origins were evaluated using the aqueous two-phase partitioning method. Each showed a specific and characteristic pattern of surface properties. Correlations between the protein surface hydrophobicities and the coverages of the proteins by lipid-coating with a synthetic detergent, dioleyl glucosyl l-glutamate, were discussed. The results indicated that the surface net hydrophobicity of each protein was indicative of the affinity of the protein for the coating detergent applied in lipid-coating.
Keywords: Aqueous two-phase system; Partitioning of lipases; Surface hydrophobicity; Lipid-coated lipases;

Comparison of biotin production by recombinant Sphingomonas sp. under various agitation conditions by Ikuo Saito; Hiroyuki Honda; Tomoyasu Kawabe; Fujio Mukumoto; Masatoshi Shimizu; Takeshi Kobayashi (129-136).
Biotin production by fermentation of recombinant Sphingomonas sp./pSP304 was investigated. A complex medium containing 60 g/l of glycerol and 30 g/l of yeast extract was suitable for biotin production. Biotin was produced in the late logarithmic or stationary phase after glycerol starvation. The optimum pH value for biotin production was 7.0. When the dissolved oxygen concentration (DO) was controlled at a constant level, the biotin concentration produced after 120 h was significantly lower than that obtained in a test tube culture. Therefore, a batchwise jar-fermentor culture with a constant agitation speed and without DO control was conducted for investigating the effect of agitation conditions on biotin production. Six types of impeller were tested: turbine-blade type, turbo-lift type, rotating mesh type (EGSTAR®), screw with draft tube type, Maxblend®type, and anchor type. With some impellers, agitation speed was also changed. Both the maximum cell concentration and biotin production varied depending on agitation conditions. Relatively high cell concentrations were attained with four of the impeller types, turbine-blade type, rotating mesh type, Maxblend® type, and anchor type. Among these impellers, the turbine-blade impeller with sintered sparger was suitable for biotin production. After 120 h, the cell concentration reached an OD660 of 43 and a biotin concentration of 66 mg/l was obtained, which was comparable with the results from the test tube culture. Morphological variation was also observed depending on the agitation conditions: oval-shaped, rod-shaped, and elongated-shaped cells. Biotin production was relatively high in slightly long rod-shape cells but low in elongated cells. The difference in morphology appeared to depend on the shear stress. It was found that biotin production was strongly correlated with cell length and the oxygen transfer coefficient (k L a); cell lengths in the range 4–7 μm and k L a values in the range 1.5–2.0/min were found to be suitable for biotin production in jar-fermentor culture.
Keywords: Biotin fermentation; Jar-fermentor; Impeller design; Agitation condition; Morphology change;

Kinetics of two-liquid-phase Taxus cuspidata cell culture for production of Taxol by Zhao-Liang Wu; Ying-Jin Yuan; Zhong-Hai Ma; Zong-Ding Hu (137-142).
The effects of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions on the cell growth and Taxol production were studied in two-liquid-phase and the two-stage culture. A kinetic model, incorporated the effects of the toxicity of organic solvents was developed for two-liquid-phase culture of Taxus cuspidata in the two-stage Taxol production. The results showed that the proposed kinetic model could fit the experimental data satisfactorily. The results also showed that Taxol production could reach the optimal value when 10−log  P was in the range of 2 to 5 and the volumetric fraction of the organic solvents at the corresponding the highest Taxol production should be lower when 10−log  P was high.
Keywords: Plant cell culture; Two-liquid-phase; Taxus cuspidata; Taxol; Kinetic model;

Efficient production of desulfurizing cells with the aid of expert system by Michimasa Kishimoto; Masaaki Inui; Takeshi Omasa; Yoshio Katakura; Ken-ichi Suga; Koichi Okumura (143-147).
An expert system was used to achieve the high production of desulfurizing cells of Rhodococcus erythropolis KA 2-5-1. By adding a proper amount of sulfur containing component with the aid of the expert system, we could avoid excess feeding which resulted in the lowering of desulfurizing activity and starvation which caused serious damage to cell growth. In order to determine the addition amount by the expert system, the data of the amount of chemical elements contained in the cells were used as a reference for comparison with the medium components present. Culture experiments were carried out using a 5 l jar fermentor with several kinds of media whose components were determined based on the inferred results with the aid of the expert system. We could restrict the amount of the sulfur component addition so that sulfur was a growth-limiting factor; in contrast, the amounts of other elements were sufficient for growth.As a result, the maximum specific production rate of 2-hydroxy biphenyl (2HBP) and the maximum cell concentration were 20 mmol kg-drycells−1  h−1 and of 45 g-drycells l−1, respectively. At 100 h of cultivation, 1 g/l of dibenzothiophene (DBT) was converted to 2HBP within 20 h, i.e., 10 mmol kg-drycells−1  h−1 of specific desulfurization activity, and the specific activity remained stable for a long period in the culture experiment.
Keywords: Expert system; Fed-batch culture; Bioreactor; Fermentation; Desulfurization; Rhodococcus;

