Biochemical Engineering Journal (v.35, #1)

The three-dimensional (3D) architecture of bone marrow (BM) supports the renewal, maintenance, proliferation and differentiation of haematopoietic stem cells (HSCs). Efforts have been made towards the reconstruction of an ex vivo HSC culture system, which could have several potential clinical applications, especially in expansion of haematopoietic stem and progenitor cells for use in bone marrow transplantation (BMT). In the present study, a numerical model has been developed to investigate the fluid flow, shear stress, and nutrient distribution within a purpose-built scaffold placed inside a rotating wall perfused bioreactor (RWPB). Furthermore, a multi-lineage cellular growth model of haematopoietic cells is incorporated to mimic the complexity of the BM. The simulation results demonstrate that: (1) the fluid dynamics inside the scaffold are qualitatively similar to those in the in vivo BM microenvironment, and (2) the inlet conditions (inoculum density), transport properties (scaffold) and reaction parameters (consumption rates) are sufficient to support the growth of haematopoietic cells required for BMT applications. Specifically, the multi-lineage cellular growth model demonstrates that the system would be able to produce the minimum number of colony forming unit-granulocyte macrophage (CFU-GM) required for transplantation in an average patient. The numerical model presented here enables the optimisation of scaffold design parameters, fluid dynamics and mass transfer environment necessary for developing a suitable ex vivo haematopoietic system.
Keywords: Computational fluid dynamics; Haematopoietic stem cells; Modelling; Bioreactors; Growth kinetics; Mass transfer;

Bioisomerization kinetics of γ-HCH and biokinetics of Pseudomonas aeruginosa degrading technical HCH by Bharat Lodha; Praveena Bhat; M. Suresh Kumar; Atul N. Vaidya; Sandeep Mudliar; Deepak J. Killedar; Tapan Chakrabarti (12-19).
Bioconversion kinetics of γ-HCH and biological growth kinetics of Pseudomonas aeruginosa degrading technical HCH was investigated in flask experiments under aerobic condition. Degradation studies for various isomers of technical HCH were also performed. The biodegradation of the most persistent β-HCH and δ-HCH isomers was also observed. At lower technical HCH concentrations (1–10 mg/l), above 99% of the degradation was observed whereas at higher concentration (20–50 mg/l), the degradation efficiency was dropped. This may be due to inhibitory effect of technical HCH at higher concentrations. The technical HCH inhibitory behavior and bacterial growth kinetics could be correlated well by simple Haldane's growth kinetic model. The value of decay and yield coefficient was also determined.Further experimental results also revealed that degradation of the γ-HCH isomer is faster (after incubation of 5 days) in the presence of other isomers of technical HCH, whereas γ-HCH alone does not degrade even after a long period of incubation (14 days), but isomerizes to α-HCH isomer by the P. aeruginosa. The rate constant for the bioisomerization was found to be 0.0474 day−1. The above estimated parameters (maximum specific growth rate, half saturation coefficient, inhibition constant, decay coefficient, yield coefficient and bioisomerization rate) are invariably required for the design and simulation of batch and continuous bioreactors treating HCH containing wastewaters.
Keywords: Biokinetics; Bioisomerization; Biodegradation; Treatment; Isomers; Hexachlorocyclohexane (HCH); Haldane's model; Monod's model;

Modelling of bioreactors employed in solid-state fermentation processes can play a crucial role in the analysis, design and development of bioprocesses based on the phenomena; which encompass a variety of applications ranging from the production of enzymes to the treatment of agro-industrial residues. In the present work, a mathematical model has been developed for a packed bed solid-state fermentation Bioreactor employing the N-tanks in series approach. The model analyses the production of protease enzyme by Aspergillus niger and is validated against an experimental study available in the literature which describes its growth during solid-state fermentation.The primary importance of employing the method is to demonstrate its successful application to the solution of model equations to situations where axial gradients dominate. As a consequence, the approach not only helps in avoiding the use of complex computational methods like orthogonal collocation but also contributes in providing insights to the design of bioreactors.
Keywords: Enzyme production; Modelling; Packed bed bioreactors; Solid-state fermentation; N-tanks in series;

A mathematical model, based on structured growth kinetics, is presented to simulate the profile of assimilated radioactive sulphur by a biofilm, after the release of calculated amounts of 35SO4 2− in the biofilm environment. The radiolabelled sulphur (35S) stands as a biomass growth indicator, thus allowing the estimation of the cellular anabolic activity (growth) profile inside the biofilm. The model predicts that the maximum rate of biomass synthesis occurs at the external layer of the biofilm. A good correlation between the model predictions and previously published experimental data is reported, indicating the applicability of the structured model to describe microbial growth in immobilized cell systems.
Keywords: Biofilms; Modelling; Structured model; Radiolabelling; Immobilised cells; Attached biomass; Microbial growth; Air lift bioreactors;

