Biochemical Engineering Journal (v.44, #2-3)

BEJ Keywords (II).

An unstructured mathematical model is proposed to describe the fermentation kinetics of growth, lactic acid production, pH and sugar consumption by Lactobacillus plantarum as a function of the buffering capacity and initial glucose concentration of the culture media. Initially the experimental data of L. plantarum fermentations in synthetic media with different buffering capacity and glucose were fitted to a set of primary models. Later the parameters obtained from these models were used to establish mathematical relationships with the independent variables tested. The models were validated with 6 fermentations of L. plantarum in different cereal-based media. In most cases the proposed models adequately describe the biochemical changes taking place during fermentation and are a promising approach for the formulation of cereal-based probiotic foods.
Keywords: Lactobacillus plantarum; Probiotic; Mathematical models; Cereal-based media; Buffering capacity; Sugar concentration;

Mycophenolic acid production in solid-state fermentation using a packed-bed bioreactor by Faiez Alani; Jason A. Grove; William A. Anderson; Murray Moo-Young (106-110).
Mycophenolic acid (MPA) was produced from Penicillium brevicompactum by solid-state fermentation (SSF) using pearl barley, and submerged fermentation (SmF) using mannitol. It was found that SSF was superior to SmF in terms of MPA concentration (1219 mg/L vs. 60 mg/L after 144 h fermentation), and the product yields were 6.1 mg/g pearl barley for SSF and 1.2 mg/g mannitol for SmF. The volumetric productivities were 8.5 and 0.42 mg/L h for SSF and SmF, respectively.The optimum solid substrate of SSF for MPA production was pearl barley, producing 5470 mg/kg compared with wheat bran (1601 mg/kg), oat (3717 mg/kg) and rice (2597 mg/kg). The optimum moisture content, incubation time and inoculum concentrations were 70%, 144 h and 6%, respectively. Neither the addition of mannitol or (NH4)2HPO4 nor adjustment of media pH within the range of 3–7 significantly enhanced MPA production.MPA production by SSF using a packed-bed bioreactor was performed and an increased maximum production of MPA 6.9 mg/g was achieved at 168 h incubation time. The higher volumetric productivity and concentrations makes SSF an attractive alternative to SmF for MPA production.
Keywords: Penicillium brevicompactum; Mycophenolic acid; Solid-state fermentation; Pearl barley; Packed-bed bioreactor;

Recovery and partial purification of penicillin G acylase from E. coli homogenate and B. megaterium culture medium using an expanded bed adsorption column by L.M. Pinotti; L.P. Fonseca; D.M.F. Prazeres; D.S. Rodrigues; E.R. Nucci; R.L.C. Giordano (111-118).
The adsorption of penicillin G acylase (PGA) from B. megaterium and from Escherichia coli on a cationic resin, Streamline SP XL, was studied using both packed and expanded beds. Stability assays showed that penicillin acylases from the two sources presented high irreversible deactivation at pH 4.0 and 4.5, but remained stable at pH 4.8. Adsorption experiments performed in a packed bed (PB), in the pH range 4.8–5.8, showed highest adsorption yields at pH 4.8, for both enzymes. Using small expanded bed adsorption (EBA) columns, PGA was directly recovered and partially purified from E. coli crude extracts, E. coli homogenates, and from B. megaterium centrifuged broth in a single unit operation. Global recovery yields of 91.0, 55.0 and 7.4% and purification factors of 4.5-, 7.5- and 12.7-fold were achieved, respectively. The elution yields of penicillin acylase obtained with these cationic EBA processes when working with E. coli homogenate and B. megaterium centrifuged medium were of 100 and 52%, respectively. The comparison of adsorption capacities of E. coli penicillin acylase from crude extracts onto Streamline SP XL showed similar results for packed-bed and for expanded-bed modes. However, PGA adsorption yields for E. coli (homogenate) and B. megaterium (centrifuged medium) were substantially lower than the values obtained for E. coli crude extract, due to the competition of cell debris and other components present in the B. megaterium medium.
Keywords: B. megaterium; E. coli; Enzymes; Purification; Adsorption; Process integration;

