Biochemical Engineering Journal (v.29, #1-2)

IFC (CO2).

Preface by Masaaki Terashima; Masahiro Goto; Teruyuki Nagamune (1).

Minimal aggregation formation between injector and column inlet and enough residence time inside the column were identified to be the key for proper proteins refolding by size exclusion chromatography (SEC). Therefore, a step change of mobile phase flow rate strategy was developed. That is, during the injected denatured protein (sample) traveling from injector to column inlet, a higher rate of mobile phase should be applied to reduce aggregation. Then, a lower flow rate should be used to allow enough time for protein to refold inside the column. The refolding of denatured lysozyme in SEC by this method was shown to reduce the aggregation and thus resulting in good refolding yields, though not completely. Combining this method together with our previously proposed chaperon solvent plug strategy could obtain complete recovery of denatured protein, both mass and activity recoveries.
Keywords: Step change of mobile phase flow rates; Chaperon solvent plug; Size exclusion chromatography; Protein refolding; Lysozyme;

Direct capture of factor IX from unclarified human plasma by IMEBAC by Yu-Kaung Chang; Jim-Tong Horng; Ren-Ze Huang; Shiuan-Yaw Lin (12-22).
The method development for direct capture of clotting factor IX (FIX) from unclarified human plasma using immobilized metal expanded bed affinity chromatography (IMEBAC) has been described. Cu(II), Ni(II), Zn(II) and Co(II) metal ions were evaluated in immobilized metal affinity chromatography for the recovery of FIX. Immobilized Cu(II) chelating adsorbent was found to be the most effective for capture of general protein and FIX. The adsorption characteristic of Cu(II)-STREAMLINE chelating adsorbent used in this work for general protein and FIX in unclarified plasma has been assessed by the measurement of equilibrium isotherm. The influence of liquid velocities between 100 and 300 cm/h on the selective adsorption of FIX from clarified plasma was investigated in small packed bed experiments. Optimal conditions for the elution of FIX were also investigated in packed bed experiments conducted with clarified plasma. On the basis of the results, IMEBAC for direct capture of FIX from unclarified plasma was established. The purification results showed that FIX was directly recovered using 20 mM glycine buffer (pH 7.0) from unclarified plasma with a purification factor of 83 and yield of 16% in a single step. Other contaminants and FIX were completely eluted by using 25 mM EDTA (pH 7.0).
Keywords: Immobilized metal affinity chromatography; Expanded bed adsorption; Clotting factor IX; Unclarified human plasma; Direct capture; Purification;

Elimination of high order nucleic acids to reduce the systematic viscosity is an important step in the separation and purification of proteins/enzymes. In this research, hydroxyl radicals produced by H2O2 was applied for the degradation of genomic nucleic acids from both the leaf of maize and recombinant Escherichia coli cells. One percent agarose gel electrophoresis analysis showed that all genomic DNA was degraded by the exposure under 10 mM H2O2 in the presence of Fe(II) for 5 min to produce 70 bp fragments. To prolong the exposure time will result in much smaller nucleotides fragments. Compared with hydrogen peroxide, other peroxides showed lower DNA degradation activity under the same conditions. Both SDS-PAGE analysis and western blotting assay showed that exposure at lower concentration (<10 mM) within 20 min, H2O2 did not impair proteins of interest, but >20 min exposure could cause damages to proteins.
Keywords: Degradation; Free radical; Genomic DNA; Hydrogen peroxide; Intracellular preparation;

