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Biochemical Engineering Journal (v.55, #1)

BEJ Keywords (pp. ii).

Editorial Board (pp. co2).

Time dependent formation of gold nanoparticles in yeast cells: A comparative study by Kamalika Sen; P. Sinha; Susanta Lahiri (pp. 1-6).

► This paper for the first time addresses the migration kinetics of nanoparticles inside a cell. ► Gold nanoparticles gradually move inward as a function of time from cell wall to cytoplasm to nucleus. ► The gamma energy of¹⁹⁸Au is solely responsible factor in the reduction of Au and formation of gold nanoparticles. ► TEM image of budding yeast shows that gold nanoparticles are not transferred to the new generation yeast cells.Two different strains of yeast, Saccharomyces cerevisiae, AP22 and CCFY-100 were studied for bioaccumulation of gold in the form of H¹⁹⁸AuCl₄. Thin sectioning and subsequent study by transmission electron microscopy (TEM) reveals that Au³⁺ was in situ reduced to Au(0) and nano sized gold particles were formed inside the cell. Very low dose γ-energy being responsible for reduction of cationic gold in the polymeric cytoplasm matrix. The formation and entry of gold nanoparticles in the yeast cells were studied as a function of time at certain intervals starting from 15min to 72h. The gold nanoparticles gradually moved inward as a function of time from cell wall to cytoplasm to nucleus and finally accumulated in the nucleolus of the cell. TEM image of budding yeast shows that gold nanoparticles are not transferred to the new generation yeast cells.

Keywords: Saccharomyces cerevisiae; TEM; Gold nano particles; H; ¹⁹⁸; AuCl; ₄; Thin sectioning; Uptake kinetics

Developing a physiological model for type II diabetes mellitus by O. Vahidi; K.E. Kwok; R.B. Gopaluni; L. Sun (pp. 7-16).

► A detailed compartmental physiological model was developed to represent a patient with type II diabetes mellitus. ► The model parameters were estimated from clinical patient measurements. ► Physiological abnormalities associated with type II diabetes mellitus were simulated. ► The simulated results were found to be clinically consistent with physiological responses of type II diabetic patients.High blood glucose levels in type II diabetic patients often result from the malfunction of different organs such as the pancreas, the liver, muscles, and adipose tissues. Accurate diagnosis of abnormalities associated with type II diabetic patients is essential for administering an effective treatment. In this paper, a model for type II diabetes has been developed based on a detailed compartmental physiological model proposed for a healthy human body. Using available clinical data from type II diabetic patients, the model parameters have been estimated by means of an optimization method for diabetic patients. The estimated model may be used to understand the behavior of different organs in type II diabetic patients. It has been shown that the proposed model can predict the relative deficiency in the pancreatic insulin production, the impaired hepatic regulatory effect on glucose concentrations, and the low peripheral glucose uptake in type II diabetic patients.

Keywords: Type II diabetes mellitus; Modeling; Compartmental model; Parameter estimation; Dynamic simulation; Nonlinear optimization

Characteristics of hydrogen production of an Enterobacter aerogenes mutant generated by a new atmospheric and room temperature plasma (ARTP) by Yuan Lu; Liyan Wang; Kun Ma; Guo Li; Chong Zhang; Hongxin Zhao; Qiheng Lai; He-Ping Li; Xin-Hui Xing (pp. 17-22).

► ARTP could be a very useful tool for the mutation breeding. ► A hydrogen producing strain of E. aerogenes as a model was mutated by ARTP. ► The hydrogen yield of a positive mutant was increased by 26.4%. ► Cellular metabolism of the mutant was changed greatly. ► The hydrogen production stability of the mutant was kept after 25 subcultures.A novel atmospheric and room temperature plasma (ARTP) which used helium as the working gas was employed to generate mutants of Enterobacter aerogenes for improving the hydrogen production. For the mutation, 50μl of the E. aerogenes culture (OD₆₀₀=2.0) was dipped onto a sterilized stainless steel plate (5mm in diameter). The plate was then treated for 3min by ARTP at the gas flow rate of 15.0slpm (standard liters per minute) and 100W of radio-frequency power input. The positive mutation rate defined by the ratio of the mutants with the increase of hydrogen productivity to the total mutants reached about 10%, and a mutant with improved cell growth and hydrogen productivity was then selected. The total hydrogen yield per mole glucose was increased by 26.4%, mainly dependent on the increase of hydrogen production by the NADH pathway. Two important parameters of the ATP yield and oxidation state balance indicated that the cellular metabolism was changed in the mutant. The genetic stability of the mutant for hydrogen production was maintained after more than 25 subcultures.

Keywords: Anaerobic fermentation; Enterobacter aerogenes; Hydrogen; Metabolic flux analysis; Mutation breeding; Atmospheric-pressure plasma

Extracted fat from lamb meat by supercritical CO₂ as feedstock for biodiesel production by Hanifa Taher; Sulaiman Al-Zuhair; Ali AlMarzouqui; Isameldin Hashim (pp. 23-31).