Release of recombinant proteins from gene-engineered Escherichia coli by applying a pulsed electric field (PEF) to a cell suspension was studied. When E. coli/pNC1, which produces β-glucosidase and accumulates it in cytoplasm, was exposed to PEF, the most effective release of this enzyme was achieved in the cell suspension of 5% glycine and 15% PEG solution under 10 kV/cm and 280 J/ml of a PEF in a needle–plate electrode chamber. However, the amount of released β-glucosidase by PEF treatment was only 26% of that by ultrasonic treatment. On the other hand, α-amylase produced by E. coli/pHI301A and accumulated in the periplasmic space could be easily released by PEF treatment. When this recombinant E. coli was suspended in 0.9% NaCl and 10% PEG solution and exposed to 10 kV/cm and 200 J/ml of a PEF in a plate–plate electrode chamber, 89% of intracellular α-amylase with nine-times higher specific activity compared with that by ultrasonic treatment was released. The release tendency of cellobiohydrolase, produced by E. coli/pNB6 and accumulated in both the cytoplasm and periplasmic space, was intermediate between those of β-glucosidase and α-amylase. In this case, 70% of cellobiohydrolase with 1.9-times higher specific activity compared with that by ultrasonic treatment could be released when E. coli/pNB6 was suspended in 15% PEG and 10 kV/cm and 200 J/ml of a PEF was applied in a needle–plate electrode chamber. These results indicated that PEF treatment could easily disrupt the outer membrane, but it was difficult to disrupt the cytoplasmic membrane simultaneously. Therefore, PEF treatment is useful for easy release of periplasmic protein with selectivity.
Keywords: Recombinant DNA; Enzyme production; Bioseparation; High-voltage; Pulsed electric field;

Light intensity distribution in the externally illuminated cylindrical photo-bioreactor and its application to hydrogen production by Rhodobacter capsulatus by Tomohisa Katsuda; Takeshi Arimoto; Koichi Igarashi; Masayuki Azuma; Jyoji Kato; Susumu Takakuwa; Hiroshi Ooshima (157-164).
The light distribution in the externally illuminated cylindrical photo-bioreactor for production of hydrogen by a photosynthetic bacterium Rhodobacter capsulatus ST-410 was estimated. The estimation was performed on the basis of the Matsuura and Smith’s diffuse model . In the diffuse model, the incident light rays are assumed to proceed in every direction and the local intensity is calculated as the sum of the intensities of light. Since Lambert–Beer’s law, extensively used in photometry, was not useful for explaining the decrease in the intensity of light by the biomass, an empirical expression was used. The measurement of the intensities from every direction was conducted in an externally illuminated cylindrical photo-bioreactor having an inner diameter of 60 mm and a working volume of 550 ml. The obtained results confirmed our estimation. The light distribution was applied to estimate the hydrogen production by R. capsulatus ST-410 using the same photo-bioreactor. The overall hydrogen-production rate was successfully estimated.
Keywords: Photosynthetic bacterium; Hydrogen production; Bioreactor; Fermentation; Batch processing; Bioprocess design;

Evaluation of kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor for wastewater treatment by Akira Hirata; Takeshi Takemoto; Kentaro Ogawa; Joseph Auresenia; Satoshi Tsuneda (165-171).
This study evaluates the kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor from the steady state values of the response of the system to step changes in inlet concentration. It was observed from the outlet biological oxygen demand (BOD5) plot of the response of the system that as the inlet BOD5 was increased, the outlet BOD5 also increased, reached a peak value and then decreased until it leveled to a new steady state value corresponding to the new inlet concentration level. The increase in BOD5 was attributed to the accumulation of substrate within the reactor as well as the decrease in biofilm substrate consumption rate as the microorganisms adjusted to the new environment. Using the substrate balance at steady state and assuming Monod kinetics, an equation relating the substrate consumption rate to substrate concentration (BOD5) and total biofilm surface area had been established. Monod kinetic parameters were found to be K=2.20 g/m2/day, K m=17.41 g/m3 and K/K m=0.13 m/day. The ratio K/K m can be taken as the indicator for biofilm substrate degradation effectiveness at low substrate concentrations.
Keywords: Biofilm reactor; Kinetic models; Step change response; Three-phase fluidized bed; Biological wastewater treatment;