The immobilized 1,3-regiospecific lipase, Mucor miehei, was employed to catalyze the trans-esterification reaction to enrich n-3 polyunsaturated fatty acid (n-3 PUFA) content of triglycerides (TG) in Menhaden oil under supercritical carbon dioxide (SCCO2). A supercritical reacting system with a syringe pump to pressurize CO2 to supercritical condition and a high-pressure liquid pump to transfer the substrate into the reactor is applied to carry out experiments. Through the saponification of Menhaden oil followed by urea inclusion, 80.1 wt% of n-3 PUFA can be concentrated with containing 29.4 wt% EPA and 41.8 wt% DHA. The profile of conversion shows a constant increase at beginning 5 h, but after which the conversion levels off. Pressure up to 103.4 bars has a significant positive effect on the conversion of n-3 PUFA onto TG. Further increasing pressure, the conversion rate decreases due to the transformation of the spatial structure of lipase and leads to deactivate. The enzyme exhibits a good performance and stability in the region of 323 K. Ethanol is used as a co-solvent and its maximal addition is 10 wt% of the total substrates. The optimal substrate molar ratio of TG to n-3 PUFA is about 1/4 by considering the TG inhibition. The conversion in SCCO2 appears 40 wt% higher than in n-hexane at ambient pressure after 5 h. The ping-pong bi-bi mechanism was proposed for the kinetic model of the reaction. The final rate equation can be simplified and expressed by a modified Michaelis–Menten equation. Based on the evaluated kinetic constants, there was 7.3% deviation of the total n-3 PUFA content between the experimental and the predicted data.
Keywords: Supercritical fluid; n-3 Polyunsaturated fatty acid; Trans-esterification; Production kinetics; Enzyme technology;

Microbial synthesis of cis,cis-muconic acid from benzoate by Sphingobacterium sp. mutants by Chun-Ming Wu; Chang-Chan Wu; Chien-Chou Su; Sung-Nung Lee; Yung-An Lee; Jiumn-Yih Wu (35-40).
The mutants used in this study, Sphingobacterium sp. M4113/M4115, were originated from a wild-type strain GCG, which was isolated from the sewage of a styrene monomer-manufacturing factory. The culturing medium contains appropriate amounts of K2HPO4, (NH4)2SO4, MgSO4·7H2O, Na-succinate, yeast extract and EDTA–FeCl3 complex. The highest conversion yield was obtained with mutant strain M4115, in which more than 0.560 g of cis,cis-muconic acid was accumulated in 28 h from a Na-benzoate solution of 2.0 g/l (∼28% yield). Growth inhibition appeared as the concentration of Na-benzoate exceeded 3.0 g/l. In addition, an increase of 17% and 6% of cis,cis-muconic acid in yield were observed for M4115 and M4113, respectively, as EDTA–FeCl3 complex was employed. It's believed that this complex can enhance the yield of catechol 1,2-dioxygenase, and therefore, the yield of cis,cis-muconic acid according to β-ketoadipate biosynthesis pathway.
Keywords: Aerobic process; Biosynthesis; Bioprocess design; Global environment; cis,cis-Muconic acid; Sphingobacterium sp;

A new immobilized lipase membrane reactor was studied for synthesis of 2-ethylhexyl palmitate. The lipase from Candida sp. 99–125 was immobilized on surfactant modified cotton membrane (130 IU/cm2). The conditions of enzymatic synthesis of 2-ethylhexyl palmitate were optimized and the optimized reaction conditions are: molar ratio of palmitic acid/hexanol 1:1.3 to 1:1.5, reaction temperature 40 °C, lipase used in immobilization 5000 IU/g membrane to 6000 IU/g membrane. The membrane reactor could be reused for at least 21 batches. The average esterification degree reached 95% under these conditions. 2-Ethylhexyl palmitate was purified by washing with water followed by evaporation of the organic solvent and the purity of 98% could be obtained.
Keywords: Membrane reactor; Lipase; Enzymatic esterification; Immobilization; Candida sp. 99–125; 2-Ethylhexyl palmitate;

The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane/water reverse micelles was significantly enhanced by pretreatment with acetone, using the hydrolysis of olive oil as a model reaction. The activity of acetone treated lipase was further enhanced when low molecular weight polyethylene glycol (PEG 400) was introduced into AOT reverse micelles. To know the effects of acetone treatment on the lipase activity in simple AOT and mixed AOT/PEG 400 reverse micelles, the influence of various parameters, such as W 0 (molar ratio of water to surfactant), pH, surfactant concentration, ionic strength and reaction temperature were investigated and compared with those for native lipase in simple AOT reverse micelles. The optimal activities of treated lipase in AOT reverse micelles with and without PEG 400 were 2.0 and 1.6 times higher respectively than that of native lipase in AOT reverse micelles. A kinetic model that considers substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was successfully used to understand the improvement of the lipase activity. The Michaelis constant (K m) and substrate adsorption equilibrium constant (K ad ) were obviously reduced compared with those for native lipase in AOT reverse micelles. The stability of the lipase in reverse micelles was also studied, and the values of half-life time (t 1/2) were determined from residual activity profiles.
Keywords: Acetone treatment; PEG 400; Reverse micelles; Chromobacterium viscosum lipase; AOT; Hydrolysis;

