Biochemical Engineering Journal (v.19, #1)

Bej Keywords (II).


Determination of protein diffusion coefficients in agarose gel with a diffusion cell by Jan Gutenwik; Bernt Nilsson; Anders Axelsson (1-7).
A diffusion cell has been used to measure the effective diffusion coefficients of proteins. The method is applied to lysozyme and BSA at different pH and ionic strength. A parameter optimization technique is used to estimate the diffusion coefficients directly from experimental data.The importance and influence of pH and ionic strength on the diffusive properties in an agarose gel have been demonstrated. A comparison with other methods showed that there is good agreement. The diffusion cell is an accurate and easy-to-use method for the measurement of protein diffusion coefficients, in spite of the long time required for a protein diffusion experiment. For smaller proteins this time can be shortened considerably by the present parameter optimization technique.
Keywords: Diffusion cell; Protein diffusion; Dynamic simulation; Modeling; Lysozyme; BSA; Agarose gel; Hindered diffusion;

Model of bioconversion of cholesterol in cloud point system by Zhilong Wang; Fengsheng Zhao; Xueqin Hao; Daijie Chen; Daotang Li (9-13).
Bioconversion of cholesterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (4-AD) in cloud point system (CPS) was studied. A mathematical model was presented based on the mass transfer rate and microbial growth rate. The calculations for cell concentration, substrate concentration, and product concentration as functions of time were made. Sensitive analysis of model was made. Some parameters, such as substrate concentration, mass transfer coefficient, and maximum product inhibition concentration, indicated that the maximum product concentration is the most significant parameter in batch process. The prediction was validated by adjusting substrate concentration, stirring speed, and surfactant concentration. It showed the batch process of cholesterol bioconversion in CPS was controlled by inhibition of product. Adjusting surfactant concentration can diminish inhibition of product and increase final product concentration. The results also indicated that CPS can increase the substrate concentration of aqueous phase in batch process.
Keywords: Model; Bioconversion; Batch process; Kinetic parameters; Nonionic surfactant; Cloud point system;

Human β-1,3-N-acetylglucosaminyltransferase-2 (β3GnT2) was produced in a baculovirus expression system as a secreted fusion protein with a green fluorescence protein variant, GFPuv, flanked by the (His)6 sequence and an enterokinase cleavage site. The expression of the β3GnT2–GFPuv fusion gene was rapidly detected using a fluorescence microscope without employing complicated assay methods. When Tn-5B1–4 cells were infected with a recombinant AcMNPV–β3GnT2–GFPuv virus at MOI 10, intracellular and extracellular β3GnT activities increased to 0.26 and 0.68 mU/ml, respectively, until 3 days post-infection (d.p.i.), and decreased markedly at 3 d.p.i. In contrast to Tn-5B1–4 cell culture medium, the extracellular β3GnT activity in Sf-9 cell culture medium increased to 0.86 mU/ml at 4 d.p.i. The fusion protein obtained from Tn-5B1–4 and Sf-9 cultures was confirmed based on the GFPuv of the fusion protein. The fusion protein was purified using a Ni2+ affinity column, and was concentrated by approximately 900-fold. The observed β3GnT activity and the specific β3GnT activity of the purified fusion protein were 77.6 mU/ml and 4.6 U/mg protein, respectively. When the purified fusion protein was treated with glycopeptidase F, its molecular weight decreased by 7–8 kDa, indicating that β3GnT2 is glycosylated.
Keywords: Insect cell culture; Baculovirus; Recombinant DNA; β-1,3-N-Acetylglucosaminyltransferase; Green fluorescence protein; Baculovirus expression system;

Lipase production by Acinetobacter radioresistens was examined in fed-batch cultures using Tween 80 as the carbon source. An optimal medium containing 3 ml/l Tween 80 and 7.5 g/l yeast extract was developed in batch cultures without pH control. Assessed from the final culture pH, this composition was regarded being balanced between the carbon and nitrogen sources. Data obtained from fed-batch cultures with DO- and pH-stat feedings showed that specific growth rate μ was the intrinsic factor that determined the efficiency of lipase synthesis. The specific lipase productivity was maximal at μ=0.1 h−1, while the optimal lipase production rate occurred around μ=0.2 h−1. Based on a growth-associated pattern for lipase formation, the production of lipase with fed-batch culture could be simulated satisfactorily.
Keywords: Lipase production; Acinetobacter radioresistens; Fed-batch culture; Tween 80;