► Simultaneous production biodiesel and healthy low-fat lean lamb was studied. ► Fat was extraction from lamb meat by supercritical CO₂. ► The extracted fat was used as a cheap raw material for biodiesel production. ► Biodiesel was produced in a bioreactor packed with immobilized lipase in supercritical CO₂ media.The feasibility of enzymatic production of biodiesel from waste animal fats using supercritical fluid technology for the extraction and reaction has been investigated. The operating conditions that resulted in the optimum extraction and biodiesel production yields were identified. The effects of extraction temperature, pressure and supercritical CO₂ (SC-CO₂) flow rate were investigated in the ranges of 35–55°C, 300–500bar and of 3–5mlmin⁻¹, respectively, and optimized using response surface methodology (RSM). In addition, the effects of reaction temperature, methanol:fat molar ratio and enzyme loading on biodiesel production yield were investigated in the ranges of 35–60°C, 3:1–6:1 and 10–50%, respectively. The optimum conditions for supercritical fluid extraction (SFE) were determined to be 45°C, 500bar and 3mlmin⁻¹; at which, 87.4% of total fat content was extracted from freeze dried meat. The statistical analysis however, showed that pressure has negligible effect on the extraction yield, which is extremely important, as it allows saving energy by using lower pressure. On the other hand, biodiesel yield of 40% was obtained using extracted fat as feedstock in SC-CO₂ medium at 50°C, 200bar, 4:1 methanol:fat molar ratio and 30% loading of lipase enzyme, after 24h of reaction. The experimental results were used to fit a suitable reaction kinetic model using non-linear regression analysis to estimate the model parameters.

Keywords: Lamb meat fat; Supercritical carbon dioxide (SC-CO; ₂; ); Biodiesel; Reaction kinetic model; Novozym 435

Development of a displacer-immobilized ligand docking scheme for displacer screening for protein displacement chromatography by Guofeng Zhao; Shu Bai; Yan Sun (pp. 32-42).

► A displacer-immobilized ligand docking scheme was developed for the prediction of displacer efficacy in protein displacement chromatography. ► The scheme was used to screen displacers for hydrophobic charge induction chromatography. ► Column displacement experiments showed that the identified displacers are efficacious. ► Combination of the docking scheme with existing techniques would greatly facilitate the discovery of high-affinity displacers for protein purification.Displacement chromatography is a powerful technique for protein purification, but the availability of high-efficacy displacers has greatly limited its applications. In this work, a displacer-immobilized ligand docking scheme was developed for the prediction of displacer efficacy and displacer screening for displacement chromatography of proteins. The structure of immobilized ligand was established by coupling a certain number of ligands to the 3D structure of agarose. A number of known cation, anion and hydrophobic displacers were docked to their respective immobilized ligands to verify the effectiveness of the scheme, and the Spearman ranking correlation coefficients of all cases were over 0.5. The scheme was then used to screen displacers for hydrophobic charge induction chromatography from over 1800 commercially available compounds. Column displacement experiments of several representative compounds showed that the identified displacers were efficacious in the displacement of single component and binary mixtures. It is expected that the combination of the docking scheme with the existing techniques for displacer discovery/design would greatly facilitate the discovery of high-affinity displacers for protein purification.

Keywords: Protein; Chromatography; Purification; Molecular docking; Displacer; Hydrophobic charge induction chromatography

Technology of protein separation from whey wastewater by two-stage foam separation by Changsuo Jiang; Zhaoliang Wu; Rui Li; Qi Liu (pp. 43-48).

► The purpose of this paper is disposing whey wastewater by foam separation technology. ► The column of the inclined foam phase was used. ► Effects of temperature, inclined angle, pH and airflow rate were studied. ► A technology of two-stage foam separation was proposed.For recovering the proteins from the whey wastewater, a two-stage foam separation technology was developed with the column of inclined foam phase. Effects of inclined angle, initial pH, volumetric air flow rate and temperature on the performances of the wastewater were investigated. The results showed that when the conditions of the first stage were protein concentration 4.0g/L, initial pH 7, inclined angle 50°, volumetric air flow rate 100mL/min and temperature 50°C, enrichment ratio of the proteins was 8.5 and the residual solution went into the second stage as the feeding stream. When the conditions of the second stage were temperature 20°C and volumetric air flow rate 200mL/min, recovery percentage of the proteins reached 70%, the foamate was added to the wastewater as the feeding stream of the first stage. Total recovery percentage of the proteins reached to 80% by the two-stage foam separation.

Keywords: Bioseparations; Adsorption; Foam separation; Waste treatment; Proteins; Inclined column

Experimental and theoretical analysis of poly(β-hydroxybutyrate) formation and consumption in Ralstonia eutropha by André Franz; Hyun-Seob Song; Doraiswami Ramkrishna; Achim Kienle (pp. 49-58).