Lysozyme was immobilized on the extrudate-shaped NaY zeolite and was used for the disruption of Micrococcus lysodeikticus cells in a well-agitated contactor. The influences of specific operating parameters on the density and activity of immobilized lysozyme were investigated. The result for the maximum activity of immobilized lysozyme was observed at pH 8 and the immobilized specific activity decreased significantly with increasing ionic strength of buffer. Simultaneously, the specific activity of immobilized lysozyme decreased with the amount of enzyme added. Based on these results, the influences of operating parameters on the efficiency of cell disruption by using lysozyme-zeolite complex were further examined. The efficiency of cell disruption was evaluated in terms of the amount of released protein and the percentage of disrupted cells, and compared with other supports and mechanical disruption methods. The results showed that the amount of released proteins and the percentage of disrupted cells in the lysates by using lysozyme-zeolite complex were much higher than those values reported in the literature by using other supports and mechanical disruption systems. After 12 successive cycles, the released protein from disrupted cells decreased and approached to the stationary value of 33.3%, as compared with the first cycle. In order to determine the reusability of lysozyme-zeolite complex, the adsorption–desorption cycle was investigated. The results showed that the density of immobilized lysozyme did not change significantly during the repeated adsorption–desorption operations.
Keywords: Extrudate-shaped NaY zeolite; Lysozyme immobilization; Micrococcus lysodeikticus; Cell disruption; Protein release;

Optimization of the Bacillus thuringiensis var. kurstaki HD-1 δ-endotoxins production by using experimental mixture design and artificial neural networks by Guilherme A. Moreira; Gabriela A. Micheloud; Alejandro J. Beccaria; Héctor C. Goicoechea (48-55).
An experimental mixture design coupled with data analysis by means of both response surface methodology (RSM) and artificial neural networks (ANNs) followed by multiple response optimization through a desirability function, was applied to the production of δ-endotoxins from Bacillus thuringiensis var. kurstaki. The composition of a culture medium was defined by testing three regional effluents: milky effluent, beer wastewater and sugar cane molasses. Both RSM and ANNs accomplished the goal pursued in this work, by predicting the optimal mixture of the effluents. ANNs provided more reliable results due to the complexity of the models to be fitted. The optimal selected blend was: 74%, 26% and 0%, respectively for each the above-mentioned effluents.
Keywords: Bacillus thuringiensis var. kurstaki; Modelling; Mixture design; Artificial neural networks (ANNs); Multiple response optimization;

Characterization of BSA adsorption on mixed mode adsorbent by Yu-Kaung Chang; Shin-Ying Chou; Jyun-Liang Liu; Jung-Chin Tasi (56-65).
STREAMLINE Direct HST (GE Healthcare Biosciences, 2004) is a newly designed mixed mode adsorbent, which contains small stainless steel particles and is based on a highly crossed-linked agarose with multi-modal functional groups. In this work, BSA (bovine serum albumin) was used as a model protein and the experiments were carried out in a well-agitated contactor under a variety of operating conditions, such as adsorption pH, conductivity, and hydrophobicity of the process liquids. The results showed that the Langmuir model seemed to successfully simulate the adsorption isotherm curves and the thermodynamic parameters (q max and K d) were little affected by the conductivity and hydrophobicity of the adsorption buffer. On the contrary, the adsorption pH had significant effects on these parameters due to the negatively charged repulsion between HST adsorbent and BSA molecule. The adsorbent for the adsorption of BSA is therefore based on the pH-dependent behavior of a dual-mode ionizable ligand. Moreover, the adsorbed BSA was eluted completely either by 10 mM sodium carbonate buffer (pH 12) or by 1 M NaCl (pH 7) and found to be more easily eluted by pH 12 buffer.
Keywords: STREAMLINE Direct HST adsorbent; Bovine serum albumin; Adsorption; Elution; Well-agitated contactor;

Enzymatic polymerization of o-phenylendiamine with cytochrome c activated by a calixarene derivative in organic media by Tatsuya Oshima; Mako Sato; Yuka Shikaze; Keisuke Ohto; Katsutoshi Inoue; Yoshinari Baba (66-70).
Cytochrome c can be dissolved in organic media through complexation with a macrocyclic compound calix[6]arene carboxylic acid derivative. Enzymatic oxidation of o-phenylenediamine with the cytochrome c complex as a catalyst was demonstrated in organic media. The cytochrome c complex displays peroxidase activity in the presence of hydrogen peroxide. Enzymatic polymerization of o-phenylenediamine was examined under optimal conditions. A polymer product was effectively obtained in hexane or chloroform; however, a monomeric compound was also in the product. In this paper, a novel solubilization method utilizing enzymes and their uses as catalysts in organic media are described.
Keywords: Enzymatic polymerization; Cytochrome c; Calixarene; Enzyme reaction; Protein extraction; Organic media;

