Biochemical Engineering Journal (v.21, #1)


BEJ Keywords (II).

An alternative way of analyzing the progress curves of enzyme-catalyzed reactions by Y.Leong Yeow; Prayad Pokethitiyook; Mee Yean Cheah; Huong D.T. Dang; Connie K.P. Law (1-10).
This paper describes a method, based on Tikhonov regularization, that converts the progress curves of enzyme-catalyzed reactions into reaction rate curves. Standard nonlinear least-squares regression methods are then used to match these curves directly with appropriately selected rate equations to obtain the numerical value of all the kinetic parameters in these equations. This technique is illustrated by applying it to a number of enzyme kinetic data sets taken from the published literature. The main advantages of this approach are highlighted. Some of the problems encountered together with possible ways of overcoming them are briefly discussed.
Keywords: Enzymes; Kinetic parameters; Modelling; Bioreactions; Progress curve; Tikhonov regularization;

The effects of the addition of various carboxylic acids on the formation of AOT-reverse micelle clusters have been studied by quantitative analysis of their percolation phenomena. The results were compared with those of various alcohols based on the functional groups, namely, the hydrocarbon group, hydroxyl group, halogen substituents, and carboxyl group. Among these groups, the hydrophobic hydrocarbon group and halogen substituents suppressed the formation of micellar clusters, depending on their chain number or species. However, the hydrophilic carboxyl and hydroxyl groups enhanced the formation of micellar clusters. Furthermore, the effect of a hydroxyl groups on the formation of micellar clusters was stronger than that of carboxyl groups. These results can be explained by the additive contribution of each carboxylic acid and alcohol molecule, and they can be interpreted as a simple correlation based on the accessible surface area (ASA). These results suggest that the formation of AOT-reverse micelle clusters by the addition of carboxylic acids and alcohols can be explained by the hydrophobicity of the molecule, as well as by the formation of micelle-like structures with both carboxylic acid and alcohol molecules.
Keywords: Percolation; Carboxylic acids; Alcohols; Micellar cluster formation; Accessible surface area; Cluster;

An effective strategy for the co-production of S-adenosyl-l-methionine and glutathione by fed-batch fermentation by Jian-Ping Lin; Jun Tian; Jian-Feng You; Zhi-Hua Jin; Zhi-Nan Xu; Pei-Lin Cen (19-25).
Both S-adenosyl-l-methionine (SAM) and glutathione (GSH) are intracellular molecules with important physiological activities and medical applications. The metabolic pathways of SAM and GSH in yeast cell are tightly connected, which leads us to develop a new strategy to produce them jointly. The aim of this work is to maximize the co-production of SAM and GSH simultaneously by high cell density culture of baker’s yeast. A strategy of two-stage fed-batch culture was developed to optimize the production. The first exponential feeding stage was aimed to reduce the formation of ethanol and to increase the cell density. For this purpose, the initial medium composition was modified to fit the high cell-density cultivation of yeast. With the modified media, the final concentration of SAM, GSH and cells obtained in experiment were increased to 8.77, 0.81 and 105 g/L, respectively. Meanwhile, the yields of SAM and GSH on glucose reached 0.039 and 0.0035 g/g, respectively, which were still relatively low due to the fermentative consumption of glucose. To enhance the yields, a constant feeding stage during the transformation phase was performed to satisfy the energy and precursor requirement for the synthesis of SAM and GSH with the absence of ethanol formation. Due to the elimination of ethanol production, the yields of SAM and GSH on glucose were enhanced up to 0.048 and 0.0038 g/g, respectively.
Keywords: S-adenosyl-l-methionine; Glutathione; Bioconversion; Fed-batch culture; Optimization; Yeast;

Response surface methodology were used to study the cumulative effect of the culture conditions and to enhance the production of extracellular ribonuclease in shake flask fermentation by Aspergillus niger. These conditions considered include initial pH, inoculum size, total carbon, ratio of glucose to corn powder, NH4NO3 and K2SO4. The relative importance of these conditions on ribonuclease production was investigated by using the Plackett–Burman experimental design. Initial pH, ratio of glucose to corn powder and NH4NO3 were found to significantly influence the ribonuclease production. For obtaining the mutual interaction between the variables and optimizing these variables, a 23 factorial central composite design using response surface methodology was employed. Initial pH has a significant positive effect on the ribonuclease production. NH4NO3 has a significant negative effect. The mutual interactions have no significant effect. The ribonuclease production was increased by 23.2% compared to that under unoptimized conditions. The fermentation time was also shortened after optimization.
Keywords: Ribonuclease; Aspergillus niger; Optimization; Response surface methodology;

The effect of different carbon sources on lactic acid production by Rhizopus oryzae was studied using glucose, sucrose, beet molasses, carob pod and wheat bran as substrate. The highest lactic acid concentration was obtained when 150 g/l glucose was present in the medium as the sole carbon source. In that case, the lactic acid yield was approximately 60% by weight based on the amount of glucose consumed. Wheat bran was found to be an unsuitable substrate for this particular fermentation. Pasteurisation of molasses increased lactic acid production rate compared to that of untreated molasses. Likewise, 58 g/l lactic acid was obtained by using the supernatant containing sugars extracted from carob pod. This medium could therefore be considered as an alternative carbon source for lactic acid production.
Keywords: Lactic acid; Rhizopus oryzae; Fermentation; Glucose; Sucrose; Carob pod;