Optimization of culture conditions for violacein production by a new strain of Duganella sp. B2 by Haisheng Wang; Peixia Jiang; Yuan Lu; Zhiyong Ruan; Ruibo Jiang; Xin-Hui Xing; Kai Lou; Dong Wei (119-124).
Duganella sp. B2 isolated from Xingjiang Municipality of China was found to be a novel violacein-producing bacterium. In this study, culture variables affecting violacein production were optimized statistically in shake flask experiments. The medium pH, culture volume, and medium components of potassium nitrate, l-tryptophan, and beef extract were identified by two-level Plackett–Burman design as the most significant factors for violacein production. The path of steepest ascent was undertaken to determine the optimal region of five significant factors. Box–Behnken design and response surface analysis were adopted to further investigate the interaction between the variables and determine the optimal values for violacein production. The optimal culture conditions for violacein production were: pH 6.71, potassium nitrate 1.18 g/L, ammonium ferrous sulfate 0.08 g/L, dipotassium hydrogen phosphate 0.25 g/L, magnesium sulfate 0.75 g/L, beef extract 1.53 g/L, l-tryptophan 0.74 g/L, soluble starch 13.0 g/L, 25 mL medium in a 250 mL flask, with an inoculum size of 10% (v/v). 1.62 g/L crude violacein was achieved using the optimized conditions after 32 h of cultivation. This proved to be much higher than any other violacein-producing strains so far reported.
Keywords: Response surface analysis; Optimization; Violacein; Duganella sp. B2; Fermentation; Biosynthesis;

The combined production of biomass, hyaluronic acid (HA) and lactic acid (LA) in a glucose fed-batch system was studied. The complex culture media used were formulated with commercial and residual peptones from fish by-products. In all cases, fed-batch fermentations increased the productive period of HA and LA. Tryptone led to the highest productions but with the peptones from shark by-products similar LA concentrations and prominent HA levels were reached. Moreover, with this residual peptone higher molecular weight of HA were achieved. On the other hand, the equations proposed adjusted with accuracy and high statistical robustness the experimental kinetic profiles.
Keywords: Streptococcus zooepidemicus; Hyaluronic acid; Lactic acid; Marine peptones; Logistic equation;

Optimization of illumination time for the production of methane using carbon felt fluidized bed bioreactor in thermophilic anaerobic digestion by Yingnan Yang; Kenichiro Tsukahara; Zhenya Zhang; Norio Sugiura; Shigeki Sawayama (131-135).
This paper reports the effects of illumination on methanogenic performance using acetate synthetic wastewater as the organic substrate. Several different illumination times such as 0 (continuous darkness), 15, 30, 45, 60, 90, 150, 240 and 480 min/day and continuous light were examined in a laboratory set-up under batch conditions. The reactors, when exposed to short illumination times such as 60 min/day, showed the greatest methane concentration, methane yield, and dissolved organic carbon (DOC) removal, which were 78.5%, 176 ml/g-VS, and 75.8%, respectively, in comparison to other conditions. Furthermore, the performances of three fluidized bed reactors operated in semi-continuous mode under conditions of continuous darkness, 60 min/day illumination, and continuous illumination were investigated. The methane yield and DOC removal was 794 ml/g-DOC added, 893 ml/g-DOC added, and 596 ml/g-DOC added, and 88.3%, 93.7%, and 60.5%, respectively. In the present investigation, illumination for 60 min/day for microbial treatment was found to be more effective than either continuous darkness or continuous illumination. Molecular and microscopic study revealed that the major methanogens in the bed reactors belonged to the genus Methanosarcina sp. Immobilized cells from the short-time illumination and continuous darkness reactors show a smooth surface membrane. However, an uneven surface membrane and destruction of cell tissue were observed in the reactor with continuous illumination.
Keywords: Illumination time; Methane production; Fluidized bed bioreactor; Thermophilic anaerobic digestion; Methanogens;

Previously, we isolated caffeine degrading Pseudomonas strain from soil of coffee plantation area, which could utilize caffeine as sole carbon and nitrogen source and could tolerate caffeine up to 20 g/L. In this study, caffeine degradation by immobilized cells of this strain was investigated. Various matrices were considered and agar–agar was chosen based on degradation rate (0.08 g/(L h)), bead stability and reusability. Further, immobilization parameters, viz., bead size (mm), agar–agar concentration % (w/v) and cell concentration (g/L) were optimized using central composite design. The optimal conditions of cell concentration, agar–agar concentration and bead size were 7.8 g/L, 5% (w/v) and 6.2 mm. Under optimal conditions, caffeine degradation rate was found to 0.15 g/(L h), which closely agrees with the model predicted values. This is the first report on caffeine degradation at high concentrations (10 g/L) by immobilized cells of Pseudomonas sp. Immobilization efficiency was 80%. Damköhler number is very much higher than 1, suggesting that mass transfer is the rate limiting process.
Keywords: Biodegradation; Immobilization; Immobilized cells; Kinetic parameters; Submerged culture; Diffusion reaction;

