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Biochemical Engineering Journal (v.40, #1)
Towards information-rich bioprocessing: Generation of spatio-temporal profiles through the use of design of experiments to determine optimal number and location of sensors—An example in thermal profiles by Mayasari Lim; Hua Ye; Emmanuel M. Drakakis; Xicai Yue; Anthony E.G. Cass; Nicki Panoskaltsis; Athanasios Mantalaris (pp. 1-7).
Stem cell and tissue bioprocessing for regenerative medicine require robust, reproducible, controllable, and optimal processes. In vivo, the body utilises real-time, on-line, in situ monitoring to deliver this level of control. Increasingly, monitoring is becoming more applicable and extremely important in tissue engineering and regenerative medicine due to the advances in monitoring techniques, including the use of biosensors. However, determination of optimal sensor location and number remains to be explored. Herein, we have presented the novel application of design of experiments methodology to address this issue. A culture chamber (equivalent to a 6-well plate) was fabricated and temperature sensors were employed in studying the temperature profile of the chamber under both static and perfused (0.01mL/min) conditions. Heating elements were used to provide a constant heat source in order to simulate the production of metabolites by the cells. The design of experiments determined the use of nine sensors at both factorial and axial locations. In comparison, two random designs utilising five and seven sensors confirmed that the design of experiments-generated data were information-rich and produced the spatial (and temporal) temperature profiles for the chamber. The implementation of this powerful technique would offer knowledge of the local concentration gradients of important culture parameters allowing for the real-time, local, ‘intelligent’ bioprocess control.
Keywords: Stem cells; Tissue engineering; Bioprocessing; Monitoring; Sensors; Design of experiments
Towards information-rich bioprocessing: Generation of spatio-temporal profiles through the use of design of experiments to determine optimal number and location of sensors—An example in thermal profiles by Mayasari Lim; Hua Ye; Emmanuel M. Drakakis; Xicai Yue; Anthony E.G. Cass; Nicki Panoskaltsis; Athanasios Mantalaris (pp. 1-7).
Stem cell and tissue bioprocessing for regenerative medicine require robust, reproducible, controllable, and optimal processes. In vivo, the body utilises real-time, on-line, in situ monitoring to deliver this level of control. Increasingly, monitoring is becoming more applicable and extremely important in tissue engineering and regenerative medicine due to the advances in monitoring techniques, including the use of biosensors. However, determination of optimal sensor location and number remains to be explored. Herein, we have presented the novel application of design of experiments methodology to address this issue. A culture chamber (equivalent to a 6-well plate) was fabricated and temperature sensors were employed in studying the temperature profile of the chamber under both static and perfused (0.01mL/min) conditions. Heating elements were used to provide a constant heat source in order to simulate the production of metabolites by the cells. The design of experiments determined the use of nine sensors at both factorial and axial locations. In comparison, two random designs utilising five and seven sensors confirmed that the design of experiments-generated data were information-rich and produced the spatial (and temporal) temperature profiles for the chamber. The implementation of this powerful technique would offer knowledge of the local concentration gradients of important culture parameters allowing for the real-time, local, ‘intelligent’ bioprocess control.
Keywords: Stem cells; Tissue engineering; Bioprocessing; Monitoring; Sensors; Design of experiments
Sorptive removal of Methylene blue from aqueous solution using palm kernel fibre: Effect of fibre dose by Augustine E. Ofomaja (pp. 8-18).
The use of palm kernel fibre, a readily available agricultural waste product for the sorption of Methylene blue from aqueous solution and the possible mechanism of sorption has been investigated at various fibre doses. The extent of dye removal and the rate of sorption were analyzed using two kinetic rate models (pseudo-first and pseudo-second-order kinetic models) and two diffusion models (intraparticle and external mass transfer models).Analysis of the kinetic data at different sorbent dose revealed that the pseudo-first order kinetics fitted to the kinetic data only in the first 5min of sorption and then deviated from the experimental data. The pseudo-second-order kinetic model was found to better fit the experimental data with high correlation coefficients at the various fibre dose used. The dye sorption was confirmed to follow the pseudo-second-order model by investigating the relationship between the amount of dye sorbed and the change in hydrogen ion concentration of the dye solution and also the dependence of dye uptake with solution temperature. It was found that the change in hydrogen ion concentration and increase in sorption temperature were directly related to the amount of dye sorbed, and activation energy was calculated to be −39.57kJ/mol, indicating that the dye uptake is chemisorption, involving valence forces through sharing or exchange of electrons between sorbent and sorbate as covalent forces.The intraparticle diffusion plots showed three sections indicating that intraparticle diffusion is not solely rate controlling. The intraparticle diffusion and mass transfer rate constants where observed to be well correlated with sorbent dose in the first 5min of sorption, indicating sorption process is complex. It was found that at low sorbent dose the mass transfer is the main rate controlling parameter. However at high sorbent dose, intraparticle diffusion becomes rate controlling.
Keywords: Sorbent dose; Sorption kinetics; Diffusion model; Change in hydrogen concentration (ΔH; +; )
Sorptive removal of Methylene blue from aqueous solution using palm kernel fibre: Effect of fibre dose by Augustine E. Ofomaja (pp. 8-18).
The use of palm kernel fibre, a readily available agricultural waste product for the sorption of Methylene blue from aqueous solution and the possible mechanism of sorption has been investigated at various fibre doses. The extent of dye removal and the rate of sorption were analyzed using two kinetic rate models (pseudo-first and pseudo-second-order kinetic models) and two diffusion models (intraparticle and external mass transfer models).Analysis of the kinetic data at different sorbent dose revealed that the pseudo-first order kinetics fitted to the kinetic data only in the first 5min of sorption and then deviated from the experimental data. The pseudo-second-order kinetic model was found to better fit the experimental data with high correlation coefficients at the various fibre dose used. The dye sorption was confirmed to follow the pseudo-second-order model by investigating the relationship between the amount of dye sorbed and the change in hydrogen ion concentration of the dye solution and also the dependence of dye uptake with solution temperature. It was found that the change in hydrogen ion concentration and increase in sorption temperature were directly related to the amount of dye sorbed, and activation energy was calculated to be −39.57kJ/mol, indicating that the dye uptake is chemisorption, involving valence forces through sharing or exchange of electrons between sorbent and sorbate as covalent forces.The intraparticle diffusion plots showed three sections indicating that intraparticle diffusion is not solely rate controlling. The intraparticle diffusion and mass transfer rate constants where observed to be well correlated with sorbent dose in the first 5min of sorption, indicating sorption process is complex. It was found that at low sorbent dose the mass transfer is the main rate controlling parameter. However at high sorbent dose, intraparticle diffusion becomes rate controlling.
Keywords: Sorbent dose; Sorption kinetics; Diffusion model; Change in hydrogen concentration (ΔH; +; )
Effect of xylanases on peroxide bleachability of eucalypt ( E. globulus) kraft pulp by Anatoly A. Shatalov; Helena Pereira (pp. 19-26).