The sorption of metal complex dyes, metal complex blue (MCB) and metal complex yellow (MCY), onto pine sawdust in a batch sorber has been studied. A model has been used for the design of a two-stage batch sorber based on pseudo second-order sorption kinetics. The model has been optimized with respect to contact time in order to minimize total contact time to achieve a fixed percentage of metal complex dyes removal. Three simplified kinetic models including pseudo first- and second-order equation, and intraparticle diffusion equation were selected to follow the sorption process. Kinetic parameters, rate constants, equilibrium sorption capacities and related correlation coefficients, for each kinetic model were calculated and discussed. It was shown that the sorption of metal complex dyes could be described by the pseudo second-order equation. The equilibrium data fit well in the Langmuir isotherm. Results of two-stage batch sorber design studies show that the required times for the 90% efficiencies removal of metal complex dyes are 45.9 and 22.4 min for MCB and MCY, respectively.
Keywords: Two-stage batch sorber; Contact time optimization; Sorption kinetics; Metal complex dyes; Sawdust;

The essential role of hydraulic settling time in the formation of aerobic granules was studied in four column sequencing batch reactors (R1–R4). Results showed that aerobic granules were successfully cultivated and became dominant only in the R4 operated at the shortest settling time of 5 min, while a mixture of aerobic granules and suspended sludge was observed in R1–R3 run at settling times of 20, 15 and 10 min, respectively. After the steady state was achieved, the respective settling times of R1–R3 was shortened from 20 to 5 min, from 15 to 2 min and from 10 to 1 min; as the result, aerobic granules completely displaced suspended sludge in these three reactors after two-week operation. It was found that cell surface hydrophobicity was improved significantly at short settling times. Analyses of the polyvalent metals contents in aerobic granules showed that the accumulation of calcium in aerobic granules was related to the settling time, however, the total content of iron, magnesium and aluminium in aerobic granules was much pretty lower than the granule calcium content and almost remained constant. It is obvious that a short settling time favours aerobic granulation, and one may expect to manipulate the formation and characteristics of aerobic granules by controlling settling time.
Keywords: Aerobic granule; SBR; Settling time; Selection pressure; Extracellular polysaccharides; Cell surface hydrophobicity; Polyvalent ions;

In this work, a 23 factorial design was performed with the aim of optimizing the bacteriocin production by Bacillus licheniformis P40, using response surface methodology (RSM). The bacteriocin production was first tested on different byproducts, like feather meal, grape bagasse, an industrial fibrous soybean residue and cheese whey. Cheese whey was the selected medium to test the effect of three variables (temperature, initial pH and whey concentration) by RSM. Statistical analysis of results showed that, in the range studied, the three variables have a significant effect on bacteriocin production. Response-surface data showed maximum bacteriocin production at initial pH between 6.5 and 7.5 and temperature between 26 and 37 °C when the cheese whey concentration was 70 g l−1. Increasing whey concentration resulted in increase of bacteriocin production, with an activity at the maximum whey concentration tested. It was not possible to determine a relation between bacteriocin production and optimal conditions for bacterial growth.
Keywords: Aerobic processes; Antibiotic; Bacteriocin; Experimental design; Modeling; Whey;

Batch experiments were performed to investigate the anaerobic degradation of crystalline cellulose of 10 g l−1 by rumen microorganisms at pH from 4.8 to 7.3. The degradation efficiency increased with pH and the highest value of about 78% was achieved at pH 6.8 and 7.3. Acetate and propionate were the major aqueous products at all pH values. With increasing pH, the molar percentage of acetate decreased, whereas that of propionate increased. A modified Gompertz equation was able to adequately model the fermentation of cellulose by rumen microorganisms. The results from this study might provide useful information for the application of rumen cultures for conversion of cellulosic wastes into value-added products.
Keywords: Cellulose; Degradation; Modelling; Rumen microorganisms; pH;

Lactic acid extraction from aqueous solutions with tri-n-octylamine dissolved in decanol and dodecane by D Yankov; J Molinier; J Albet; G Malmary; G Kyuchoukov (63-71).
The paper deals with the lactic acid extraction by tri-n-octylamine dissolved in decanol and docecane. The extraction efficiency depends on the initial lactic acid concentration and the initial pH value of the solution. In case of lactic acid extraction from simulated fermentation broth, the partially loading of extractant with HCl leads to increasing of the distribution coefficient. The extraction equilibrium constants have been determined. The strong influence of diluents on the values of the graphically determined extraction constant and the number of extractant molecules in the acid–amine complex has been shown. A mathematical model of extraction, taking into account formation of (1:1) (2:1) and (1:2) acid/amine complexes, has been composed and solved. The model fits very well the experimental results.
Keywords: Lactic acid; Liquid–liquid extraction; Extraction kinetics; Modelling; Separation; Modifier influence;