A model was developed to represent the biooxidation by Acidithiobacillus ferrooxidans of a refractory gold concentrate in a continuous stirred tank reactor (CSTR). The model accounts for the growth of attached and planktonic cells, their role in mineral solubilisation, and the contribution of ferric ions to the process. Predicted solubilisation rates increase with increasing solids dilution rates until a maximum, in agreement with the experimental results. Concentration of suspended cells was more affected by changes in solids dilution rates than attached cells did, behaviour well represented by the model. The model was also able to predict the observed increase in cell growth and mineral solubilisation due to the decrease in particle size and the enrichment of the air with CO2. Finally, the model was used to predict the optimal configuration of a continuous biooxidation system that minimises the residence time required to attain a defined degree of mineral solubilisation. This configuration was found to be a two-stage system consisting of a CSTR followed by a plug flow reactor. The latter can be replaced by a series of smaller equal-size CSTRs.
Keywords: Acidithiobacillus ferrooxidans; Bioreactors; Continuous biooxidation; Heterogeneous biocatalysts; Kinetic parameters; Modelling;

Activation of manganese peroxidase in an organic medium using a mediator by Junji Michizoe; Yoichi Uchimura; Hirofumi Ichinose; Tatsuo Maruyama; Noriho Kamiya; Hiroyuki Wariishi; Shintaro Furusaki; Masahiro Goto (43-46).
We found that both unsaturated fatty acids (UFAs) and 1-hydroxybenzotriazole (HBT) enhance the enzymatic activity of manganese peroxidase in an organic medium. The effects of hydrophobic unsaturated fatty acids directly dissolved in an organic medium and hydrophilic HBT encapsulated in reverse micelles on the oxidation activity of a surfactant–manganese peroxidase (MnP) complex were investigated. The addition of UFAs or HBT (mediator) using reverse micelles improved the oxidation of 2,4-dichlorophenol in toluene up to 3-fold the oxidation without each mediator. This study presents, for the first time, the possibility of MnP-catalyzed oxidation coupled with mediators in organic media. The capability of the latter system using HBT was further assessed by the oxidative conversion of environmental pollutants, i.e. 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, and bisphenol A (100, 84, 91, and 100% conversions at 12 h of reaction, respectively).
Keywords: Biodegradation; Bioremediation; Enzymatic oxidation; Mediator; Non-aqueous solvent; Surfactant–enzyme complex;

Ammonium lactate production by Lactobacillus lactis BME5-18M in pH-controlled fed-batch fermentations by DongMei Bai; ZhiHui Yan; Qiang Wei; XueMing Zhao; XinGang Li; ShiMin Xu (47-51).
A process for efficient production of ammonium lactate by Lactobacillus lactis BME5-18M in pH-controlled fed-batch fermentation was developed. Effects of pH on the production of biomass and ammonium lactate in batch culture were studied at first. When the pH of the medium was maintained at 6.5, the highest biomass concentration and ammonium lactate production was obtained. The ammonium lactate production was remarkably improved by continuous feeding of glucose, when its residual concentration was between 5 and 10 g l−1. Controlling the pH at 6.5 during the fed-batch fermentation under a constant feeding rate of 25 ml h−1, the final concentration of 161.2 g ammonium lactate per liter was obtained, and the maximal cell dry weight and average ammonium lactate productivity obtained were 2.15 g DW l−1 and 2.02 g l−1  h−1, respectively.
Keywords: Ammonium lactate; Fed-batch; Fermentation; Glucose feeding; Lactobacillus; pH control;

Inverse flux analysis (IFA) was utilized to investigate the effect of gene-knockout on the metabolic flux distribution of Escherichia coli. In particular, it was shown from IFA that if pyk was knocked-out, then the flux through ppc and TCA cycle pathway increased, while the acetate production flux reduced. To verify this IFA result, we cultivated pykF E. coli and compared the fermentation data of E. coli K12. The enzyme activities of G6PDH, 6PGDH, Pgi, Pfk, Pyk, Mez, MDH, Ppc, and Pyk were also measured. Those data well coincided with the IFA result. The effects of other gene-knockout such as pgi, zwf, etc., were also investigated. From the present research, it was shown that the theoretical approach using computer shows some powers which needs careful attention.
Keywords: Metabolic flux distribution; Inverse flux analysis; Gene-knockout; Escherichia coli; Enzyme activity;

Simple prediction of oxygen penetration depth in biofilms for wastewater treatment by Kazuaki Hibiya; Jun Nagai; Satoshi Tsuneda; Akira Hirata (61-68).
The distribution of oxygen in a biofilm is very important in oxidation processes such as simultaneous carbon oxidization and nitrification using a fluidized bed biofilm reactor. In this study, attention was paid to biofilm thickness and biofilm density which significantly affect the oxygen distribution in biofilms. Using an oxygen microelectrode that we fabricated, the oxygen distribution in biofilms of different thicknesses was measured, and subsequently oxygen penetration depth and ratio were determined. As a result, oxygen penetration ratio decreased gradually with increasing biofilm thickness. Moreover, the kinetic parameters of a Monod-type reaction and the effective diffusion coefficient were computed based on the oxygen distribution in the biofilms. Using the obtained biofilm dry density, kinetic parameters and effective diffusion coefficient, the oxygen distribution in biofilms was successfully fitted to the results of a microelectrode analysis. The oxygen distribution was simulated by the finite difference method using the kinetic parameters and effective diffusion coefficient. Therefore, oxygen penetration ratio can be predicted at various biofilm thicknesses and oxygen concentrations in a bulk solution.
Keywords: Biofilm density; Biofilm thickness; Fluidized bed reactor; Microelectrode; Nitrification; Organic carbon oxidation; Oxygen penetration ratio;