Spodoptera frugiperda (Sf21) insect cells were grown in the microspheres that were prepared using sodium-cellulosesulfate (NaCS) and poly-diallyldimethylammoniumchloride (PDADMAC). The highest Sf21 cell density was 1 × 108  cells/ml in the microshperes. The immobilised Sf21 cells were infected with Autograpa californica multiple nuclear polyhedrosis virus (AcMNPV) at a “theoretical” MOI of 1.0 and a TOI of 3.9 × 107  cells/ml in the microspheres. The AcMNPV polyhedral inclusion bodies (PIBs) titer reached was 1.76 × 1010  PIBs/ml in the microspheres, which was 58 times higher than that (2.75 × 107  PIBs/ml) in a suspension condition. The immobilised Sf21 cells were infected with a recombinant baculovirus, Ac-omega-GFP baculovirus, and the highest concentration of green fluorescent protein (GFP) produced was 0.0048 mg/ml in suspension culture and 0.159 mg/ml in the microspheres.
Keywords: Immobilisation; NaCS; PDADMAC; Ac-omega-GFP baculovirus; PIBs; GFP;

Azadirachtin, obtained from Azadirachta indica (neem) has emerged as wonderful natural biopesticide for pest control due to its efficacy, biodegradability and minimum side effects. To overcome constraints associated with its limited, variable and unreliable supply from natural trees, plant cell suspension culture of A. indica was developed (obtained from callus of seeds). The kinetics of cell growth/azadirachtin formation and substrate consumption of A. indica suspension culture was established in bioreactor with low shear setric impeller under optimized culture conditions (statistically optimized independently). The culture featured a growth of 15.5 and 0.05 g/l azadirachtin in 10 days of cultivation after which azadirachtin concentration declined. To prevent any inhibition caused by the excess feeding of substrate during fed-batch cultivation, inhibition of the carbon, nitrate and phosphate were separately established. Based on batch kinetics/inhibitory data a mathematical model was proposed and model parameters were evaluated. The model was extrapolated to fed-batch cultivation and nutrient feeding strategy (with respect to carbon, nitrate and phosphate) were computer simulated for a suitable fed-batch cultivation of A. indica cells to enhance growth and product formation. Fed-batch cultivation demonstrated a growth of 20.06 g/l biomass and 0.082 g/l of azadirachtin in 14 days of cultivation. A 30% increase in cell growth and 83% increase in azadirachtin production were obtained when a computer simulated feeding strategy was experimentally implemented in 3 l bioreactor.
Keywords: Azadirachtin; Growth kinetics; Substrate inhibition; Bioreactor; Mathematical modeling; Fed-batch cultivation;

Enzymatic synthesis of sorbitan methacrylate: Comparison of methacrylic acid and vinyl methacrylate by Gwi-Taek Jeong; Ki-Young Byun; Woo-Tai Lee; Hwa-Won Ryu; Changshin Sunwoo; Hae-Sung Kim; Don-Hee Park (69-74).
“Biomaterials” is a generic term which refers to a variety of medical material which is designed to be in direct contact with living tissue. Clearly, biomaterials must be carefully and microscopically fabricated for optimal acceptance within the living organism, in both functional and structural senses. In this study, the enzymatic synthesis of sorbitan methacrylate from 1,4-sorbitan via the manipulation of an immobilized biocatalyst (Novozym 435) and acryl donors (methacrylic acid and vinyl methacrylate) was evaluated. The conversion yield of sorbitan methacrylate was found to be 80.4% under the following experimental conditions: 1:3 molar ratio of 1,4-sorbitan and vinyl methacrylate, reaction temperature of 50 °C, initial sorbitan concentration of 50 g/L, and 3% (w/v) Novozym 435.
Keywords: Immobilized enzyme; Bioconversion; Optimization; Biocatalysis; Sorbitan; Esterification;