Industrial eucalypt ( E. globulus L.) kraft pulp was treated with two commercial xylanase preparations Ecopulp® TX-200A and Pulpzyme® HC (endo-1,4-β-xylanase activity; EC 3.2.1.8) and bleached by totally chlorine-free (TCF) three-stage hydrogen peroxide bleaching sequence, without oxygen pre-delignification. The effect of enzymatic stage on pulp properties and bleachability has been studied and compared with reference (control) pulps, processed without enzyme addition. The similar mode of enzymatic action was noted for both xylanase preparations. Final brightness of 86% ISO was achieved after complete bleaching. Direct bleaching effect caused pulp brightening (by 1.2–1.5% ISO) and delignification (by 7–10%) immediately after the enzymatic stage. The maximal bleach boosting was shown after the first peroxide stage and then diminished, despite the progressive increase in delignification over the control. The loss in efficiency of xylanase treatment by the end of peroxide bleaching was associated with specific behavior of xylan-derived chromophores, i.e., hexenuronic acids.
Keywords: Xylanase bleaching; Eucalypt kraft pulp; Biocatalysis; Enzymes; Enzyme biocatalysis; Enzyme technology
Effect of xylanases on peroxide bleachability of eucalypt ( E. globulus) kraft pulp by Anatoly A. Shatalov; Helena Pereira (pp. 19-26).
Industrial eucalypt ( E. globulus L.) kraft pulp was treated with two commercial xylanase preparations Ecopulp® TX-200A and Pulpzyme® HC (endo-1,4-β-xylanase activity; EC 3.2.1.8) and bleached by totally chlorine-free (TCF) three-stage hydrogen peroxide bleaching sequence, without oxygen pre-delignification. The effect of enzymatic stage on pulp properties and bleachability has been studied and compared with reference (control) pulps, processed without enzyme addition. The similar mode of enzymatic action was noted for both xylanase preparations. Final brightness of 86% ISO was achieved after complete bleaching. Direct bleaching effect caused pulp brightening (by 1.2–1.5% ISO) and delignification (by 7–10%) immediately after the enzymatic stage. The maximal bleach boosting was shown after the first peroxide stage and then diminished, despite the progressive increase in delignification over the control. The loss in efficiency of xylanase treatment by the end of peroxide bleaching was associated with specific behavior of xylan-derived chromophores, i.e., hexenuronic acids.
Keywords: Xylanase bleaching; Eucalypt kraft pulp; Biocatalysis; Enzymes; Enzyme biocatalysis; Enzyme technology
Biomass production and carbon dioxide fixation by Aphanothece microscopica Nägeli in a bubble column photobioreactor by Eduardo Jacob-Lopes; Lucy Mara Cacia Ferreira Lacerda; Telma Teixeira Franco (pp. 27-34).
The objective of the present study was to evaluate the growth kinetics of Aphanothece microscopica Nägeli under different conditions of temperature, light intensity and CO2 concentration. The growth kinetics of the microorganism and carbon biofixation were evaluated using a central composite design, considering five different temperature levels (21.5, 25, 30, 35 and 38.5°C), light intensities (0.96, 3, 6, 9 and 11klux) and carbon dioxide concentrations (3, 15, 25, 50 and 62%). The results obtained showed the effects of temperature, light intensity and CO2 concentration ( p<0.05) on the photosynthetic metabolism of the microorganism. Response surface methodology was adequate for process optimisation, providing a carbon fixation rate to the order of 109.2mgL−1h−1 under conditions of 11klux, 35°C and 15% carbon dioxide, representing an increase of 58.1% as compared to the conditions tested initially.
Keywords: Carbon dioxide sequestration; Photobioreactor; Cyanobacteria
Biomass production and carbon dioxide fixation by Aphanothece microscopica Nägeli in a bubble column photobioreactor by Eduardo Jacob-Lopes; Lucy Mara Cacia Ferreira Lacerda; Telma Teixeira Franco (pp. 27-34).
The objective of the present study was to evaluate the growth kinetics of Aphanothece microscopica Nägeli under different conditions of temperature, light intensity and CO2 concentration. The growth kinetics of the microorganism and carbon biofixation were evaluated using a central composite design, considering five different temperature levels (21.5, 25, 30, 35 and 38.5°C), light intensities (0.96, 3, 6, 9 and 11klux) and carbon dioxide concentrations (3, 15, 25, 50 and 62%). The results obtained showed the effects of temperature, light intensity and CO2 concentration ( p<0.05) on the photosynthetic metabolism of the microorganism. Response surface methodology was adequate for process optimisation, providing a carbon fixation rate to the order of 109.2mgL−1h−1 under conditions of 11klux, 35°C and 15% carbon dioxide, representing an increase of 58.1% as compared to the conditions tested initially.
Keywords: Carbon dioxide sequestration; Photobioreactor; Cyanobacteria
Folding-like-refolding of heat-denatured MDH using unpurified ClpB and DnaKJE by Rui Nian; Lihan Tan; Woo-Seok Choe (pp. 35-43).
The Escherichia coli heat-shock protein ClpB can efficiently solubilize protein aggregates and refold them into active proteins in cooperation with the DnaK–DnaJ–GrpE chaperone (DnaKJE) system. However, the application of this bichaperone system at a large-scale was restricted because of the difficulties and high cost to express and purify each of these molecular chaperones. In this study, we constructed a plasmid encoding ClpB with a 6xHis-tag at its C-terminus (His-ClpB) to facilitate its purification through Immobilized Metal Affinity Chromatography (IMAC). A different plasmid capable of expressing the DnaKJE was used to obtain a cell extract containing unpurified DnaKJE. The effect of purified His-ClpB and unpurified DnaKJE on the refolding of heat-denatured malate dehydrogenase (MDH) was investigated, and proved to be highly efficient for MDH refolding. Furthermore, the use of both unpurified His-ClpB and DnaKJE available in the cell extract enabled highly successful refolding of the heat-denatured MDH with efficacy comparable to the case where the purified His-ClpB was used. To the best of our knowledge, this is the first attempt to apply a refolding cocktail comprising unpurified bichaperone system to the refolding of a heat-denatured protein, providing a practical and economically viable way of implementing a large-scale folding-like-refolding strategy.
Keywords: Molecular chaperone; ClpB; DnaK–DnaJ–GrpE; Protein aggregate; Refolding; MDH
Folding-like-refolding of heat-denatured MDH using unpurified ClpB and DnaKJE by Rui Nian; Lihan Tan; Woo-Seok Choe (pp. 35-43).
The Escherichia coli heat-shock protein ClpB can efficiently solubilize protein aggregates and refold them into active proteins in cooperation with the DnaK–DnaJ–GrpE chaperone (DnaKJE) system. However, the application of this bichaperone system at a large-scale was restricted because of the difficulties and high cost to express and purify each of these molecular chaperones. In this study, we constructed a plasmid encoding ClpB with a 6xHis-tag at its C-terminus (His-ClpB) to facilitate its purification through Immobilized Metal Affinity Chromatography (IMAC). A different plasmid capable of expressing the DnaKJE was used to obtain a cell extract containing unpurified DnaKJE. The effect of purified His-ClpB and unpurified DnaKJE on the refolding of heat-denatured malate dehydrogenase (MDH) was investigated, and proved to be highly efficient for MDH refolding. Furthermore, the use of both unpurified His-ClpB and DnaKJE available in the cell extract enabled highly successful refolding of the heat-denatured MDH with efficacy comparable to the case where the purified His-ClpB was used. To the best of our knowledge, this is the first attempt to apply a refolding cocktail comprising unpurified bichaperone system to the refolding of a heat-denatured protein, providing a practical and economically viable way of implementing a large-scale folding-like-refolding strategy.
