Biochemical Engineering Journal (v.28, #1)

BEJ Keywords (IV).

IFC (CO2).

Equilibrium study of immobilized lysozyme on the extrudate-shaped NaY zeolite by Yu-Kaung Chang; Ren-Ze Huang; Shiuan-Yaw Lin; Shwu-Jer Chiu; Juan-Chin Tsai (1-9).
Studies of adsorption isotherms were used as a method of characterizing the adsorption behavior of the extrudate-shaped NaY zeolite for lysozyme in a reciprocal shaking water bath system. In equilibrium isotherm studies, the experimental data obtained either at different pH or with different ionic strength of buffer were fitted reasonably well by the Langmuir model. In parallel experiments, the influences of changes in temperature on the equilibrium isotherms were further investigated. With increasing temperature, different patterns of isothermal adsorption behavior for lysozyme were observed. When the temperature was lower than 310 K, equilibrium isotherms were well correlated by the Langmuir model. However, at temperature higher than 323 K, equilibrium isotherms were well correlated by the Freundlich model. In addition, thermodynamic parameters ( Δ G ads o , Δ H ads o , and Δ S ads o ) for the adsorption process were also determined by using equilibrium association constant and van’t Hoff relationship to analyze the experimental data. The results revealed that the use of extrudate-shaped NaY zeolite may be an ideal and promising support for the adsorption of lysozyme because of its low cost and simplicity of its preparation method.
Keywords: Lysozyme; Adsorption; Extrudate-shaped NaY zeolite; Equilibrium isotherm model; van’t Hoff equation;

In this study, we have established a suitable model to describe the dynamic characteristic of an aspartase-overproducing Escherichia coli strain. A traditional Monod model and power-law models are respectively applied to describe the dynamic behaviors. When the model structure was selected, the parameter values of the model should be determined by the global/local optimization method. There are two major challenges, numerical integration for differential equations and finding global parameter values. In this study, we introduce hybrid differential evolution, which is a variant of genetic algorithms, to avoid obtaining a premature solution. In addition, we apply a modified collocation method to avoid numerical integration. The Monod's model could only predict the growth characteristic of the recombinant E. coli, qualitatively. The S-system representation could suit for constructing the model structure of the microbial growth.
Keywords: Modeling; Biokinetics; Growth kinetics; Kinetic parameters; Fermentation; Parameter estimation;

Recombinant Escherichia coli with self-disruption characteristic was developed. The cell harbored a d-amino acid oxidase (DAAO) expressing plasmid resulted in an obvious self-disruption. When T7 lysozyme instead of DAAO was used as a lysis gene, no appreciable self-disruption was observed. In the presence of two compatible plasmids, which expressed DAAO and T7 lysozyme, respectively, E. coli BL21(DE3) showed a significant self-disruption. A lysis plasmid containing both inducible DAAO and constitutive T7 lysozyme genes were, therefore, constructed to make the transformed cells self-disrupt more effectively. Green fluorescent protein (GFP), a model target protein, was co-expressed in this self-disruptive E. coli cell. About 60% of the cells were self-disrupted when the lysis genes were induced by isopropyl thiogalactoside (IPTG). The cell disruption efficiency reached to 90% after freeze/thawing treatment.
Keywords: Escherichia coli; Self-disruption; d-amino acid oxidase; T7 lysozyme; Green fluorescent protein;

Electrolytic stimulation of bacteria Enterobacter dissolvens by a direct current by Peng She; Bo Song; Xin-Hui Xing; Mark van Loosdrecht; Zheng Liu (23-29).
The effects of a weak electric field on the growth and metabolic behavior of Enterobacter dissolvens were investigated using glucose as the sole carbon source. A direct current (DC) was applied using salt bridge electrodes and platinum electrodes. The best stimulating effects in terms of cell growth and the dehydrogenase activity (DHA) were obtained when a DC of 10 mA was applied for 12 h via the platinum electrodes. In this case, the electrolysis of water was the major electrode reaction, as determined by cyclic voltammetry. The presence of the hydrogen generated a strong reductive environment and led to a reduction of NAD/NADH ratio from 7 to 3. The specific activity of dehydrogenase and glucose consumption increased 2- and 1.5-fold, respectively. The application of the DC via the platinum electrodes also led to accelerated cell death during the later stationary phase. This is possibly due to the presence of anodic intermediates including H2O2, OH• and O2 •. These results provide more details for understanding the effect of a DC on E. dissolvens, a strain with potential application in the electro-remediation of PAHs contaminated soil.
Keywords: Electrolytic stimulation; Enterobacter dissolvens; Direct current; Dehydrogenase activity; Glucose; Redox potential;