A recombinant vector was constructed to overproduce a glutathione S-transferase (GST) tagged N-acetyl-d-neuraminic acid aldolase (Neu5Ac aldolase) fusion protein in Escherichia coli. A solid-phase cloning method was proposed for the recombination of a GST-containing plasmid and Neu5Ac aldolase gene amplified from E. coli K12. Although the over-expressed fusion protein was largely present as aggregate or inclusion body, soluble GST-Neu5Ac aldolase fusion protein possessing the enzymatic activity could be purified to homogeneity by the affinity of GST tag. Results from sequencing the reconstruction of Neu5Ac aldolase gene fusion and gel electrophoresis of its expressing product suggested that the insertion of DNA was in the proper reading frame. Using N-acetyl-d-neuraminic acid (Neu5Ac) as the substrate, the purified fusion protein showed a K m value of 2.8 mM that is close to those reported in the literature for Neu5Ac aldolase, indicating that the addition of GST tag in the N terminus did not change the affinity of Neu5Ac aldolase for substrate. The GST-Neu5Ac aldolase fusion protein, which was applied even in a very small dosage, could effectively catalyze the production of 2-keto-3-deoxy-d-glycero-d-galacto-nonopyranulosonic acid (KDN) from d-mannose and pyruvic acid.
Keywords: Fusion protein; N-acetyl-d-neuraminic acid aldolase; KDN; GST tag;

A hybrid model combining hydrodynamic and biological effects is developed to describe the cultivation of Acetobacter xylinum for bacterial cellulose production in a modified airlift reactor with wire-mesh draft tubes. The hydrodynamic part is essentially a modified tanks-in-series model whose parameters are determined using a tracer response method. The modified tanks-in-series model is based on that the materials in the riser and downcomer are exchangeable across the wire-mesh draft tube. In addition, the effect of oxygen transfer is also taken into consideration for the tanks-in-series model. An artificial neural network (ANN) model based on the data of a batch cultivation in a stirred tank reactor combined with system equations is utilized as the biological part. Simulation is carried out to demonstrate that the proposed hybrid model represents the cultivation system very well.
Keywords: Modeling; Airlift bioreactors; Wire-mesh draft tubes; Cellulose; Acetobacter xylinum; Biokinetics;

Fabrication and characterization of a PDMS–glass hybrid continuous-flow PCR chip by Jeong Ah Kim; Ji Youn Lee; Shimyoung Seong; Seung Hwan Cha; Seung Hwan Lee; Jae Jeong Kim; Tai Hyun Park (91-97).
A continuous-flow (CF) microchip was fabricated using polydimethylsiloxane (PDMS)/glass laminate for efficient polymerase chain reaction (PCR). The temperatures of the three separate zones, corresponding to the three PCR thermal cycling steps were controlled using an external PID temperature controller. A PCR mixture was introduced to the CF microchip using a syringe pump. The hydrophobic surface of the PDMS channel which adsorbs enzymes was modified by adding 2.5% polyvinylpyrrolidone (PVP) to the PCR mixture. The effect of flow rate in the CF chip on DNA amplification was examined and its PCR efficiency was compared with that of conventional PCR. The microchip requires only 8–30 min to produce detectable amounts of PCR products as determined by slab gel separation, whereas a conventional thermocycler requires 90–150 min over 30 amplification cycles to achieve the same result.
Keywords: Adsorption; DNA; Bioreactions; Heat transfer; Continuous-flow (CF) PCR; Polyvinylpyrrolidone (PVP);

Colony lift immunoassay utilizing antibody-coupled liposomes encapsulating HRP by Yoichi Kumada; Masumi Maehara; Naoki Minami; Masao Nogami; Shigeo Katoh (98-102).
The liposome immunoblotting assay was applied to colony lift immunoassay for detection of colonies expressing target protein (scFv) with higher signal intensity. Only edges of blotted samples on the membrane were developed by a direct blotting method, because cells adsorbed on the surface of the membrane plugged openings of pores and physically prevented interaction between the antigen and immunoliposomes. By an indirect blotting method, uniform signals with higher density were successfully observed from blotted membranes by use of immunoliposomes. Immunoliposome of large unilamellar vesicles (immuno-LUVs) showed the highest S/N ratio among three kinds of liposomes. In the colony lift imunoassay utilizing immuno-LUVs, the signal density was three times higher, compared with the conventional assay, while their background levels were almost same. Consequently, a higher S/N ratio could be attained for detection of colonies secreting active scFv. Thus, this method is useful to screen colonies expressing target protein with higher sensitivity.
Keywords: Immunoliposomes; Blotting assay; Colony lift immunoassay;