Keywords: Molecular chaperone; ClpB; DnaK–DnaJ–GrpE; Protein aggregate; Refolding; MDH
Extraction of defatted rice bran by subcritical water treatment by Shigeru Hata; Jintana Wiboonsirikul; Atsushi Maeda; Yukitaka Kimura; Shuji Adachi (pp. 44-53).
Defatted rice bran was treated with subcritical water in the temperature range of 180–280°C for 5min using 117mL and 9mL vessels to produce the extracts. The total sugar and protein contents and radical scavenging activity of the extracts were then estimated for both vessels. The total sugar concentration of ca. 0.3g/L-extract was the highest for the extracts at 200°C, and it significantly decreased at the higher temperatures. The protein concentration and radical scavenging activity were higher at the higher temperatures. Extraction was also done at 200°C and 260°C for various times using the small vessel. The total sugar concentration decreased with the increasing extraction time, while the protein concentration and radical scavenging activity only slightly depended on the extraction time. The extracts at 200°C or lower temperatures using the large vessel possessed the emulsifying and emulsion-stabilizing activities. The HPLC analysis of the extract at 260°C for 5min using the small vessel indicated that it contained both hydrophilic and hydrophobic substances. The hydrophilic fraction of the extract mainly contained low-molecular-mass substances.
Keywords: Antioxidative ability; Extraction; Rice bran; Subcritical water
Extraction of defatted rice bran by subcritical water treatment by Shigeru Hata; Jintana Wiboonsirikul; Atsushi Maeda; Yukitaka Kimura; Shuji Adachi (pp. 44-53).
Defatted rice bran was treated with subcritical water in the temperature range of 180–280°C for 5min using 117mL and 9mL vessels to produce the extracts. The total sugar and protein contents and radical scavenging activity of the extracts were then estimated for both vessels. The total sugar concentration of ca. 0.3g/L-extract was the highest for the extracts at 200°C, and it significantly decreased at the higher temperatures. The protein concentration and radical scavenging activity were higher at the higher temperatures. Extraction was also done at 200°C and 260°C for various times using the small vessel. The total sugar concentration decreased with the increasing extraction time, while the protein concentration and radical scavenging activity only slightly depended on the extraction time. The extracts at 200°C or lower temperatures using the large vessel possessed the emulsifying and emulsion-stabilizing activities. The HPLC analysis of the extract at 260°C for 5min using the small vessel indicated that it contained both hydrophilic and hydrophobic substances. The hydrophilic fraction of the extract mainly contained low-molecular-mass substances.
Keywords: Antioxidative ability; Extraction; Rice bran; Subcritical water
Joint effect of nitrogen and phosphorous on glucose oxidase production by Aspergillus niger: Discussion of an experimental design with a risk of co-linearity by J. Mirón; J.A. Vázquez; M .P. González; M.A. Murado (pp. 54-63).
The combined effects of nitrogen and phosphorous on the production of glucose oxidase and gluconic acid by Aspergillus niger cannot be adequately described with Monod-type model, neither do they fit well to linear equations with interactions N× P, nor quadratic with N2 and P2 terms. On the other hand, the interactions of type N2 P and NP2, although common in real cases such as enzymatic kinetics in the presence of inhibitors, should be verified – if included in empiric models – by means of designs that can lead to artefactual results derived from the co-linearity. To avoid this risk we propose a procedure, based on the ‘bootstrap’ algorithm, which provided consistent results in the mentioned bioproductions. Applied together with methods of response surface and gradient, said procedure allowed to optimize the enzyme production as a function of the concentrations of N and P, to quintuple the initially obtained levels, and to explain other culture behaviours related with the sources of these nutrients.
Keywords: GOD production; Gluconic acid production; Bootstrap algorithm; Factorial design
Joint effect of nitrogen and phosphorous on glucose oxidase production by Aspergillus niger: Discussion of an experimental design with a risk of co-linearity by J. Mirón; J.A. Vázquez; M .P. González; M.A. Murado (pp. 54-63).
The combined effects of nitrogen and phosphorous on the production of glucose oxidase and gluconic acid by Aspergillus niger cannot be adequately described with Monod-type model, neither do they fit well to linear equations with interactions N× P, nor quadratic with N2 and P2 terms. On the other hand, the interactions of type N2 P and NP2, although common in real cases such as enzymatic kinetics in the presence of inhibitors, should be verified – if included in empiric models – by means of designs that can lead to artefactual results derived from the co-linearity. To avoid this risk we propose a procedure, based on the ‘bootstrap’ algorithm, which provided consistent results in the mentioned bioproductions. Applied together with methods of response surface and gradient, said procedure allowed to optimize the enzyme production as a function of the concentrations of N and P, to quintuple the initially obtained levels, and to explain other culture behaviours related with the sources of these nutrients.
Keywords: GOD production; Gluconic acid production; Bootstrap algorithm; Factorial design
Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix by Nadir Dizge; Okan Gunaydin; Faruk Yilmaz; Aziz Tanriseven (pp. 64-71).
A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55°C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The Km values were 35 and 38mM for free and immobilized enzyme, respectively. The Vmax values were 29 and 24mgglucose/mgenzymemin for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.
Keywords: Poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM/PTAA); Immobilization; Conductive polymer; Invertase; Carbodiimide
Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix by Nadir Dizge; Okan Gunaydin; Faruk Yilmaz; Aziz Tanriseven (pp. 64-71).
A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55°C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The Km values were 35 and 38mM for free and immobilized enzyme, respectively. The Vmax values were 29 and 24mgglucose/mgenzymemin for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.
Keywords: Poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM/PTAA); Immobilization; Conductive polymer; Invertase; Carbodiimide
Mathematical modeling and study of mass transfer parameters in supercritical fluid extraction of fatty acids from Trout powder by Hamzeh Zakizadeh Nei Nei; Shohreh Fatemi; Mohammad Reza Mehrnia; Alireza Salimi (pp. 72-78).