Production of methane gas from Japanese cedar chips pretreated by various delignification methods by Harumi Take; Yoshifumi Andou; Yoshitoshi Nakamura; Fumihisa Kobayashi; Yasuji Kurimoto; Masaaki Kuwahara (30-35).
Methane fermentation of woody waste was carried out using various pretreatment methods for delignification in order to evaluate the efficient pretreatment method for producing methane gas from woody waste. Japanese cedar (Cryptomeria japonica) chips were used as sample of woody waste. As pretreatment methods, refiner treatment, steaming treatment, steaming treatment followed by refiner treatment, basidiomycete fungi treatment, and steam explosion treatment were attempted and the methane gas evolved from treated wood chips were compared. The steam explosion treatment resulted in the most effective pretreatment method for methane fermentation from woody waste among the pretreatment methods. The steam explosion at a steam pressure of 4.51 MPa and a steaming time of 5 min was the optimal pretreatment method for obtaining a large amount of methane gas in energy saving manner. The strong correlation between the amount of methane gas evolved and the ratio of amount of Klason lignin was observed. The study confirmed the necessity of pretreatment for delignification of cedar wood waste.
Keywords: Delignification; Japanese cedar; Methane gas; Pretreatment method; Woody waste;

Mathematical modeling to investigate temperature effect on kinetic parameters of ethanol fermentation by Muenduen Phisalaphong; Nuttapan Srirattana; Wiwut Tanthapanichakoon (36-43).
A mathematical model was developed to describe the effects of temperature on the kinetic parameters of ethanol fermentation by the flocculating yeast, Saccharomyces cerevisiae M30, using cane molasses as the substrate. Three state variables, biomass, ethanol and the substrate and 12 kinetics parameters were used to describe the phenomenon. The kinetic parameters of the model were determined by using the least-square method. The influence of temperature and initial sugar concentration on cell activities was investigated and quantified. Arrhenius relationships between operating temperature and the maximum specific growth rate, specific production rate, specific death rate were then established. The activation energy for growth, ethanol production and death rate were 3.461 × 104, 3.496 × 104 and 1.777 × 105  kJ/kmol, respectively. Polynomial equations were established for the effects of temperature on the other kinetic parameters. A high temperature led to a decrease in the ethanol and cell yields but an increase in the inhibition effect of ethanol and sugar on cell growth and ethanol production. In addition, an inhibition effect of the initial sugar concentration on cell growth was clearly observed. The adopted mathematical model could describe very well the dynamics of ethanol fermentation from the beginning up to the stationary phase.
Keywords: Ethanol; Fermentation; Kinetics; Modeling; Temperature; Yeast;

The local composition models, such as M-Wilson, NRTL and M-NRTL models have been used to predict the activity coefficients of amino acids and simple peptides in water. In this study, the binary interaction parameters of the local composition models for water–amino acid and amino acid–water molecules have been calculated. The results obtained from the models have been compared with the experimental data of activity coefficients and the deviations of all those models have been reported, too. It is shown that M-NRTL model can more accurately predict the activity coefficients. The models have been also used to predict the solubility of amino acids in water. The results show that the M-Wilson model can accurately predict the solubility of amino acid at a wide range of temperatures. Finally, the values of Δh, Δs and Δg of the solutions have been calculated.
Keywords: Thermodynamic; Amino acid; Peptide; Local composition model; Water;

Effect of 19 different medium components on chitinase production by marine isolate Pantoea dispersa was studied by Plackett–Burman design. Each component was screened on the basis of ‘p’ (probability) value, which was at 95% or above of confidence level. In the initial chitinase production medium containing five medium components chitinase production of 108 units/ml was obtained. In the Plackett–Burman method, significant factors influencing the chitinase production were screened. 4.21-fold increase in chitinase production was observed in 22nd medium of Plackett–Burman experimental design. Increase in production of endochitinase (3.95-fold) and chitobiase (2.31-fold) was observed.
Keywords: Biomass; Chitinase; Pantoea dispersa; pH; Plackett–Burman;