QCM-based DNA biosensor for detection of genetically modified organisms (GMOs) by İrem Karamollaoğlu; Hüseyin Avni Öktem; Mehmet Mutlu (142-150).
Development of a mass sensitive quartz crystal microbalance (QCM)-based DNA biosensor for the detection of the hybridization of CaMV 35S promoter sequence (P35S) was investigated for the screening of genetically modified organisms (GMOs). Attention was focused on the choice of the coating chemistry that could be used for the immobilization of probe sequences on the gold surface of the quartz crystal. Two immobilization procedures were tested and compared considering the amount of the immobilized P35S probe and the extent of the hybridization reaction with the target oligonucleotide. In wet chemistry procedure, the interaction between the thiol and gold for the immobilization of a thiolated probe was employed. Direct surface functionalization of piezoelectric quartz crystals were achieved in 13.56 MHz plasma polymerization reactor utilising ethylenediamine (EDA) precursors for the immobilization of amined probes. Results indicated that immobilization of a thiolated probe provides better immobilization characteristics and higher sensitivity for the detection of the hybridization reaction. The thiolated probe was used for the detection of P35S sequence in PCR-amplified DNAs and in real samples of pflp (ferrodoxin like protein)-gene inserted tobacco plants. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and ultrasonication. The results obtained from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM-based DNA biosensor system. The developed QCM-based DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of GMOs.
Keywords: DNA-based biosensors; Genetically modified organisms (GMOs); CaMV 35S promoter; Immobilization; Plasma polymerization; Pflp (ferrodoxin like protein)-gene inserted tobacco plants;

Surface chemistry evaluation of some solid wastes from olive-oil industry used for lead removal from aqueous solutions by M.A. Martín-Lara; F. Hernáinz; M. Calero; G. Blázquez; G. Tenorio (151-159).
In this paper three different olive oil wastes used for heavy metal removal from aqueous solutions: olive stone (OS), the two-phase olive mill solid (OMS) and olive tree pruning (OTP) were compared about chemical characterization and lead biosorption capacities.A detailed description of the surface acidity/basicity of all the samples was studied by continuous potentiometric titrations. The acid–base properties of the solids were well described by simplified chemical equilibrium models, in terms of discrete sites and continuous distributions. That study evidenced that carboxylic group is the main active site present in these olive oil wastes and constitutes the highest percentage of titratable sites (greater than 50%).The Langmuir isotherm model was used to characterize the interaction of Pb(II) ions with the olive oil wastes and a realistic uptake mechanism which can be used to assess and predict the binding (adsorption) capacity of the materials studied was formulated.
Keywords: Biosorption; Adsorbents; Heavy metals; Waste treatment; Acid–base properties; Mechanism of biosorption;

Hypothermic preservation of hepatocytes has been widely studied for potential storage and transportation of bioartificial liver (BAL), but the liver-specific functions of hepatocytes are severely impaired by hypothermic treatment. A miniaturized gel entrapment-based BAL without circulation system was used to screen protectants from Chinese herbal medicines in this paper. Although anisodamine (ANI), matrine (MAT) and schisandrin B (Sch B) individually enhanced, to some extent, cell viability and liver-specific functions of hypothermically preserved hepatocytes, glycyrrhizic acid (GA), performed superior to these three extracts. The multieffect of GA on enhancement of mitochondrial membrane potential and inhibition of oxidative stress as well as lipid accumulation might determine its protection on hepatocytes from hypothermia-induced cell death. Furthermore, cell viability and intracellular glutathione (GSH) content decreased more dramatically at 6 h of the rewarming compared to those immediately after hypothermic preservation, indicating the aggravated cell injury by rewarming treatment. Therefore, gel entrapped hepatocytes in this study could be proposed for the throughput screening of desired conditions for hypothermic preservation of BAL.
Keywords: Hypothermic preservation; Rat hepatocytes; Bioartificial liver (BAL); Gel entrapment culture; Chinese herbal medicine; Glycyrrhizic acid (GA);