Mathematical modeling of fatty acids (FAs) extraction from Trout powder by supercritical carbon dioxide was performed in the present work. Trout powder with its low cost contains high amount of essential FAs and it is commonly available as a proper source of FAs. The effect of process parameters, such as pressure (25, 28, 31, 34 and 37MPa) and temperature (310, 318 and 326K) of extraction and void fraction of the bed (0.25, 0.35 and 0.45, v/v) on the yield of FAs extraction was examined in a series of experiments conducted in a laboratory scale apparatus. The results indicated a significant increase of extraction yield with an increase of pressure from 25 to 34MPa, but working at the higher pressure (37MPa) caused reduction of the extract. Increasing the temperature higher than 318K revealed significant reduction of the FAs yield and increasing the bed void fraction from 0.25 to 0.45 showed enhancement of the extraction.The mathematical model was developed considering diffusion-controlled regime in the particle and film mass transfer resistance around the particle with axial dispersion of the bulk phase at dynamic conditions. Henry law was used to describe the equilibrium state of solid and fluid phases. The proposed mass balance equations were numerically solved using implicit finite difference method and the model parameters were correlated using the experimental results of the outlet FAs concentration in the oil extracted at dynamic conditions. Well-known Nelder–Mead method was applied to estimate the four parameters of the model, namely, mass transfer coefficient ( kf), axial dispersion coefficient ( Dax) in the bulk phase, effective diffusivity ( Deff) into the pores and Henry coefficient ( H). In the range of studied conditions, the higher extraction efficiency with higher pressure resulted lower correlated H, although the temperature increasing which showed a retrograde phenomena in the FAs yield, revealed H passing though a minimum.
Keywords: Mathematical modeling; Supercritical fluid extraction; Trout; powder; Fatty acids; Mass transfer coefficient
Mathematical modeling and study of mass transfer parameters in supercritical fluid extraction of fatty acids from Trout powder by Hamzeh Zakizadeh Nei Nei; Shohreh Fatemi; Mohammad Reza Mehrnia; Alireza Salimi (pp. 72-78).
Mathematical modeling of fatty acids (FAs) extraction from Trout powder by supercritical carbon dioxide was performed in the present work. Trout powder with its low cost contains high amount of essential FAs and it is commonly available as a proper source of FAs. The effect of process parameters, such as pressure (25, 28, 31, 34 and 37MPa) and temperature (310, 318 and 326K) of extraction and void fraction of the bed (0.25, 0.35 and 0.45, v/v) on the yield of FAs extraction was examined in a series of experiments conducted in a laboratory scale apparatus. The results indicated a significant increase of extraction yield with an increase of pressure from 25 to 34MPa, but working at the higher pressure (37MPa) caused reduction of the extract. Increasing the temperature higher than 318K revealed significant reduction of the FAs yield and increasing the bed void fraction from 0.25 to 0.45 showed enhancement of the extraction.The mathematical model was developed considering diffusion-controlled regime in the particle and film mass transfer resistance around the particle with axial dispersion of the bulk phase at dynamic conditions. Henry law was used to describe the equilibrium state of solid and fluid phases. The proposed mass balance equations were numerically solved using implicit finite difference method and the model parameters were correlated using the experimental results of the outlet FAs concentration in the oil extracted at dynamic conditions. Well-known Nelder–Mead method was applied to estimate the four parameters of the model, namely, mass transfer coefficient ( kf), axial dispersion coefficient ( Dax) in the bulk phase, effective diffusivity ( Deff) into the pores and Henry coefficient ( H). In the range of studied conditions, the higher extraction efficiency with higher pressure resulted lower correlated H, although the temperature increasing which showed a retrograde phenomena in the FAs yield, revealed H passing though a minimum.
Keywords: Mathematical modeling; Supercritical fluid extraction; Trout; powder; Fatty acids; Mass transfer coefficient
Statistical description of influence of biogenic compounds on process reduction of organic substance from municipal sewage in functioning treatment plant by S. Masiuk; R. Rakoczy; M. Kordas (pp. 79-91).
The subject of this paper is the statistical analysis and mathematical description of the influence of biogenic compounds on the process reduction of organic substance from a municipal sewage. The proposed considerations are based on the industrial database obtained from the real functioning sewage treatment plant. The discrete-time linear problems are estimated by using the adequate statistical estimators. The correlation and spectral density functions are applied to analyse of the process purification of municipal sewages. The mathematical analysis of this process is attained by means of the matrices at the time and frequency domains.
Keywords: Correlation and spectral analysis; Biogenic compounds; BOD; Mathematical description
Statistical description of influence of biogenic compounds on process reduction of organic substance from municipal sewage in functioning treatment plant by S. Masiuk; R. Rakoczy; M. Kordas (pp. 79-91).
The subject of this paper is the statistical analysis and mathematical description of the influence of biogenic compounds on the process reduction of organic substance from a municipal sewage. The proposed considerations are based on the industrial database obtained from the real functioning sewage treatment plant. The discrete-time linear problems are estimated by using the adequate statistical estimators. The correlation and spectral density functions are applied to analyse of the process purification of municipal sewages. The mathematical analysis of this process is attained by means of the matrices at the time and frequency domains.
Keywords: Correlation and spectral analysis; Biogenic compounds; BOD; Mathematical description
Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris by Zhuge Bin; Du Guo-Cheng; Shen Wei; Zhuge Jian; Chen Jian (pp. 92-98).
The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65°C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg2+ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.
Keywords: Bacillus subtilis; WSHB04-02; Expression; Pectate lyase; Pichia pastoris
Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris by Zhuge Bin; Du Guo-Cheng; Shen Wei; Zhuge Jian; Chen Jian (pp. 92-98).
The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65°C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg2+ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.
Keywords: Bacillus subtilis; WSHB04-02; Expression; Pectate lyase; Pichia pastoris
Anaerobic digestion of solid slaughterhouse waste (SHW) at laboratory scale: Influence of co-digestion with the organic fraction of municipal solid waste (OFMSW) by María José Cuetos; Xiomar Gómez; Marta Otero; Antonio Morán (pp. 99-106).
Mesophilic anaerobic digestion of slaughterhouse waste (SHW) and its co-digestion with the organic fraction of municipal solid waste (OFMSW) have been evaluated. These processes were carried out in a laboratory plant semi-continuously operated and two set-ups were run. The first set-up, with a hydraulic retention time (HRT) of 25 days and organic loading rate (OLR) of 1.70kgVSm−3day−1 for digestion, and 3.70kgVSm−3day−1 for co-digestion, was not successful. The second set-up was initiated with an HRT of 50 days and an OLR of 0.9kgVSm−3day−1 for digestion and 1.85kgVSm−3day−1 for co-digestion. Under these conditions, once the sludge had been acclimated to a medium with a high fat and ammonia content, it was possible to decrease the HRT while progressively increasing the OLR to the values used in the first set-up until an HRT of 25 days and OLRs of 1.70 and 3.70kgVSm−3day−1, for digestion and co-digestion, respectively (the same conditions of the digesters failures previously). These digesters showed a highly stable performance, volatile fatty acids (VFAs) were not detected and long chain fatty acids (LCFAs) were undetected or only trace levels were measured in the analyzed effluent. Fat removal reached values of up to 83%. Anaerobic digestion was thus found to be a suitable technology for efficiently treating lipid and protein waste.
Keywords: Anaerobic processes; Biogas; Co-digestion; Process inhibition; Solid slaughterhouse waste; Waste treatment
Anaerobic digestion of solid slaughterhouse waste (SHW) at laboratory scale: Influence of co-digestion with the organic fraction of municipal solid waste (OFMSW) by María José Cuetos; Xiomar Gómez; Marta Otero; Antonio Morán (pp. 99-106).