Enhanced production of an extracellular protease from Beauveria bassiana by optimization of cultivation processes by Yerra Koteswara Rao; Shih-Chin Lu; Bing-Lan Liu; Yew-Min Tzeng (57-66).
Statistics-based experimental design, response surface methodology was employed to investigate the effect of medium components on the production of extracellular protease from Beauveria bassiana A1. The optimum medium composition for protease production was found to be (in %, w/v): 0.72 shrimp shell powder, 0.60 soy powder, 0.19 sucrose and 0.68 yeast extract. This medium was projected to produce, theoretically, 281.14 U/ml. By using this medium, an experimental maximum protease activity of 280.72 U/ml in 7 days cultivation verified the applied methodology. Further studies on bioreactor production of protease were investigated in a 5 l stirred-tank and 30 l airlift bioreactors. In 5 l stirred-tank bioreactor, a maximum protease activity of 238.77 U/ml was attained in 6 days of cultivation when aeration rate and agitation speed were controlled at 0.6 vvm and 150 rpm, respectively. On the other hand, in 30 l airlift bioreactor the maximum protease activity of 283.84 U/ml was found with the controlled aeration rate of 0.9 vvm operated in a bubble column mode. This activity in the 30 l airlift bioreactor was close to that obtained from the shaker-flask cultivation study (280.72 U/ml). However, the duration of cultivation period using 30 l airlift bioreactor was shorter than the corresponding duration obtained from the shaker-flask cultivation by 4 days.
Keywords: Beauveria bassiana; Protease; Shaker-flask cultivation; Response surface methodology; Airlift bioreactor;

Efficient production of recombinant protein in immobilized insect cell culture using serum-free basal media after baculovirus infection by Hideki Yamaji; Toshitaka Manabe; Akinori Kitaura; Eiji Izumoto; Hideki Fukuda (67-72).
To develop an efficient biological production process using the baculovirus–insect cell system, recombinant protein production was investigated in an immobilized cell culture with medium replacement. Sf9 insect cells were naturally entrapped within reticulated polyvinyl formal resin biomass support particles (BSPs; 2 mm × 2 mm × 2 mm; pore diameter 30–50 μm) in a 2.5-l stirred-tank bioreactor. The immobilized cells were grown to a density of over 107  cells/cm3-BSP, with the medium of 10% fetal bovine serum (FBS)-supplemented TNM-FH replaced at appropriate intervals, before infection with a recombinant baculovirus carrying the β-galactosidase gene. When serum-free TNM-FH was used instead of 10% FBS-supplemented TNM-FH for medium replenishment on and after post-infection day 1, the immobilized cells showed a high β-galactosidase yield comparable to that obtained in a culture using the serum-supplemented medium throughout. This finding indicates that immobilized insect cell culture allows not only intensification of cell culture but also recombinant protein production in protein-free basal media.
Keywords: Insect cell culture; Baculovirus; Immobilization; Biomass support particles; Bioreactors; Recombinant protein production;

Effect of cereal extracts and cereal fiber on viability of Lactobacillus plantarum under gastrointestinal tract conditions by Haruhito Michida; Sriappareddy Tamalampudi; Severino S. Pandiella; Colin Webb; Hideki Fukuda; Akihiko Kondo (73-78).
New probiotic foods are expected to develop out of the increasing demand for a health-oriented society. Fermented cereal has recently been looked to as a way of delivering lactic acid bacteria. The present study investigated the effect of cereal extracts and cereal fiber on the viability of the lactic acid bacterium Lactobacillus plantarum under gastrointestinal tract conditions, with simulated gastric juice and bile juice used for the evaluation of gastrointestinal tolerance. The cells were cultured using cereal media that contained cereal extract and cereal fiber to immobilize the cells. Malt and barley were used as cereal. Malt and barley extracts were also used to examine the effect of cereal extracts on gastrointestinal tolerance. Regarding gastric tolerance, the addition of cereal extracts significantly improved the viability of L. plantarum while immobilization within cereal fiber slightly improved its viability. The effect of malt extract was more positive than that of barley extract. Meanwhile, immobilization within cereal fiber played a major role in bile tolerance and the presence of cereal extracts further enhanced the tolerance of L. plantarum to bile juice. In both cereal media, cell immobilization within cereal fiber and the presence of cereal extracts had a synergistic effect on the gastrointestinal tolerance.
Keywords: Cereals; Fermentation; Immobilized cells; Viability; Gastrointestinal tolerance; Functional foods;