We have developed a new strategy to construct a colorimetric biosensor on a miniaturized platform. Polydiacetylene (PDA), which is widely known to change color by external stimuli, was employed as a sensing material. To construct a gel-based portable colorimetric biosensor using PDA, PDA was incorporated into poly(ethylene glycol) (PEG)-based hydrogel upon photopolymerization and PDA-embedded PEG hydrogel matrix was patterned within PDMS-based microfluidic channels. The resulting intrachannel hydrogel matrix containing PDA was reacted with α-cyclodextrin as a model target sample, which causes a color change of PDA from blue to red. Parallel analyses were performed inside microchannels for multiple solutions containing different amounts of α-cyclodextrin and color change gradients were successfully demonstrated depending on the contacting time and amount of α-cyclodextrin.
Keywords: Biosensors; Scale-down; Polydiacetylene; Hydrogel; Immobilization; Optimization;

Simulation and real-time monitoring of polymerase chain reaction for its higher efficiency by Ji Youn Lee; Hee-Woong Lim; Suk-In Yoo; Byoung-Tak Zhang; Tai Hyun Park (109-118).
Polymerase chain reaction (PCR) is an important molecular biological tool for the amplification of nucleic acids. PCR process can be divided into three phases according to the amplification rate: exponential, quasi-linear, and plateau. We investigated the cause of the plateau phenomenon through real-time monitoring of the amplification profile and computerized simulation. Possible limiting components, such as Taq DNA polymerase, primer pair, and dNTPs were added during quasi-linear phase, after which the differences in the amplification profiles were monitored. Modeling and computerized simulations were performed to look into the complex mechanism of the reactions, such as renaturation of templates during temperature transition from denaturation to annealing step and effective enzyme concentration profiles within the cycle progress. The decrease of effective polymerase concentration due to heat inactivation and product accumulation caused the PCR plateau. Addition of polymerase during the quasi-linear phase could increase the final product amount; however, the PCR process still reached the plateau phase in spite of polymerase addition. Simulation results suggest that renaturation of templates before competitive annealing reaction and decrease of effective enzyme concentration by non-specific binding of polymerase to double-stranded DNA is the main contribution to plateau forming.
Keywords: Bioreactions; DNA; Enzyme inactivation; Modeling; Polymerase chain reaction (PCR); Plateau;

Detection of Escherichia coli in the sewage influent by fluorescent labeled T4 phage by Kazuhiko Miyanaga; Tomonori Hijikata; Chiaki Furukawa; Hajime Unno; Yasunori Tanji (119-124).
For a precise estimation of sanitary condition, Escherichia coli detection system using E. coli-specific bacteriophage T4 was constructed. To facilitate E. coli detection, T4e phage which did not produce the lysozyme was constructed and green fluorescent protein (GFP) was displayed on T4e small outer capside (SOC) protein. This T4e/GFP can detect E. coli K12 without cell lysis. In this study, we applied T4e/GFP to the detection of E. coli in the sewage influent.Chromocult® coliform (CC) agar plates are generally used for simultaneous detection of total coliforms and E. coli. We investigated the number of coliforms and E. coli in the municipal sewage influent by using CC agar plates for 1 year. There were 105–106  CFU/ml of total coliforms and 104–105  CFU/ml of E. coli throughout the year. More than 20 strains of E. coli selected from CC agar were infected by T4e/GFP. The ratio of clear plaque forming E. coli was different every month and very low (annual average 8%). Most of the E. coli showed turbid plaque or no plaque. E. coli formed the turbid plaque showed no-fluorescence, fluorescence only on cell surface due to phage adsorption, or heterogeneous fluorescence among the cells, while E. coli formed clear plaque could be detected because of T4e/GFP amplification in the cell.
Keywords: Escherichia coli; Fluorescent labeled phage; Sewage influent; Chromocult® coliform agar;