Mesophilic anaerobic digestion of slaughterhouse waste (SHW) and its co-digestion with the organic fraction of municipal solid waste (OFMSW) have been evaluated. These processes were carried out in a laboratory plant semi-continuously operated and two set-ups were run. The first set-up, with a hydraulic retention time (HRT) of 25 days and organic loading rate (OLR) of 1.70kgVSm−3day−1 for digestion, and 3.70kgVSm−3day−1 for co-digestion, was not successful. The second set-up was initiated with an HRT of 50 days and an OLR of 0.9kgVSm−3day−1 for digestion and 1.85kgVSm−3day−1 for co-digestion. Under these conditions, once the sludge had been acclimated to a medium with a high fat and ammonia content, it was possible to decrease the HRT while progressively increasing the OLR to the values used in the first set-up until an HRT of 25 days and OLRs of 1.70 and 3.70kgVSm−3day−1, for digestion and co-digestion, respectively (the same conditions of the digesters failures previously). These digesters showed a highly stable performance, volatile fatty acids (VFAs) were not detected and long chain fatty acids (LCFAs) were undetected or only trace levels were measured in the analyzed effluent. Fat removal reached values of up to 83%. Anaerobic digestion was thus found to be a suitable technology for efficiently treating lipid and protein waste.
Keywords: Anaerobic processes; Biogas; Co-digestion; Process inhibition; Solid slaughterhouse waste; Waste treatment
Modeling of batch fermentation kinetics for succinic acid production by Mannheimia succiniciproducens by Hyohak Song; Seh Hee Jang; Jong Myoung Park; Sang Yup Lee (pp. 107-115).
Kinetic models are proposed for the batch production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. The models include terms accounting for both substrate and product inhibitions. Experimental data collected from a series of batch fermentations with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the parameters were approximated by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of M. succiniciproducens could be expressed by a modified Monod model incorporating inhibitions of glucose and organic acids accumulated in the culture broth. The Luedeking–Piret model was able to describe the formation of organic acids as the fermentation proceeded, in which succinic, acetic, and formic acids followed a mixed-growth-associated pattern. However, unexpectedly, lactic acid fermentation by M. succiniciproducens was nearly nongrowth-associated. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of M. succiniciproducens during efficient succinic acid production from glucose. These models thus can be employed for the development and optimization of biobased succinic acid production processes.
Keywords: Mannheimia succiniciproducens; Succinic acid; Fermentation; Kinetic models; Inhibition
Modeling of batch fermentation kinetics for succinic acid production by Mannheimia succiniciproducens by Hyohak Song; Seh Hee Jang; Jong Myoung Park; Sang Yup Lee (pp. 107-115).
Kinetic models are proposed for the batch production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. The models include terms accounting for both substrate and product inhibitions. Experimental data collected from a series of batch fermentations with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the parameters were approximated by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of M. succiniciproducens could be expressed by a modified Monod model incorporating inhibitions of glucose and organic acids accumulated in the culture broth. The Luedeking–Piret model was able to describe the formation of organic acids as the fermentation proceeded, in which succinic, acetic, and formic acids followed a mixed-growth-associated pattern. However, unexpectedly, lactic acid fermentation by M. succiniciproducens was nearly nongrowth-associated. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of M. succiniciproducens during efficient succinic acid production from glucose. These models thus can be employed for the development and optimization of biobased succinic acid production processes.
Keywords: Mannheimia succiniciproducens; Succinic acid; Fermentation; Kinetic models; Inhibition
Continuous production of monoacylglycerols from palm olein in packed-bed reactor with immobilized lipase PS by Aran H-Kittikun; Wiphum Kaewthong; Benjamas Cheirsilp (pp. 116-120).
A packed-bed reactor (PBR) system using immobilized lipase PS as biocatalyst was developed for continuous monoacylglycerols (MAG) production. The condition for continuous MAG production using immobilized lipase PS (IM-PS) of 1.5g (550U) in PBR (0.68cm i.d., 25cm long) was optimized. The effect of molar ratio of glycerol to palm olein, water content in glycerol and residence time on MAG production was investigated. The optimal glycerol to palm olein molar ratio and water content in glycerol were 12:1 and 10% (w/w), respectively. The yield of MAG increased with increasing residence time. At a residence time of 7.5h gave the highest yield of MAG of 60%. The long-term operation gave the highest yield of MAG 61.5% at 24h of the operation time with the productivity of 1.61gMAG/day. A half-life of the long-term process was 35 days of the operation time with the productivity of 0.81gMAG/day. Furthermore, the large scale of MAG production was performed continuously with IM-PS of 15g (5500U) in PBR (1.5cm i.d., 50cm long). The highest yield of MAG in large-scale operation of 70.1% and the 11-fold increasing in productivity of 18.3gMAG/day were obtained at 24h of the operation time.
Keywords: Continuous production; Immobilized lipase; Monoacylglycerols; Packed-bed reactor; Palm olein
Continuous production of monoacylglycerols from palm olein in packed-bed reactor with immobilized lipase PS by Aran H-Kittikun; Wiphum Kaewthong; Benjamas Cheirsilp (pp. 116-120).
A packed-bed reactor (PBR) system using immobilized lipase PS as biocatalyst was developed for continuous monoacylglycerols (MAG) production. The condition for continuous MAG production using immobilized lipase PS (IM-PS) of 1.5g (550U) in PBR (0.68cm i.d., 25cm long) was optimized. The effect of molar ratio of glycerol to palm olein, water content in glycerol and residence time on MAG production was investigated. The optimal glycerol to palm olein molar ratio and water content in glycerol were 12:1 and 10% (w/w), respectively. The yield of MAG increased with increasing residence time. At a residence time of 7.5h gave the highest yield of MAG of 60%. The long-term operation gave the highest yield of MAG 61.5% at 24h of the operation time with the productivity of 1.61gMAG/day. A half-life of the long-term process was 35 days of the operation time with the productivity of 0.81gMAG/day. Furthermore, the large scale of MAG production was performed continuously with IM-PS of 15g (5500U) in PBR (1.5cm i.d., 50cm long). The highest yield of MAG in large-scale operation of 70.1% and the 11-fold increasing in productivity of 18.3gMAG/day were obtained at 24h of the operation time.
Keywords: Continuous production; Immobilized lipase; Monoacylglycerols; Packed-bed reactor; Palm olein
Evaluation method for the drying performance of enzyme containing formulations by Jakob Sloth; Poul Bach; Anker D. Jensen; Søren Kiil (pp. 121-129).
A method is presented for fast and cheap evaluation of the performance of enzyme containing formulations in terms of preserving the highest enzyme activity during spray drying. The method is based on modeling the kinetics of the thermal inactivation reaction which occurs during the drying process. Relevant kinetic parameters are determined from differential scanning calorimeter (DSC) experiments and the model is used to simulate the severity of the inactivation reaction for temperatures and moisture levels relevant for spray drying. After conducting experiments and subsequent simulations for a number of different formulations it may be deduced which formulation performs best. This is illustrated by a formulation design study where 4 different enzyme containing formulations are evaluated. The method is validated by comparison to pilot scale spray dryer experiments.