Conformational transitions of bovine β-lactoglobulin A (β-LG) induced by carboxylic acid were systematically studied by steady-state tryptophan (Trp) fluorescence. The behavior of β-LG denaturation depends upon the species and concentration of carboxylic acid, as well as on the pH of solutions. The order of the effectiveness of the respective carboxylic acids was described as follows: MeCOOH < EtCOOH < PrCOOH < ClEtCOOH < TFA <  iBuCOOH <  nBuCOOH < PFPA. The conformational change of β-LG through the carboxylic acid-induced transitions of the β-LG conformation were analyzed assuming a two-state mechanism between unfold and native states in order to obtain the m value, a measure of the dependence of the free energy change on the concentration of carboxylic acid. The m values of various carboxylic acids were compared with those of various alcohols based on the role of each group constituting the carboxylic acid and alcohol molecules, namely, the hydrocarbon group, hydroxyl group, halogen substituents, and the carboxyl group. Among these groups, the hydrophobic hydrocarbon groups and halogen substituents contributed positively to the m value, whereas the hydrophilic carboxyl and hydroxyl group contributed negatively. The present results can therefore be interpreted as a simple correlation based on the accessible surface area (ASA) of each groups of carboxylic acids and alcohols. These results suggest that the conformational transition of the protein due to the addition of carboxylic acids and alcohols can be explained both by hydrophobicity as well as clustering effects of each carboxylic acid and alcohol molecule.
Keywords: β-Lactoglobulin; Unfolding; Carboxylic acid; Alcohol; Accessible surface area; Cluster formation;

Study on acid-hydrolysis of spent cells for lactic acid fermentation by Min-tian Gao; Makoto Hirata; Eiichi Toorisaka; Tadashi Hano (87-91).
To reduce nutrient cost for lactic acid production, spent cells, a by-product in fermentation processes, were used as a nutrient source and the treatments of spent cells were investigated in this study. Untreated spent cells did not show significant effect on lactic acid production. After acid-hydrolysis, spent cells gave an increase in productivity to some extent, but the low glucose consumption indicated a nutrient limitation. The nutrient limitation was overcome easily by supplementing a small amount of yeast extract (YE). The combination of 5 g/L YE and the spent cell hydrolyzate (SCH) had high performance in lactic acid production relative to 15 g/L YE. Moreover, two kind of acid-hydrolysis methods, diluted acid based hydrolysis and concentrated acid based hydrolysis, were also compared. The diluted acid based hydrolysis had the advantages over the concentrated acid based one in lactic acid production due to its less nutrient destruction and lower salt inhibition. When diluted acid based SCH was supplemented, YE supplementation could be cut down to 20% with no significant decrease in productivity and yield.
Keywords: Spent cells; Reuse; Yeast extract; Acid-hydrolysis; Lactic acid fermentation;

The cell physiology and metabolic flux distribution of the osmophilic yeast Candida krusei under hyperosmotic (NaCl as the osmoticum) and normal conditions (as a control) were compared. For the cells in exponential phase (12th hour) under hyperosmotic condition, the glycerol concentration was doubled than the control and the trehalose content reached 0.124 g/g dry cell weight. Metabolic flux analysis indicated that the carbon flux flowed toward glycerol-3-phosphate (G3P) to form glycerol from dihydroxyacetone phosphate (DHAP) and the glyceraldehydes-3-phosphate (GAP) node increased 67.2% while that toward TCA cycle decreased 26.9%. At the same time, the carbon flux flowed toward trehalose synthesis from glucose-6-phosphate (G6P) node was 2.6-fold that of the control. The metabolic energy flux analysis also suggested that the hyperosmotic condition efficiently improved the cellular glycerol and trehalose metabolism. Furthermore, the hyperosmotic condition also altered key enzyme activities. Compared with the control, G3P dehydrogenase (ctGPD) activity showed a rapid increase under hyperosmotic condition. For the activity of trehalose-6-phosphate synthase (T-6-P synthase), it slightly decreased but could keep at a higher level in the late stage of fermentation. This work is considered helpful to the further understanding of the metabolism of the osmophilic yeast under osmotic stress environment and also useful for the metabolic flux control of C. krusei for efficient production of glycerol and trehalose.
Keywords: Candida krusei; Enzyme activity; Glycerol; Hyperosmotic condition; Metabolic flux; Trehalose;

A new way to cultivate aerobic granules in the process of papermaking wastewater treatment by Wang Hailei; Yu Guangli; Liu Guosheng; Pan Feng (99-103).
A new way to cultivate aerobic granules using papermaking wastewater was presented in this paper. By seeding superior mixed flora (SMF) to sequencing batch reactor (SBR), aerobic granules appeared on day 19. The formation process was studied by microscopes. The results showed that granulation process consisted of five stages, i.e. microbe multiplication phase, floc appearance phase, floc cohesion phase, mature floc phase and aerobic granule phase; SMF was the key reason that aerobic granules could shape and filamentous microbes played a crucial role in granulation process. Furthermore, some factors affecting granule formation were discussed.
Keywords: Aerobic granule; Papermaking wastewater; SBR; Filamentous microbes;

List of Reviewers (104-107).