Biochemical Engineering Journal (v.25, #2)

BEJ Keywords (II).

IFC (CO2).

The experimental data showed that, in glutamate fermentation with strain Corynebacterium glutamicum S9114, glutamate produced in different ways with different patterns of OUR, CER and lactate accumulation at different DO levels. A metabolic network model combined with the linear programming optimization was thus developed to on-line predict glutamate production under different DO levels, with only OUR and CER be on-line measured. The results indicated the advantages of the metabolic network model over the traditional unstructured models, in terms of easy parameter identification, prediction accuracy, and the universal abilities. The proposed model potentially supplies an alternative way for on-line control and optimization of fermentation processes.
Keywords: Fermentation; Linear programming; Metabolic network; Modeling; On-line bioprocess monitoring;

Reliability of methods for the determination of specific substrate consumption rates in batch culture by Johann F. Görgens; Willem H. Van Zyl; Johannes H. Knoetze (109-112).
Four methods for the calculation of specific substrate consumption rates in batch cultivation were compared. Calculations based on a simple mathematical model resulted in the most reliable rate predictions. The application and accuracy of a novel, Logarithmic Method for rate estimations directly from experimental data were also demonstrated.
Keywords: Batch culture; Substrate consumption rate; Modelling;

Hydraulic modeling and axial dispersion analysis of UASB reactor by Y. Zeng; S.J. Mu; S.J. Lou; B. Tartakovsky; S.R. Guiot; P. Wu (113-123).
In this work, the axial distribution of a conservative tracer in the upflow anaerobic sludge bed (UASB) reactor was modelled by a two-compartment model describing sludge bed and liquid zones. The orthogonal collocation algorithm was used for numerical solution of the model. The model parameters were estimated using on-line fluorescence-based measurements of an inert tracer rhodamine. Rhodamine concentrations were simultaneously measured at the bottom, middle and top of the sludge bed, as well as in the reactor effluent. The model used either a constant or variable axial dispersion coefficient in the sludge bed zone. A significantly better agreement between model outputs and observed tracer distribution was obtained when the dispersion coefficient was proportional to a constant raised to the power of the reactor height. In addition, a near linear dependence of the dispersion coefficient on the liquid upflow velocity was found. These findings, along with high efficiency of the orthogonal collocation algorithm used for model solution, enable future development of a comprehensive model, which accounts for both hydraulics and biokinetics of the anaerobic digestion process.
Keywords: Upflow anaerobic sludge bed (UASB); Axial dispersion; Modeling; Bioreactor; Fluorometry; Dynamic simulation; Orthogonal collocation;

A packed-bed chromatographic model developed in this study includes adsorption isotherms considering steric mass action (SMA) as well as non-linearity in liquid/solid phases, adsorption kinetics and mass transport. For solving the nonlinear and nonequilibrium adsorption model described by a partial differential algebraic equation (PDAE) system, a fast and accurate numerical method (i.e., conservation element/solution element (CE/SE) method), is proposed. Sensitivity and elasticity of the model parameters (e.g., steric/shape factors, adsorption heat coefficient, effective protein charge, equilibrium constant, mass transfer coefficient, axial dispersion coefficient and bed voidage) are analyzed for a BSA–salt system in a low protein concentration range.Within a low concentration range of bovine serum albumin (BSA) where linear adsorption isotherms are shown, the adsorption heat coefficient, shape and steric factors have little effect on adsorption isotherms and the retention time. However, the effective protein charge which is related to pH and ionic strength affects the retention time significantly even if the change is slight. The mass transfer coefficient (i.e., adsorption rate coefficient) has an effect on the peak height of the elution curve at the same retention time. Sensitivity of the retention time to the bed voidage is relatively large, since the bed voidage changes the fluid velocity of the mobile phase.Comparing the proposed model with experimental elution curves obtained at different salt concentrations, it is proposed that the effective protein charge could depend upon the salt concentration (or ionic strength). The reason for this dependence may be a steric hindrance of protein binding sites combined with a salt shielding effect neutralizing the surface charges of the protein.
Keywords: Chromatography; Adsorption; Modeling and simulation; Protein ion-exchange; Ionic strength; Effective protein charge;

A temperature stimuli responsive polymer, β-CD-grafted-PNIPAAm(β-CD-g-PNIPAAm), was prepared by radical polymerization using cerium ammonium nitrate as the initiator. The use of β-CD-g-PNIPAAm as the stripper and cetyltrimethylammonium bromide (CTAB) as the capturer for protein refolding was tested using lysozyme as the model protein. The stripping of CTAB from the denatured lysozyme-CTAB complex was accomplished by the β-CD segment of β-CD-g-PNIPAAm. The interactions between lysozyme in different states with CTAB and β-CD-g-PNIPAAm were characterized by fluorescence emission spectroscopy. Based on these results, a temperature stimuli responsive “artificial chaperone” composed of CTAB and β-CD-g-PNIPAAm was proposed for protein refolding, in which stripper, β-CD-g-PNIPAAm, displayed dual functions in terms of stripping CTAB via β-CD segment and inhibiting the formation of protein aggregate. The latter was accomplished by the PNIPAAm segment. This leads to an improved refolding yield particularly at high temperatures, as compared to that obtained by using β-CD as the stripper.
Keywords: Protein; Refolding; Artificial chaperone; β-CD-g-PNIPAAm; Temperature stimuli responsive polymer; Lysozyme;