Keywords: Downstream processing; Enzyme deactivation; Enzyme production; Kinetic parameters; Formulation design; Differential scanning calorimetry
Evaluation method for the drying performance of enzyme containing formulations by Jakob Sloth; Poul Bach; Anker D. Jensen; Søren Kiil (pp. 121-129).
A method is presented for fast and cheap evaluation of the performance of enzyme containing formulations in terms of preserving the highest enzyme activity during spray drying. The method is based on modeling the kinetics of the thermal inactivation reaction which occurs during the drying process. Relevant kinetic parameters are determined from differential scanning calorimeter (DSC) experiments and the model is used to simulate the severity of the inactivation reaction for temperatures and moisture levels relevant for spray drying. After conducting experiments and subsequent simulations for a number of different formulations it may be deduced which formulation performs best. This is illustrated by a formulation design study where 4 different enzyme containing formulations are evaluated. The method is validated by comparison to pilot scale spray dryer experiments.
Keywords: Downstream processing; Enzyme deactivation; Enzyme production; Kinetic parameters; Formulation design; Differential scanning calorimetry
Application of ion exchange to purify acarbose from fermentation broths by Juan F. Rodriguez; Antonio De Lucas; Manuel Carmona; Fátima Cañas (pp. 130-137).
Acarbose is conventionally used to reduce the insuline consumption of the diabetic patients. This compound is an oligosaccharide with the general formulae C25H43NO18 and obtained from fermentation processes by certain strains of Actinoplanes Utahensis. After the fermentation process, the acarbose has to be isolated from the fermentation broth where is accompanied of a large amount of substances, such as substrates, intermediate metabolites, proteins and different salts.Four strong acid resins considering geliform and macroporous matrix types in aqueous and organic media have been tested in order to reach an easy and selective separation process. According to the experimental data, the Finex CS10GC (a gel strong cationic ion exchanger) presented the maximum acarbosa uptake and also the highest rate of ion exchange in water. The best behavior in non-aqueous media was observed with the Purolite CT151 (macroporous ion exchanger) but its maximum capacity of ion exchange was really lower than that exhibited by the Finex CS10GC resin in aqueous media. These results suggest that the acarbose removal from fermentation broths must be carried out in aqueous media to ensure the maximum usage of the resin uptake capacity. The results obtained provide a significant insight into the main equilibrium phenomena that takes place depending on the characteristics of the liquid phase. Finally, the elution of acarbose from the resin can be accomplished of a selectivity way by using a solution of 2.25N of HCl. The proposed separation method seems to be technically and economically feasible.
Keywords: Acarbose; Ion exchange; Adsorption; Kinetics; Regeneration
Application of ion exchange to purify acarbose from fermentation broths by Juan F. Rodriguez; Antonio De Lucas; Manuel Carmona; Fátima Cañas (pp. 130-137).
Acarbose is conventionally used to reduce the insuline consumption of the diabetic patients. This compound is an oligosaccharide with the general formulae C25H43NO18 and obtained from fermentation processes by certain strains of Actinoplanes Utahensis. After the fermentation process, the acarbose has to be isolated from the fermentation broth where is accompanied of a large amount of substances, such as substrates, intermediate metabolites, proteins and different salts.Four strong acid resins considering geliform and macroporous matrix types in aqueous and organic media have been tested in order to reach an easy and selective separation process. According to the experimental data, the Finex CS10GC (a gel strong cationic ion exchanger) presented the maximum acarbosa uptake and also the highest rate of ion exchange in water. The best behavior in non-aqueous media was observed with the Purolite CT151 (macroporous ion exchanger) but its maximum capacity of ion exchange was really lower than that exhibited by the Finex CS10GC resin in aqueous media. These results suggest that the acarbose removal from fermentation broths must be carried out in aqueous media to ensure the maximum usage of the resin uptake capacity. The results obtained provide a significant insight into the main equilibrium phenomena that takes place depending on the characteristics of the liquid phase. Finally, the elution of acarbose from the resin can be accomplished of a selectivity way by using a solution of 2.25N of HCl. The proposed separation method seems to be technically and economically feasible.
Keywords: Acarbose; Ion exchange; Adsorption; Kinetics; Regeneration
Engineering characterisation of a single well from 24-well and 96-well microtitre plates by Hu Zhang; Sally R. Lamping; Samuel C.R. Pickering; Gary J. Lye; Parviz Ayazi Shamlou (pp. 138-149).
The detailed engineering characterisation of shaken microtitre-plate bioreactors will enhance our understanding of microbial and mammalian cell culture in these geometries and will provide guidance on the scale-up of microwell results to laboratory and pilot scale stirred bioreactors. In this work computational fluid dynamics (CFD) is employed to provide a detailed characterisation of fluid mixing, energy dissipation rate and mass transfer in single well bioreactors from deep square 24-well and 96-well microtitre plates. The numerical predictions are generally found to be in good agreement with experimental observation of the fluid motion and measured values of the key engineering parameters. The CFD simulations have shown that liquid mixing is more intensive in 96-well than in 24-well bioreactors due to a significant axial component to the fluid velocity. Liquid motion is strongly dependent on the orbital shaking amplitude which generally has a greater impact than the shaking frequency. Average power consumptions of 70–100Wm−3 and 500–1000Wm−3, and overall mass transfer coefficient, kL a, values of 0.005–0.028s−1 and 0.056–0.10s−1 were obtained for 24-well and 96-well bioreactors respectively at an orbital shaking amplitude of 3mm and shaking frequencies ranging from 500rpm to 1500rpm. The distribution of energy dissipation rates within each bioreactor showed these to be greatest at the walls of the well for both geometries. Batch culture kinetics of E. coli DH5α showed similar maximum specific growth rates and final biomass yields in shaken 24-well and shake flask bioreactors and in stirred miniature and 20L bioreactors at matched kL a values. The CFD simulations thus give new insights into the local and overall engineering properties of microwell bioreactor geometries and further support their use as high throughput tools for the study and optimisation of microbial and mammalian cell culture kinetics at this scale.
Keywords: Shaken bioreactor; Fluid mechanics; Mass transfer; Microtitre plates; Computational fluid dynamics
Engineering characterisation of a single well from 24-well and 96-well microtitre plates by Hu Zhang; Sally R. Lamping; Samuel C.R. Pickering; Gary J. Lye; Parviz Ayazi Shamlou (pp. 138-149).