A marine green alga, Platymonas subcordiformis, was demonstrated to photobiologically evolve hydrogen (H2) after the first stage of photosynthesis, when subjected to a two-phase incubation protocol in a second stage of H2 production: anaerobic incubation in the dark followed by the exposure to light illumination. The anaerobic incubation induced hydrogenase activity to catalyse H2 evolution in the following phase of light illumination. H2 evolution strongly depended upon the duration of anaerobic incubation, deprivation of sulphur (S) from the medium and the medium pH. An optimal anaerobic incubation period of 32 h gave the maximum H2 evolution in the second phase in the absence of sulphur. Evolution of H2 was greatly enhanced by 13 times when S was deprived from the medium. This result suggests that S plays a critical role in the mediation of H2 evolution from P. subcordiformis. A 14-fold increase in H2 production was obtained when the medium pH increased from 5 to 8; with a sharp decline at pH above eight. H2 evolution was enhanced by 30–50% when supplementing the optimal concentrations of 25 mM acetate and 37.5 mM glucose.
Keywords: Photo-biological; Green algae; Platymonas subcordiformis; Sulphur-deprivation; Hydrogen; Hydrogenase;

An anaerobic digestion technique was applied to azo-reactive dye aqueous solutions and cotton textile wastewater aiming at the color elimination. A batch-mode water-jacketed anaerobic reactor and acclimatized acetate-consuming bacteria initially derived from the anaerobic digester sludge of a municipal wastewater treatment plant were used for this study. Acetic acid solution and a pH-controller were utilized to maintain the pH at the desired level (6.6–7.2) while the temperature was kept constant at 37 °C using an external water-bath. Acetic acid also served as an external substrate (electron-donor) supply for the bacteria metabolism. Complete decolorization of all dye solutions was succeeded in 4–5 days of experimental run. The biodegradation ability of cotton textile wastewater was also examined without the addition of external substrate supply (acetic acid) resulting to poor decolorization results. However, anaerobic digestion of the same wastewater using the acetate-consuming bacteria and acetic acid as an external substrate supply lead to the complete decolorization of the wastewater in 4 days of experiment.
Keywords: Acetobacter; Acetic acid; Azo-reactive dyes; Anaerobic processes; Decolorization; Wastewater treatment;

Unsteady-state kinetics of lipolytic hydrolysis of palm oil in a stirred bioreactor by Sulaiman Al-Zuhair; K.B. Ramachandran; Masitah Hasan (81-86).
An unsteady-state kinetic model has been proposed for the hydrolysis of palm oil by lipase in a stirred batch bioreactor. The hydrolysis model was derived from a reaction mechanism and simplified for conditions of low enzyme concentration. From it, the regions where the quasi-steady-state assumption for the intermediates is applicable is identified and verified experimentally. The experimental results showed that the hydrolysis of palm oil by lipase in a well-stirred reactor has a short induction time for the penetration of enzyme at the interface and for the formation of the enzyme–substrate complex. This indicates that the quasi-steady-state assumption for the intermediates is applicable under the operating conditions used in this study. It is also shown that increasing the agitation speed results in reduced induction time.
Keywords: Lipase; Palm oil; Enzyme; Bioreactor; Agitation; Kinetic parameters;

On-line monitoring is a valuable tool for designing operation strategies. In this work, oxygen uptake rate (OUR) was measured on-line in infected insect cell cultures. OUR increased up to 80% when cultures were infected and glucose was present. In the absence of glucose, OUR did not increase and low yields (9.7×10−6  U/cell) of recombinant rotavirus VP8 were obtained. Restoration of the initial glucose concentration at the time of infection increased 10 times the VP8 yield. Glutamine feeding further increased VP8 yields. Glucose or glutamine depletion after infection caused a sudden decrease in OUR. Such a decrease was 19 times faster when glutamine was depleted than when glucose was depleted, indicating that OUR can be utilized to assess which and when either of these two nutrients is exhausted. Feeding strategies were implemented based on OUR measurements. This work shows that OUR can be utilized to take corrective actions for reestablishing recombinant protein yields. Selective glucose and glutamine feeding resulted in recombinant protein yields similar to those obtained by complete medium exchange upon infection, but at reduced expenses and simplified operation.
Keywords: Oxygen uptake rate; Insect cell; Baculovirus; Fed-batch culture; Bioprocess monitoring; Glucose;