Modeling and simulation of airlift bioreactors by H. Znad; V. Báleš; J. Markoš; Y. Kawase (73-81).
A tanks-in-series model was applied for mathematical modeling of the unsteady state performance of a semi batch operation in a 10.5 dm3 internal loop airlift bioreactor for the production of gluconic acid by fermentation. A set of first order differential equations for the material balances of micro-organism, substrate, product, and dissolved oxygen around the hypothetical well mixed stages in the riser and the downcomer was solved simultaneously using the Athena software package. The kinetic model used considers the effect of two substrates (glucose and dissolved oxygen) on the growth rate.Both the effect of airflow rate and the height of the airlift bioreactor on the gluconic acid production were investigated. The model has been validated with experimental data. The model is simple enough to be used in design studies and it can be adapted to airlift system configurations and fermentation systems other than gluconic acid fermentation.
Keywords: Airlift bioreactor; Gluconic acid; Dissolved oxygen; Modeling; Bioreactors;

Power consumption and heat transfer resistance in large rotary shaking vessels by Yoshihito Kato; Cyril P. Peter; Ali Akgün; Jochen Büchs (83-91).
The application of disposable equipment is a new trend in bioprocessing. Liu and Hong (C.M. Liu, L.N. Hong, Development of a Shaking Bioreactor System for Animal Cell Cultures, Biochem. Eng. J. 7 (2) (2001) 121–125) have shown that the cultivation of animal cell cultures in large plastic shaking vessels of up to 56 L volume resulted in good performance in terms of product concentration. In order to characterize this type of reactor from a chemical engineering view point, the power consumption of a large rotary shaking vessel (20 L) by a temperature method was measured. It was shown that heat insulation of the large vessel is not required to measure power consumption and, therefore, a simple experimental setup could be employed. The power consumption per unit volume in the large vessel was found to be in the same range (≤10 kW/m3) as that in small shaking flasks (<2 L). The dependency of power consumption on shaking frequency was less pronounced than that reported in previous papers within the experimental conditions investigated. Since the gas side heat transfer coefficient was the limiting step of heat transfer through the vessel wall, the overall heat transfer coefficient could be estimated using the power consumption per unit volume and the liquid filling volume. The temperature difference between the liquid and the surrounding air in case of the non-insulated vessel was found to be in the range of 5 K.
Keywords: Disposable equipment; Shaking vessel; Power consumption; Rotary shaker; Heat transfer; Temperature method;

A mathematical model of direct ethanol production from starch in immobilized recombinant amylase-producing yeast culture was proposed for estimating the dynamic behavior of cell growth, starch degradation, glucose accumulation, ethanol production, and glucoamylase synthesis by immobilized yeast. The application of the model was compared to the experiments of ethanol production from starch in batch and continuous cultures. The calculated values agreed well with experimental data, regarding concentration of cells, starch, ethanol, and glucoamylase. The profiles of concentration of starch, glucoamylase, cells, and ethanol in the gel bead for estimating the efficient ethanol production were calculated by the model. By comparing the ethanol productivity and the washout dilution rate in an immobilized cell culture with those in a free cell culture, it was confirmed that the immobilized cell culture had a higher ethanol productivity than the free cell culture and produced ethanol continuously with a dilution rate of 0.5 h−1, which is about 10 times larger than the washout rate in a free cell culture.
Keywords: Immobilized cell culture; Mathematical model; Recombinant yeast; Ethanol production; Starch; Glucoamylase;

Enzymatic hydrolysis by immobilized lipase applied to a new prototype anti-asthma drug by Juliana Vaz Bevilaqua; Jose Carlos Pinto; Lidia M. Lima; Eliezer J. Barreiro; Tito L.M. Alves; Denise M.Guimaraes Freire (103-110).
The last step of the production of 4-ethyl-(2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl))-phenoxy acetic acid (LASSBio 482), designed to act as an anti-asthma drug, was performed by enzymatic hydrolysis of the methyl ester. Reactions were performed in a three-phase system with a solid biocatalyst (Lipozyme® RM IM), an organic solvent phase (ethyl acetate) and an aqueous phase (saturated Na2CO3 solution). In order to optimize the reaction conditions, experimental design techniques were used. The variables studied were the amount of enzyme, the reaction temperature and the total volume of saturated Na2CO3 solution. The experimental results were analysed statistically and the main effects of the variables were computed and analysed. Dynamic experiments were then performed under optimised reaction conditions for different initial enzyme concentrations (0.5, 0.9 and 1.4 UH Lipozyme/mL solvent). An empirical nonlinear model was then proposed to describe the process, and experimental data were used to estimate the model parameters. The optimised reaction conditions were 20 mg Lipozyme® (0.9 UH/mLsolvent), 5.0 mL Na2CO3 (sat), a temperature of 40 °C and batch reaction time of 6 h. The results showed that enzymatic hydrolysis is a promising technique for the synthesis of the desired product.
Keywords: Enzyme biocatalysis; Immobilized enzymes; Lipase; Hydrolysis; Organic media; Optimisation;