The detailed engineering characterisation of shaken microtitre-plate bioreactors will enhance our understanding of microbial and mammalian cell culture in these geometries and will provide guidance on the scale-up of microwell results to laboratory and pilot scale stirred bioreactors. In this work computational fluid dynamics (CFD) is employed to provide a detailed characterisation of fluid mixing, energy dissipation rate and mass transfer in single well bioreactors from deep square 24-well and 96-well microtitre plates. The numerical predictions are generally found to be in good agreement with experimental observation of the fluid motion and measured values of the key engineering parameters. The CFD simulations have shown that liquid mixing is more intensive in 96-well than in 24-well bioreactors due to a significant axial component to the fluid velocity. Liquid motion is strongly dependent on the orbital shaking amplitude which generally has a greater impact than the shaking frequency. Average power consumptions of 70–100Wm−3 and 500–1000Wm−3, and overall mass transfer coefficient, kL a, values of 0.005–0.028s−1 and 0.056–0.10s−1 were obtained for 24-well and 96-well bioreactors respectively at an orbital shaking amplitude of 3mm and shaking frequencies ranging from 500rpm to 1500rpm. The distribution of energy dissipation rates within each bioreactor showed these to be greatest at the walls of the well for both geometries. Batch culture kinetics of E. coli DH5α showed similar maximum specific growth rates and final biomass yields in shaken 24-well and shake flask bioreactors and in stirred miniature and 20L bioreactors at matched kL a values. The CFD simulations thus give new insights into the local and overall engineering properties of microwell bioreactor geometries and further support their use as high throughput tools for the study and optimisation of microbial and mammalian cell culture kinetics at this scale.
Keywords: Shaken bioreactor; Fluid mechanics; Mass transfer; Microtitre plates; Computational fluid dynamics
Modeling the yield of activated sludge in the presence of 2,4-dinitrophenol by Guo-Wei Chen; Han-Qing Yu; Peng-Ge Xi; De-Qian Xu (pp. 150-156).
In this work, a model was developed to evaluate the yield of activated sludge in the presence of 2,4-dinitrophenol (dNP). Experimental results show that the presence of dNP substantially reduced the sludge yield at all solid retention times tested, but that it had no negative effect on the bioactivity and settleability of activated sludge. With the established model, the maximum observed sludge yield(Y′max) and specific endogenous respiration rate ( kd) for the sludge without dNP dose were estimated as 0.79g-MLSS/g-COD and 0.042d−1, respectively, whereas for the sludge dosed with dNP the corresponding values were calculated as 0.56g-MLSS/g-COD and 0.056d−1, respectively. In the presence of dNP at 5mg/l, the energy uncoupling coefficient was calculated as 0.29.
Keywords: 2,4-Dinitrophenol; Modeling; Sludge growth; Solids retention time (SRT); Yield
Modeling the yield of activated sludge in the presence of 2,4-dinitrophenol by Guo-Wei Chen; Han-Qing Yu; Peng-Ge Xi; De-Qian Xu (pp. 150-156).
In this work, a model was developed to evaluate the yield of activated sludge in the presence of 2,4-dinitrophenol (dNP). Experimental results show that the presence of dNP substantially reduced the sludge yield at all solid retention times tested, but that it had no negative effect on the bioactivity and settleability of activated sludge. With the established model, the maximum observed sludge yield(Y′max) and specific endogenous respiration rate ( kd) for the sludge without dNP dose were estimated as 0.79g-MLSS/g-COD and 0.042d−1, respectively, whereas for the sludge dosed with dNP the corresponding values were calculated as 0.56g-MLSS/g-COD and 0.056d−1, respectively. In the presence of dNP at 5mg/l, the energy uncoupling coefficient was calculated as 0.29.
Keywords: 2,4-Dinitrophenol; Modeling; Sludge growth; Solids retention time (SRT); Yield
Dynamics of positional enrichment: Theoretical development and application to carbon labeling in Zymomonas mobilis by Fernando Alvarez-Vasquez; Yusuf A. Hannun; Eberhard O. Voit (pp. 157-174).
Positional enrichment analysis has become an important technique for assessing detailed flux distributions and the fates of specific atoms in metabolic pathway systems. The typical approach to positional enrichment analysis is performed by supplying specifically labeled substrate to a cell system, letting the system reach steady state, and measuring where label had arrived and accumulated. The data are then evaluated mathematically with the help of a linear stoichiometric flux distribution model. While this procedure has proven to yield new and valuable insights, it does not address the transient dynamics between providing label and its ultimate steady-state distribution, which is often of great interest to the experimentalist (pulse labeling experiments). We show here that an extension of a recent mathematical method for dynamic labeling analysis is able to shed light on these transitions, thereby revealing insights not obtained with traditional positional enrichment analyses. The method traces the dynamics of one or more carbons through fully regulated metabolic pathways, which, in principle, may be arbitrarily complex. After a brief review of the earlier method and description of the theoretical extension, we illustrate the method with an analysis of the pentose phosphate pathway in Zymomonas mobilis, which has been used for traditional positional enrichment analyses in the past. We show how different labeling schemes result in distinctly different transients, which nevertheless eventually lead to a steady-state labeling profile that coincides exactly with the corresponding profile from traditional analysis. Thus, over the domain of commonality, the proposed method leads to results equivalent to those from state-of-the-art existing methods. However, these steady-state results constitute only a small portion of the insights obtainable with the proposed method. Our method can also be used as an “inverse” technique for elucidating the topology and regulation of pathway systems, if appropriate time series data are available. While such dynamic data are still rather rare, they are now being generated with increasing frequency and we believe it is desirable, and indeed necessary, to accompany this trend with an adequate, rigorous method of analysis.
Keywords: Atom positional labeling; Isotopomer; Dynamic labeling; BST; Mathematical model; Pentose phosphate pathway; Positional enrichment; Zymomonas mobilis
Dynamics of positional enrichment: Theoretical development and application to carbon labeling in Zymomonas mobilis by Fernando Alvarez-Vasquez; Yusuf A. Hannun; Eberhard O. Voit (pp. 157-174).
Positional enrichment analysis has become an important technique for assessing detailed flux distributions and the fates of specific atoms in metabolic pathway systems. The typical approach to positional enrichment analysis is performed by supplying specifically labeled substrate to a cell system, letting the system reach steady state, and measuring where label had arrived and accumulated. The data are then evaluated mathematically with the help of a linear stoichiometric flux distribution model. While this procedure has proven to yield new and valuable insights, it does not address the transient dynamics between providing label and its ultimate steady-state distribution, which is often of great interest to the experimentalist (pulse labeling experiments). We show here that an extension of a recent mathematical method for dynamic labeling analysis is able to shed light on these transitions, thereby revealing insights not obtained with traditional positional enrichment analyses. The method traces the dynamics of one or more carbons through fully regulated metabolic pathways, which, in principle, may be arbitrarily complex. After a brief review of the earlier method and description of the theoretical extension, we illustrate the method with an analysis of the pentose phosphate pathway in Zymomonas mobilis, which has been used for traditional positional enrichment analyses in the past. We show how different labeling schemes result in distinctly different transients, which nevertheless eventually lead to a steady-state labeling profile that coincides exactly with the corresponding profile from traditional analysis. Thus, over the domain of commonality, the proposed method leads to results equivalent to those from state-of-the-art existing methods. However, these steady-state results constitute only a small portion of the insights obtainable with the proposed method. Our method can also be used as an “inverse” technique for elucidating the topology and regulation of pathway systems, if appropriate time series data are available. While such dynamic data are still rather rare, they are now being generated with increasing frequency and we believe it is desirable, and indeed necessary, to accompany this trend with an adequate, rigorous method of analysis.