Dependence upon ionic strength in the immobilization of probing oligonucleotides onto streptavidin-modified probe surfaces by Ryujiroh Yamasaki; JongMin Kim; HoSub Jung; HeaYeon Lee; Tomoji Kawai (125-128).
For the production of reliable DNA chip systems, it is imperative that the probe DNA be properly immobilized upon the electrode surface. The ionic strength in solution is the one of the most important factors for the proper immobilization of DNA. We have observed the dependence upon ionic strength (I) in buffer solution by quartz crystal microbalance (QCM) when probe ssDNA molecules were immobilized upon gold electrode surfaces by utilizing the characteristics of streptavidin–biotin interaction. When utilizing a Tris–HCl buffer solution with I  = 0.01, biotinylated probe ssDNA was not successfully immobilized upon the streptavidin-modified gold surface. Conversely, when a combination of Tris–HCl and TBS buffer with I  = 0.16 was utilized; we were successful in immobilizing biotinylated probe ssDNA upon the streptavidin-modified surface. This is a strong indication that ionic strength in buffer solution is an important factor in the stability of the immobilization process of biomolecular reagents onto the gold electrode surface.
Keywords: Streptavidin monolayer; Streptavidin–biotin interaction; QCM; Quantitative analysis; Gene hybridization;

A kinetic study on the aggregation behavior of β-amyloid peptides in different initial solvent environments by Steven S.-S. Wang; Ya-Ting Chen; Po-Han Chen; Kuan-Nan Liu (129-138).
β-Amyloid peptide (Aβ) is the major proteinacious constituent of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. This work is aimed at determining the effect of solvent environment on the aggregation kinetics of Aβ peptides. Prior to dilution into phosphate buffer saline, we have used three different initial solvent systems, 0.1% (v/v) trifluoroacetic acid in deionized water, 100% (v/v) dimethylsulfoxide, and 8 M urea to solubilize Aβ peptides. Our research shows that the increase in ThT fluorescence intensity or absorbance elicited by aggregated species of Aβ peptides exhibited a solvent environment-dependent behavior. Results from Aβ aggregation in trifluoroacetic acid–phosphate buffer saline and dimethylsulfoxide–phosphate buffer saline systems suggested the involvement of the seeding effect. Moreover, with the aid of three proposed reaction schemes, the effect of this solvent environment-dependent behavior was quantitatively analyzed. We believe that a better understanding of how Aβ species and its derivatives aggregate/self-assemble will shed light on the design and analysis of future work in this area.
Keywords: Alzheimer's disease; β-Amyloid; Aggregation; Kinetics; Solvent;

Influence of hypothermic conditions on primary porcine hepatocyte-entrapped hollow fiber bioreactors by Chi-Chang Lin; Kuo-Chen Hung; Tai-Horng Young; Yaw-Sen Chen; Chee-Chien Yong; Eiji Kobayashi; Chien-Hung Wu; Chin-Hsiung Yang; Chao-Long Chen; Chih-Chi Wang (139-148).
Transportation of bioartificial liver (BAL) device with viable cells and higher metabolic functions are necessary. The aim of this study is to evaluate hypothermic (4 °C) influence on the performance of hepatocyte-entrapped hollow fiber bioreactors, as well as the effects on cell function with or without medium supplement. Oxygen consumptions were stable at average of 15–25 mmHg per cartridge during 8 h normothermic incubation after cold-perfusion and cold-non-perfusion. All groups showed increase in glutamic oxaloacetic transaminase (GOT) and lactate dehydrogenase (LDH) level. Urea production and albumin synthesis were only slightly higher in cold-perfusion groups than that of the cold-non-perfusion groups and the control. There were no significant differences in the metabolic functions of bioreactors between each study groups during normothermic operation. Our study suggests that the use of a preliminary cold-storage step prior to normal culture condition or clinical therapy can prolong the transportation time without changing various functions of the BAL device.
Keywords: Porcine hepatocytes; Cold storage; Hypothermic; Bioartificial liver;