Catalase encapsulated in mesoporous silica and its performance by Tetsuji Itoh; Ryo Ishii; Shun-ichi Matsuura; Satoshi Hamakawa; Takaaki Hanaoka; Tatsuo Tsunoda; Junko Mizuguchi; Fujio Mizukami (167-173).
Catalase was confined in a nanospace of about 7 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a catalase-FSM conjugate. That the enzyme was encapsulated in the pores of mesoporous materials was proven by the direct visualization technique. The catalase-FSM showed not only an activity similar to that of the native catalase in the decomposition of H2O2 but also much higher stability: specifically, the catalase immobilized in FSM still retained its original activity after being applied 50 times to the decomposition, whereas the native lost its activity after 20 times recycling. Furthermore, the catalase-FSM showed catalase activity even in an organic media in which the native catalase had almost no catalytic activity.
Keywords: Catalase; Mesoporous silica;

Performances of various bioreactors under different operating conditions were evaluated with respect to hexavalent chromium (Cr(VI)) reduction and COD removal. Continuous reactor studies were carried out with (i) aerobic suspended growth system, (ii) aerobic attached growth system, and (iii) anoxic attached growth system, using both synthetic and actual industrial wastewater. Arthrobacter rhombi-RE (MTCC7048), a Cr(VI) reducing strain enriched and isolated from chromium contaminated soil, was used in all the bioreactors for Cr(VI) biotransformation and COD removal. Aerobic and anoxic batch experiments were conducted to evaluate the bio-kinetic parameters. The bio-kinetic parameters for aerobic system were: μ max  = 2.34/d, K s  = 190 mg/L (as COD), K i  = 3.8 mg/L of Cr(VI), and Y T  = 0.377. These parameters for anoxic conditions were: μ max  = 0.57/d, K s  = 710 mg/L (as COD), K i  = 8.77 mg/L of Cr(VI), and Y T  = 0.13. Aerobic attached growth system, operated at a hydraulic retention time (HRT) of 24 h and an organic loading rate (OLR) of 3 kg/m3/d, performed better than aerobic suspended and the anoxic attached growth systems operated under identical conditions, while treating synthetic wastewater as well as industrial effluent.
Keywords: Arthrobacter rhombi-RE; Cr(VI) reduction; COD removal; Kinetic parameter; Bioreactor;

Strain XA05 and FG03 with high biodegradation activity of phenol were isolated from the activated sludge and phenol-contaminated soils in Northwest of China, respectively. DNA sequencing and homologous analysis of 16s rRNA gene identified that XA05 belonged to an Acinetobacter sp. and FG03 was closely related to the Sphingomonas sp. Strain XA05 and FG03 were mixed at the ratio of 1:1, and polyvinyl alcohol (PVA) was used as a gel matrix to immobilize mixed cells of two strains by repeated freezing and thawing. The removal efficiency of phenol by free and immobilized cells and factors affecting phenol degradation were investigated, the stability of the immobilized cells was also reported. Experimental values indicated that both free suspended and immobilized cells showed high phenol degradation efficiencies, higher than 95% within 35 h with an initial concentration of 800 mg/l phenol, and the immobilized cells showed better performance than that of the suspended cells.
Keywords: Phenol biodegradation; Immobilized cells; Free suspended cells; Acinetobacter sp. XA05; Sphingomonas sp. FG03;

In this study we examined the influence of the treatment time and addition of oxygen on the efficiency of a laccase mediator system (L) applied to flax pulp at atmospheric pressure. The redox potential and the dissolved oxygen concentration during L tests are measured. After L stage, an alkaline extraction (E) is carried out. The pulp properties (kappa number, brightness and viscosity) and the effluents properties (color and COD) were measured in order to evaluate the environmental impact of this enzymatic treatment.The biotreatment involves two distinct stages in both L and LE sequences; in the beginning the pulp exhibits a fast delignification and a slow viscosity decrease that is followed by slow delignification in the second. Pulp brightness changed differently during L stage and LE sequence. Initially brightness after the L stage decreased with respect to the initial pulp; then, it increased rapidly and eventually leveled off. After the LE sequence, brightness increased rapidly in the beginning and more gradually afterwards. The results show that supplying the medium with oxygen and increasing the oxygen concentration in it, influence the kinetics of the process.Based on CIE L*a*b* color coordinates study, the enzyme treatment not only removes lignin, but also alters the structure of the pulp by causing the formation of chromophoric groups giving color. Such groups are removed in an E stage.
Keywords: Delignification; Effluents; Flax pulp; 1-Hydroxybenzotriazole; Laccase; Optical properties; Oxygen; Reaction time; CIE L*a*b* coordinates; COD; Color; Viscosity; Chromophore;

Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl-beta-cyclodextrin (MbCD)) on the production of Molony murine leukemia virus (MoMuLV) retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than 2-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors.
Keywords: Lipid rafts; Cholesterol; Infectivity; Retroviral vector; Lentiviral vector; Viral vector production;

A comparative study of microbial reduction of nitrate ions by immobilized Pseudomonas denitrificans cells in the presence and in the absence of a constant electric field was carried out. The experiments were carried out in a continuous stirred tank reactor at a broad range of dilution rates. The cathode potential was maintained constant around the standard potential value of the nitrate/nitrite redox couple. The residual outlet concentrations of nitrate, when the electric field was applied, were much lower than those obtained without the electric field, all other conditions being equal. It was demonstrated that the electric field had a significant effect on the nitrate reduction rate allowing the use of much higher inlet flow rates compared to the flow rate in the absence of an electric field.
Keywords: Bioremediation; Denitrification; Stirred tank bioreactor; Continuous process; Immobilized cells; Constant electric field;

Influence of the operating conditions on lipase-catalysed synthesis of ricinoleic acid estolides in solvent-free systems by A. Bódalo; J. Bastida; M.F. Máximo; M.C. Montiel; M.D. Murcia; S. Ortega (214-219).
In this work, the synthesis of ricinoleic acid estolides, also named polyricinoleic acid (PR), in two different solvent-free reaction systems, catalysed by immobilised Candida rugosa lipase is described. First, the esterification was performed in an open air jacketed batch reactor and the influence of amount of enzyme, temperature and initial water content was tested. The best results were obtained when 5 g of soaked immobilised derivative was used as biocatalyst, and the reaction was carried out at 40 °C. It was observed that environmental relative humidity plays an important role in the enzymatic synthesis of ricinoleic acid estolides and, given that this parameter takes on a wide range of values depending on the season, it is considered the main cause of the poor reproducibility achieved in the open air reactor. To solve this problem, the ricinoleic acid estolides were synthesised under controlled atmosphere in a vacuum reactor with dry air intake. The optimum drying time of 7 h was selected. In these conditions, PR with an acid value of 57.5 mg KOH/g was synthesised in 48 h of reaction and the results were entirely reproducible.
Keywords: Enzyme; Immobilised; Bioreactor systems; Stirred tank; Ricinoleic acid estolide; Solvent-free;

In this study, the methyl esters of the long chain fatty acids (biodiesel) were synthesized by methanolysis of canola oil by immobilized lipase. Lipase from Thermomyces lanuginosus was immobilized by both physical adsorption and covalent attachment onto polyglutaraldehyde activated styrene–divinylbenzene (STY–DVB) copolymer, which is synthesized by using high internal phase emulsion (polyHIPE). Two different STY–DVB copolymers were evaluated: STY–DVB copolymer and STY–DVB copolymer containing polyglutaraldehyde (STY–DVB–PGA). Lipase from T. lanuginosus was immobilized with 60% and 85% yield on the hydrophobic microporous STY–DVB and STY–DVB–PGA copolymer, respectively. Biodiesel production using the latter lipase preparation was realized by a three-step addition of methanol to avoid strong substrate inhibition. Under the optimized conditions, the maximum biodiesel yield was 97% at 50 °C in 24 h reaction. The immobilized enzyme retained its activity during the 10 repeated batch reactions.
Keywords: Biofuels; Immobilized; Biocatalyst preparation; Enzyme; Styrene–divinylbenzene copolymer; Polyglutaraldehyde;