Keywords: Atom positional labeling; Isotopomer; Dynamic labeling; BST; Mathematical model; Pentose phosphate pathway; Positional enrichment; Zymomonas mobilis
Study of the effects of temperature and pH on lactic acid production from fresh cassava roots in tofu liquid waste by Streptococcus bovis by Suripto Dwi Yuwono; Takao Kokugan (pp. 175-183).
We report a study of the effects of temperature and pH on the kinetics of lactic acid production from fresh cassava roots (FCR) by Streptococcus bovis in new media: tofu liquid waste (TLW); TLW with 2wt% concentrated maguro waste (TLW+CMW2); and in a standard trypto-soya broth (TSB) compared with a standard medium (glucose in TSB). The results showed that 39°C and pH 5.5 were optimal for fermentation properties (lactic acid concentration, productivity and specific growth rate) in all media, including the standard. The energy of activation ( Ea) and the energy of deactivation ( Ed) of lactic acid fermentation in all media were calculated using the Arrhenius relation. The Ea and Ed values increased in the order FCR in TLW
Keywords: Kinetics; Lactic acid; Fresh cassava roots; Streptococcus bovis; Activation energy; Inhibition models
Study of the effects of temperature and pH on lactic acid production from fresh cassava roots in tofu liquid waste by Streptococcus bovis by Suripto Dwi Yuwono; Takao Kokugan (pp. 175-183).
We report a study of the effects of temperature and pH on the kinetics of lactic acid production from fresh cassava roots (FCR) by Streptococcus bovis in new media: tofu liquid waste (TLW); TLW with 2wt% concentrated maguro waste (TLW+CMW2); and in a standard trypto-soya broth (TSB) compared with a standard medium (glucose in TSB). The results showed that 39°C and pH 5.5 were optimal for fermentation properties (lactic acid concentration, productivity and specific growth rate) in all media, including the standard. The energy of activation ( Ea) and the energy of deactivation ( Ed) of lactic acid fermentation in all media were calculated using the Arrhenius relation. The Ea and Ed values increased in the order FCR in TLW
Keywords: Kinetics; Lactic acid; Fresh cassava roots; Streptococcus bovis; Activation energy; Inhibition models
Effect of gamma-ray on activity and stability of alcohol-dehydrogenase from Saccharomyces cerevisiae by Najla Ben Akacha; Amel Zehlila; Sonia Mejri; Taieb Jerbi; Mohamed Gargouri (pp. 184-188).
The effect of γ-ray irradiation on alcohol-dehydrogenase activity of yeast was investigated. The results suggested that low doses of γ-ray (10 and 20Gy) significantly increased the enzyme activity. This work also describes the impact of irradiation on immobilization efficiency of biocatalyst entrapped on to alginate gel beads. When yeast irradiated to a dose of 20Gy was immobilized, ADH stability was improved up to 1.4 times at 45°C compared to the immobilized non-irradiated cells. Also, the irradiated biocatalyst, when immobilized, can be reused more than eight times in oxidation reaction of ethanol. This preparation also permitted to reach high yields of immobilization (79%) and activity (88%).
Keywords: Alcohol-dehydrogenase; Entrapment; Immobilization; γ-ray; Saccharomyces cerevisiae
Effect of gamma-ray on activity and stability of alcohol-dehydrogenase from Saccharomyces cerevisiae by Najla Ben Akacha; Amel Zehlila; Sonia Mejri; Taieb Jerbi; Mohamed Gargouri (pp. 184-188).
The effect of γ-ray irradiation on alcohol-dehydrogenase activity of yeast was investigated. The results suggested that low doses of γ-ray (10 and 20Gy) significantly increased the enzyme activity. This work also describes the impact of irradiation on immobilization efficiency of biocatalyst entrapped on to alginate gel beads. When yeast irradiated to a dose of 20Gy was immobilized, ADH stability was improved up to 1.4 times at 45°C compared to the immobilized non-irradiated cells. Also, the irradiated biocatalyst, when immobilized, can be reused more than eight times in oxidation reaction of ethanol. This preparation also permitted to reach high yields of immobilization (79%) and activity (88%).
Keywords: Alcohol-dehydrogenase; Entrapment; Immobilization; γ-ray; Saccharomyces cerevisiae
Enhancement of the isolation selectivity of isoflavonoid puerarin using oligo-β-cyclodextrin coupled polystyrene-based media by Li Yang; Tianwei Tan (pp. 189-198).
The isolation selectivity of the isoflavonoid puerarin, a well-known traditional Chinese medicine, was studied when native and oligo-β-cyclodextrin (CDP) coupled polystyrene-based macroporous resins were used as adsorbents by static tests. The research results indicated that the CDP coupled resin HPD-100-CDP offered the best adsorption and desorption capacity for puerarin than others and its equilibrium adsorption data at 25°C fit best to the Freundlich isotherm. The performance of separation of puerarin on HPD-100-CDP column in one step was evaluated. Based on the above experimental data, a novel medium PS-CDP was synthesized and its chromatographic retention behaviors were also explored. ESI-MS/MS,1H NMR spectroscopy and UV absorption spectrum were used for the detection and characterization of puerarin in isolated fraction. Under the optimum mobile phase, methanol/acetic acid/water=5.0/6.6/88.4 (v/v/v), the purity and recovery of puerarin were 95.3% and 86.7%, respectively, by HPLC analysis. In conclusion, the PS-CDP medium can enhance the isolated selectivity of puerarin and it can be applied in preparative scale operations.
Keywords: Oligo-β-cyclodextrin; Puerarin; Polystyrene-based media; Coupled; Chromatography; Isolation
Enhancement of the isolation selectivity of isoflavonoid puerarin using oligo-β-cyclodextrin coupled polystyrene-based media by Li Yang; Tianwei Tan (pp. 189-198).
The isolation selectivity of the isoflavonoid puerarin, a well-known traditional Chinese medicine, was studied when native and oligo-β-cyclodextrin (CDP) coupled polystyrene-based macroporous resins were used as adsorbents by static tests. The research results indicated that the CDP coupled resin HPD-100-CDP offered the best adsorption and desorption capacity for puerarin than others and its equilibrium adsorption data at 25°C fit best to the Freundlich isotherm. The performance of separation of puerarin on HPD-100-CDP column in one step was evaluated. Based on the above experimental data, a novel medium PS-CDP was synthesized and its chromatographic retention behaviors were also explored. ESI-MS/MS,1H NMR spectroscopy and UV absorption spectrum were used for the detection and characterization of puerarin in isolated fraction. Under the optimum mobile phase, methanol/acetic acid/water=5.0/6.6/88.4 (v/v/v), the purity and recovery of puerarin were 95.3% and 86.7%, respectively, by HPLC analysis. In conclusion, the PS-CDP medium can enhance the isolated selectivity of puerarin and it can be applied in preparative scale operations.
Keywords: Oligo-β-cyclodextrin; Puerarin; Polystyrene-based media; Coupled; Chromatography; Isolation