Kinetic study of glycerolysis of palm olein for monoacylglycerol production by immobilized lipase by Benjamas Cheirsilp; Wiphum Kaewthong; Aran H-Kittikun (71-80).
Kinetic study of monoacylglycerol production by glycerolysis of palm olein using immobilized lipase has been investigated. A mathematical model took into account mechanism of glycerolysis reaction using palm olein as substrate was developed. The kinetic parameters were estimated by fitting experimental data of glycerolysis reaction of palm olein by immobilized lipase PS on Accurel EP-100. There was a good agreement between experimental results and those predicted by proposed model equations under various enzyme, water, glycerol and palm olein concentrations. From proposed model equations, the synergic effect of glycerol and palm olein concentrations was simulated. The simulation results show that the behavior of high substrate concentrations obtained high initial production rates but low yields of monoacylglycerol. Thus, at high concentrations of palm olein and glycerol of 7.16 and 19.14 mM, respectively, high initial production rate of monoacylglycerol of 0.338 mM h−1 and acceptable yield of 82% were obtained. On the other hand, the highest yield of monoacylglycerol of 100% was obtained at comparatively low palm olein concentration of 2.39 mM and excess glycerol concentration of 19.14 mM.
Keywords: Glycerolysis; Immobilized lipase; Kinetic; Modeling; Monoacylglycerol; Palm olein;

Monitoring of biofilm in cooling water system by measuring lactic acid consumption rate by Yasunori Tanji; Takaaki Nishihara; Kazuhiko Miyanaga (81-86).
Biofilm formation in cooling water systems causes many problems such as increase of the frictional resistance in tubes and decrease in the heat exchange capacity of heat exchanger. Monitoring of biofilm formation in the system is necessary to avoid biofilm related problems and optimize biocide addition. However, detection of biofilm formation in the whole system is difficult. To solve this problem, a novel and simple method for monitoring biofilm was developed in this study. Biofilm consists of living microorganisms, and nonliving organic and inorganic substances. Microorganisms in the biofilm consume substrate in the circulating cooling water. Therefore substrate consumption rate reflects biofilm activity in the system. Experiments were scaled up stepwisely; i.e. beaker-scale, bench-scale, and operating plant experiments. The beaker-scale experiment was conducted to select suitable substrate. Among the eight substrates examined, lactic acid was the most bio-consumable one by the biofilm formed on carbon steel coupon. The lactic acid consumption rate (R lac) was estimated at 273 mg/(m2  h). The R lac value was compared before and after the hydrogen peroxide wash of the bench-scale plant. The R lac before the wash was 36.3 mg/(m2  h). On the other hand, the R lac after the wash was negligible. An experimental study was next conducted in the operating cooling water plant. A rapid decrease of lactic acid concentration in the circulating water of a relatively unclean system was shown. On the other hand, R lac in a clean system was low. This simple method provided us information on biologically active biofilm formation in the system.
Keywords: Biofilm; Monitoring; Cooling water; Fouling;

Stability of a nitrifying activated sludge reactor by J.L. Campos; J.M. Garrido; A. Mosquera-Corral; R. Méndez (87-92).
A nitrifying activated sludge unit operated in stable conditions was subjected to different instability actuations as overloads due to a sudden increase of the inlet ammonia concentration or a quick change of the inflow rate, pH shocks and limitations of dissolved oxygen (DO) concentration. The response of the unit to these different actuations was evaluated. During concentration overloads there were transitory nitrite accumulations but not during hydraulic overloads. However, efficiencies obtained for both kinds of overloads with respect to ammonia oxidation were very similar. The normal efficiency of the system was always restored and biomass was not washed out in spite of the low values of hydraulic retention time (HRT) used. The pH shocks of 9 and 10 had no appreciable effects, but the pH shock of 11 caused an irreversible loss of efficiency of 60%. An increase of the total organic carbon (TOC) in the effluent after this shock showed that biomass underwent a partial lysis. The specific activity of ammonia-oxidizers decreased from 0.6 to 0.3 g NH4 +-N/(g VSS day) but the nitrite-oxidizing specific activity remained practically constant. Finally, the unit was operated at different DO levels, ammonia being fully oxidized to nitrate at DO levels higher than 1 mg O2/L. Different proportions of ammonia and nitrite appeared when the reactor was operated at DO values of 0.4 and 0.6 mg O2/L.
Keywords: Aerobic processes; Chemoautotrophes; Nitrification; Shocks; Substrate inhibition; Wastewater treatment;

Equal weights of cuttlebone chitosan and activated clay were mixed and prepared as the wet (without freeze-drying) and dried (with freeze-drying) beads. The resulting composite beads were then cross-linked with glutaraldehyde as the supports for β-glucosidase immobilization. The immobilization conditions including enzyme loading, immobilization time, cross-linking time, and the amount of glutaraldehye were optimized. The properties of the enzyme immobilized on wet and dried composites were compared. It was shown that the storage stability of immobilized enzyme was higher than that of free enzyme. The activity of immobilized enzyme on dried composite was higher those on wet chitosan beads and wet composite after being repeatedly used for 50 times. The pH and thermal stabilities of free and immobilized enzymes were also investigated.
Keywords: β-Glucosidase; Immobilization; Chitosan–clay composite; Stability;