Cineolic essential oils are used in medicinal, perfumery and flavour preparations. 1,8-Cineole, being an ecofriendly compound, has the potential to replace the ozone depleting industrial solvents. Optimized process parameters for hydrodistillation and production of essential oil enriched with 1,8-cineole from Eucalyptus cinerea grown in the mid-hills of western Himalaya, were reported. The effect of drying of the foliage prior to distillation with respect to oil composition and content was studied. The first order kinetic and Langmuir adsorption models were evaluated to simulate hydrodistillation of E. cinerea oil. The Langmuir model parameters that simulate the hydrodistillation process were determined. GCMS analysis revealed that the oil produced from fresh foliage contained higher 1,8-cineole content (84.4%) than the dried foliage (77.6%). The other major constituents were limonene and α-terpineol.
Keywords: Production kinetics; Hydrodistillation; Modelling; Optimization; 1,8-Cineole; Separation;

Trace-level sensing of dopamine in real samples using molecularly imprinted polymer-sensor by Bhim Bali Prasad; Shrinkhala Srivastava; Khushaboo Tiwari; Piyush Sindhu Sharma (232-239).
Induced-fit responsive dopamine (DA) imprinted polymer, poly (melamine-co-chloranil), was used as a suitable coating material for the modification of a hanging mercury drop electrode. The zwitterionic conformation of the imprinted polymer responded differential pulse, cathodic stripping voltammetric current, without any false-positive or false-negative contributions of non-specific sorptions, in aqueous environment of complex matrices. The limit of detection (3σ) of dopamine was found to be as low as 0.148 ng mL−1, by the proposed sensor that could be considered a sensitive marker of dopamine depletion in Parkinson's disease (PD).
Keywords: Bioseparations; Membrane; Mass-transfer; Affinity; Dopamine; Molecularly imprinted polymer;

Effects of arcA and arcB genes knockout on the metabolism in Escherichia coli under aerobic condition by Syed Asif Nizam; Jiangfeng Zhu; Pei Yee Ho; Kazuyuki Shimizu (240-250).
The effects of arcA and arcB genes knockout on the metabolism in Escherichia coli were investigated for the aerobic batch and continuous cultivations based on gene expressions, enzyme activities, and the metabolic flux distributions obtained by 13C-labeling experiment. The integration of the different levels of information indicates that arcB gene knockout activated TCA cycle by gene and enzyme levels regulation as compared with the wild type BW25113 under aerobic condition. The TCA cycle was further activated for arcA gene knockout mutant as compared with arcB mutant. Since the TCA cycle is the source of energy for the cell, its activation together with up-regulation of cyoA gene expression is attractive for the improvement of the cell growth and useful metabolite production, etc. The present research shows the reduction of acetate production in arcA/B mutants, which is the common obstacle for useful metabolite production using E. coli. The activation of TCA cycle together with limited respiratory capacity caused NADH accumulation, which in turn repress the TCA cycle activity by enzyme level regulation. This could be released by adding nicotinic acid.
Keywords: Escherichia coli; arcA and arcB genes knockout; Nicotinic acid; Metabolic regulation;

Application of metabolically engineered Saccharomyces cerevisiae to extractive lactic acid fermentation by Min-Tian Gao; Takashi Shimamura; Nobuhiro Ishida; Haruo Takahashi (251-255).
In this study, Saccharomyces cerevisiae OC-2T T165R, metabolically engineered to produce optically pure L(+)-lactic acid, was used to develop a high performance extractive fermentation process. Since the transgenic yeast could produce lactic acid efficiently even at lower than pH 3.5, high extractive efficiency was achieved when tri-n-decylamine (TDA), a tertiary amine, was used as the extractant. Separation of microorganisms by means of a hollow fiber module could not only improve the total amount of lactic acid produced but also increase the lactic acid concentration in the solvent. Moreover, pH had a significant effect on extractive fermentation. The highest rate of recovery of lactic acid could be obtained on pH-uncontrolled fermentation (pH 2.5); however, the lowest amount of lactic acid was produced. Taking into account the trade-off between the fermentation and extraction efficiencies, the optimum pH value was considered to be 3.5, with which the largest amount of lactic acid was produced and the highest lactic acid concentration in the solvent was obtained. The results show promise for the use of the transgenic yeast for extractive fermentation.
Keywords: Extractive fermentation; Lactic acid; Saccharomyces cerevisiae; Oligomerization;