► We model PHB degradation as well as consumption in R. eutropha. ► We extended the HCM approach to take internal metabolites explicitly into account. ► Possibility of multiple steady states in continuous bio reactor were investigated. ► Influence of PHB consumption is analyzed and found to be crucial at low dilution rate.In this paper a mathematical model is presented to describe poly(β-hydroxybutyrate) (PHB) formation and consumption in Ralstonia eutropha. The model is based on the hybrid cybernetic modeling approach, which was introduced by Kim et al. and which allows a systematic derivation of the model equations from elementary mode analysis. An extension of this approach is presented to allow for non quasi-stationary metabolites, i.e. PHB. The model is shown to be in good agreement with experimental data for PHB formation and consumption. The model is used afterwards to discuss the occurrence of multiple steady states in a continuous bio reactor. It is shown that the multiplicity region predicted by the model is rather small and it is argued that multiple steady states are therefore unlikely to occur in practice for this specific system.Due to various desirable features such as accounting for cellular regulation at network level and dynamics of intracellular metabolites with a moderate complexity, it is believed that the constructed model is most suitable for control, optimization and monitoring of industrial PHB production processes.

Keywords: Ralstonia eutropha; Cybernetic modeling; Bifurcation analysis; Biopolymers; Metabolic regulation; Elementary mode

Enhanced recombinant human erythropoietin production by Pichia pastoris in methanol fed-batch/sorbitol batch fermentation through pH optimization by Elif Şükran Soyaslan; Pınar Çalık (pp. 59-65).

► In this study, pH operation strategy was developed for rHuEPO production by P. pastoris. ► Four phase operation was conducted to obtain higher product amount. ► Sorbitol batch/methanol fed-batch feeding was applied in production step. ► At pH≥5.0 same cell growth profiles were obtained in the first two phases of the operation. ► In the production phase, the highest cell and rHuEPO concentration was obtained at pH=4.5.The effects of pH on recombinant human erythropoietin (rHuEPO) production by Pichia pastoris was investigated at pH=4.0, 4.5, 5.0, 5.5 and 6.0, where sorbitol and methanol were supplied to the bioreactor in batch and fed-batch mode, respectively, in the recombinant protein production phase (PP). For the first three phases, glycerol batch (GB), glycerol fed-batch (GFB) and methanol transition (MT), the growth profiles were almost the same for pH≥5.0; however, at pH<5.0 the growth rate was lower. The highest cell concentration was obtained as 81.4gL⁻¹ where GB and GFB were operated at pH=5.0 and MT and PP were operated at pH=4.5. Sorbitol added at the beginning of PP, was totally consumed within 15h, at pH≤5.0; however, at pH>5.0 sorbitol consumption rate decreased. The highest rHuEPO concentration was achieved at pH=4.5 as 0.16gL⁻¹, where the overall cell and rHuEPO yield on substrate, were 0.5gg⁻¹ and 1.5mgg⁻¹. pH did not have a significant effect on extracellular protease activity. The highest specific AOX activity (110Ug⁻¹ CDW) was attained at pH=4.5 parallel to the highest rHuEPO production. At pH=4.5, higher oxygen demand of the cells resulted in higher oxygen K_La, 0.11s⁻¹, values.

Keywords: Pichia pastoris; Erythropoietin; pH; Sorbitol; Methanol; AOX

Enzymatic packed-bed reactor integrated with glycerol-separating system for solvent-free production of biodiesel fuel by Shinji Hama; Sriappareddy Tamalampudi; Ayumi Yoshida; Naoki Tamadani; Nobuyuki Kuratani; Hideo Noda; Hideki Fukuda; Akihiko Kondo (pp. 66-71).

► A practical bioreactor design for continuous biodiesel production. ► The developed model incorporates a packed-bed reactor and glycerol-separating system. ► Flow rate is an important factor for glycerol removal from the column. ► Long-term methanolysis can be achieved, obtaining a product meeting the standard. ► For both production and purification, no wastewater treatment is necessary.From the viewpoint of process engineering in enzymatic biodiesel production, this paper provides a practical basis for development of the packed-bed reactor (PBR) on a bench scale. A key component of this work is the separation of glycerol byproduct in continuous operation. To facilitate measuring and separating the byproduct, we constructed the PBR integrated with a glycerol-separating system. Methyl ester content in the effluent increased with increasing pass number of the reaction mixture, whereas the efficiency of glycerol removal was affected significantly by the flow rate in PBR. Further optimization revealed that the PBR could be operated over a long period realizing both a high methyl ester content and an efficient glycerol removal, obtaining a product that meets the biodiesel standard for residual glycerides. To remove soluble glycerol present in the biodiesel, adsorptive purification using ion-exchange resin was applied to the PBR system. The initial glycerol content of approximately 0.053wt% was reduced to less than 0.003wt%, which was maintained for more than 140 bed volumes and repeated after regeneration of the resin using methanol. Therefore, this developed model incorporating the enzymatic PBR and glycerol-separating system is promising for practical biodiesel production, in which no wastewater treatment is necessary.

Keywords: Lipase; Biodiesel fuel; Methanolysis; Glycerol; Bioreactors; Scale-up

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Biochemical Engineering Journal (v.55, #1)