Culture and characterization of human hepatocytes isolated from hepatitis C virus infected liver: Effect of collagen-coated surface and FBS-supplemented medium by Chih-Chi Wang; Chi-Chang Lin; Tai-Horng Young; Yaw-Sen Chen; Chee-Chien Yong; Eiji Kobayashi; Chien-Hung Wu; Chin-Hsiung Yang; Chao-Long Chen; Kuo-Chen Hung (149-156).
The aim of this study is to maintain and prolong the viability and metabolic functions of hepatocytes isolated from hepatitis C virus (HCV)-infected human livers. Collagen-coated surface and 10% fetal bovine serum (FBS) supplemented culture media were studied. Metabolic functions and attachment ability of human hepatocytes in these conditions were compared with normalized conditions (Petri dish, medium without FBS). Our results showed that collagen-coated dishes with 10% FBS-supplemented culture medium increased urea production and albumin synthesis. Urea concentration remained stable (30 pg/cell/h) during the first 3-days when human hepatocytes were cultured in collagen-coated dishes. These hepatocytes showed higher urea production when compared with hepatocytes cultured in normal Petri dishes producing 36.4 and 31.7 pg/cell/h concentrations of urea, respectively. Albumin secretion rate was maximal on day 3 after plating (0.86 pg/cell/h). When using culture medium supplemented with 10% FBS, the metabolic functions of hepatocytes were higher compared to when using normal culture medium. This study demonstrates that human hepatocytes can be isolated from HCV-infected livers via surgical biopsies with high cell yield rate, and their metabolic functions remain stable when cultured in collagen-coated dishes with FBS-rich medium for several days. Base on these results, HCV-infected human hepatocytes can be used in the study of clinical diseases and in the performance of physiological and pharmacological liver experiments in vitro.
Keywords: Collagen; Hepatitis C virus; Human hepatocytes; Fetal bovine serum;

Degradation of 2,4-dinitrophenol by a mixed culture of photoautotrophic microorganisms by Takashi Hirooka; Hiroyasu Nagase; Kazumasa Hirata; Kazuhisa Miyamoto (157-162).
2,4-Dinitrophenol (2,4-DNP) is frequently used as a starting material in the production of pesticides, herbicides, and dyes. This compound is toxic to animals, plants, and microorganisms because it inhibits respiration in mitochondria and photophosphorylation in chloroplasts. Anabaena variabilis showed a high ability to remove 2,4-DNP in the concentration range of 5–150 μM under continuous light. However, 2-amino-4-nitrophenol (2-ANP), which is a degradation product of the 2,4-DNP removal by A. variabilis, accumulated in the culture. Although 2,4-DNP was completely removed when incubation was with a light and dark cycle, accumulation of 2-ANP was observed. By screening various microalgae and cyanobacteria for their ability to remove 2-ANP, we found that Chlamydomonas reinhardtii and Anabaena cylindrica showed the highest ability. However, a high concentration of 2-ANP remained when C. reinhardtii was cultivated with A. variabilis. On the other hand, a mixed culture of A. variabilis and A. cylindrica could remove 2,4-DNP completely, and only a low concentration of 2-ANP was detected compared to a culture of A. variabilis. Therefore, a cyanobacterial mixed culture of A. variabilis and A. cylindrica is expected to be useful for the removal of 2,4-DNP from industrial wastewater.
Keywords: 2,4-Dinitrophenol; 2-Amino-4-nitrophenol; Anabaena variabilis; Anabaena cylindrica; Mixed culture;

Effect of environmental factors on performance of immobilized consortium system for degradation of carbendazim and 2,4-dichlorophenoxyacetic acid in continuous culture by Hiroyasu Nagase; Anchana Pattanasupong; Eiko Sugimoto; Katsuji Tani; Masao Nasu; Kazumasa Hirata; Kazuhisa Miyamoto (163-168).
As a model process for bioaugmentation, a microbial consortium obtained from soil of paddy fields was immobilized on loofa sponge. The immobilized consortium had the ability to degrade carbendazim (methyl-2-benzimidazole carbamate; MBC) and 2,4-dichlorophenoxyacetic acid (2,4-D), which are fungicide and herbicide, respectively, used in paddy fields in large quantities and are often detected from environmental water, during the 2-month operation period using a column type reactor for the continuous treatment of runoff water from paddy fields. For success of bioaugmentation, it is important to investigate the effect of environmental factors on degradation ability for hazardous chemicals. The degradation ability of the immobilized consortium for MBC and 2,4-D was also maintained at various flow rates (the mean residence time of up to 0.1 day), and at low concentrations of MBC, 2,4-D, ammonium, and phosphate estimated condition of runoff water. The degradation ability was also stably maintained up to at least 6 months by simple preservation methods. From these results, valuable information to establish the treatment system for actual environmental conditions was obtained. This study may be useful as a model research for solving problems of pollution by hazardous organic compounds.
Keywords: Environmental factor; Consortium; Carbendazim; 2,4-Dichlorophenol (2,4-D);