Optimization of Saccharomyces cerevisiae culture in alginate–chitosan–alginate microcapsule by Qi Wen-tao; Yu Wei-ting; Xie Yu-bing; Ma Xiaojun (151-157).
Microencapsulation is one of the promising methods for microorganism immobilization. The parameters of microencapsulation have impact on the growth and performance of microencapsulated microorganism. In this paper, the effects of microcapsules core state (solid or liquid), initial cell density (1.5 × 107, 3 × 106 and 3 × 105  cells/ml of microcapsules), microcapsule diameter (200, 500, 600 and 700 μm) and membrane formation times (0, 5, 15 and 30 min) on cell growth, including proliferating capacity, metabolic activity and product secretion of Saccharomyces cerevisiae, cultured in alginate–chitosan–alginate (ACA) microcapsule, were investigated. The results showed that there was no significant difference in cell growth of microencapsulated cells, whether the core of the microcapsules was solid or liquefied. Increase in inoculate cell density shortened the lag phase time of cell growth, while cell density obtained 25 times of the initial density of 3 × 106  cells/ml of microcapsule, which was the highest. Increase in microcapsules size had no significant impact on cell proliferation, metabolism and product secretion, but the leakage of the bacteria was the least when the microcapsule size was 600 μm in diameter. The increase in membrane formation time reduced the leakage of cells. It was demonstrated that the optimized parameters for microencapsulated S. cerevisiae culture were initial cell density of 3 × 106  cells/ml of microcapsule, microcapsule size of 600 μm in diameter, and membrane formation time of 15 min. This study provides useful information for intestinal delivery of therapeutic agents from genetically modified food-grade microorganisms, using microencapsulation.
Keywords: Microencapsulation; ACA microcapsule; Saccharomyces cerevisiae; Cell growth;

Production of lactic acid by continuous electrodialysis fermentation with a glucose concentration controller by Makoto Hirata; Min-tian Gao; Eiichi Toorisaka; Hirokazu Takanashi; Tadashi Hano (159-163).
In this study, the system of electrodialysis fermentation (EDF) was improved by a glucose concentration controller (GC controller) which could control glucose concentration stable and low in fermentation broth. Under the control of the GC controller, there were little inhibitory effects of the end product and substrate on bacteria so as to result in high activity of bacteria and, further, in high productivity of lactic acid. Moreover, the low glucose concentration in the fermentation broth brought about little glucose leakage to the recovery solution and hence high yield of lactic acid. Compared with the EDFs carried out in the previous studies, the continuous EDF with a glucose concentration controller (CEDF with GC controller) was higher in productivity, yield and conversion ratio of lactic acid, indicating that it was a system more efficient to produce lactic acid.
Keywords: Lactic acid, Electrodialysis fermentation, Glucose concentration controller, Level meter;

Chitosan was depolymerized either by HCl hydrolysis or enzymatic degradation with a commercial preparation Pectinex Ultra Spl. The chitooligosaccharides released by both methods were selectively precipitated in methanol solutions and characterized using MALDI-TOF mass spectrometry. Differences between the two methods were detected and concerned the degrees of polymerization of the fragments produced and their acetylation. The enzymatic method yielded shorter fragments with a higher proportion of fully deacetylated chitooligomers. Conversely, acid hydrolysis of the starting chitosan resulted in fragments with degrees of polymerization up to sixteen and more monoacetylated residues than with the enzymatic procedure.
Keywords: Chitooligosaccharides; Enzyme technology; MALDI-TOF-MS; Enzymatic hydrolysis; Acid hydrolysis; Viscosity;

Modeling the growth of Corynebacterium glutamicum under product inhibition in l-glutamic acid fermentation by Noor Salam Khan; Indra Mani Mishra; R.P. Singh; Basheshwer Prasad (173-178).
In the fermentation of l-glutamic acid by Corynebacterium glutamicum, the growth inhibition by the substrate (glucose) at higher concentrations, and by the product at almost all concentrations seem to occur. In order to identify the range of concentrations for substrate limitation/inhibition, the experiments were conducted separately with different initial glucose concentrations. Proof of growth inhibition by the product was established by analyzing the data obtained from the time course of batch fermentation. Based on the experimental observations, a product inhibition model has been developed by modifying the Monod's kinetic equation for cell growth. This model simulates the growth satisfactorily. The same model is also able to describe the experimental data for growth of C. glutamicum obtained from different investigators.
Keywords: l-Glutamic acid; Fermentation; Corynebacterium glutamicum; Monod's equation; Substrate limitation; Product inhibition; Modeling;

This paper presents the development and application of a knowledge-based software controller for the external electron-donor supply in the anaerobic denitrification stage. The basic principle of the control concept is based on the continuous measurement of the methane concentration in the off-gas. Since under anoxic conditions and incomplete NO x removal the denitrifying species out-compete efficiently the methanogens for the carbon source, the appearance of methane at a significant concentration (≥0.3%) in the gas phase indicates the complete denitrification and the increasing consumption of the C-source by the methanogens. Thus, a software controller that uses the methane concentration as variable was designed for process control and tested in different reactor types (CSTR, Fluidized bed). The necessary amount of the external electron-donor can be kept at the optimal level without any undesired accumulation of nitrite or nitrate.
Keywords: Wastewater treatment; Denitrification; Electron-donor supply; Software controller; Methane concentration;