The effectiveness of lipase immobilized on ceramic beads, in the production of biodiesel from simulated waste cooking oil in organic solvent system, was compared to that of free lipase. Experimental determination of the effect of concentrations of methanol on the rate of the enzymatic transesterification was experimentally determined. In addition, the effectiveness of lipases from bacterial and yeast sources for biodiesel production from simulated waste cooking oil was compared. A kinetic model was developed to describe the system, taking into consideration the mass transfer resistances of the reactants. Inhibition effects by both substrates on the interfacial reaction were also considered. The experimental results were used to determine the kinetic parameters of the proposed model and to determine the effect of mass transfer. On the other hand, it was shown that biodieasel can be produced in considerable amounts, with yield reaching 40%, in absence of organic solvent using immobilized lipase from P. cepacia on ceramic beads.
Keywords: Biodiesel; Immobilized lipase; Mass transfer resistance; Kinetic model;

Mesoporous SBA-15 was synthesized under acidic condition at 40 °C with a non-ionic triblock copolymer (P123) as the template. The synthesis gel composition used was 1 SiO2:0.017 P123:2.9 HCl:202.6 H2O. Functionalization of SBA-15 with 3-aminopropyltriethoxysilane (APTES) by post-synthesis method was performed under reflux for 2 h. The mesoporous samples were characterized using Fourier transform infrared (FT-IR), nitrogen adsorption, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). They were then utilized as supports for the immobilization of lipase to be subsequently used for the esterification of citronellol and lauric acid. Leaching and reusability tests were also conducted on the immobilized enzymes. Functionalization resulted in about 10% improvement in enzyme loading, leading to higher activity. The immobilized enzyme was also more stable to low pH and high temperature while showing better retention (up to 95%) of enzyme molecules. Immobilized lipase maintained 90% of its esterification activity in non-aqueous system even after 4 cycles of use. The improvements were associated with enhanced surface hydrophobicity, changes in pore shapes and stronger enzyme–support interactions with minimal effects to the enzymatic activity.
Keywords: Biocatalyst preparation; Solid-state; Functionalized SBA-15; Enzyme; Immobilized; Citronellyl laurate;

Synthesis of 2-monoacylglycerols (2-MAG) by enzymatic alcoholysis of fish oils using different reactor types by Luis Esteban; María del Mar Muñío; Alfonso Robles; Estrella Hita; María J. Jiménez; Pedro A. González; Belén Camacho; Emilio Molina (271-279).
This paper studies the synthesis of 2-monoacylglycerols (2-MAGs) by alcoholysis of cod liver oil and tuna oil, catalyzed by lipases, in stirred tank (STR) and packed bed (PBR) type reactors, operating in discontinuous and continuous modes. Firstly, several lipases were tested (DF from Rhizopus oryzae, Palatase 20000L from Mucor miehei and Novozym 435 from Candida antarctica), and although the highest 2-MAG yield was obtained with lipase DF, Novozym 435 was selected due to its greater stability. 2-MAGs were then produced with this lipase in the above-mentioned reactors. Using Novozym 435 2-MAGs can be obtained by ethanolysis of TAGs, and the major operational variable is the treatment intensity (TI, lipase amount × reaction time/oil amount). The highest 2-MAG yields (63–65%) were obtained in the STR operated in discontinuous mode. For TI of over approximately 1 g lipase × h/g oil, the 2-MAGs were degraded to glycerol. This system was scaled up to 100 times the initial volume, achieving the same yield at the same TI (1 g lipase × h/g oil). Operating in continuous mode, the 2-MAG yields obtained (53–54%) were roughly 15% less in both reactors at this TI. 2-MAGs in the final reaction mixture were separated from the ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); 2-MAG recovery yield (g 2-MAGs extracted/100 g of 2-MAGs in the reaction mixture) and 2-MAG purity in the target product (g 2-MAGs/100 g of total product) were approximately 90%. The fatty acid profile of the 2-MAGs produced was similar to the fatty acid profile in position 2 of the original oils. The two major n-3 polyunsaturated fatty acids (n-3 PUFAs) of 2-MAGs produced were eicosapentaenoic acid (EPA) and docosohexaenoic acid (DHA), and their combined contents were about 40% and 45% for cod liver oil and tuna oil, respectively.
Keywords: Bioreactors; Enzyme; Packed bed reactor (PBR); Stirred tank reactor (STR); 2-Monoacylglycerol (2-MAG); Alcoholysis; Extraction;

Model-based evaluation of cell retention by crossflow ultrafiltration during fed-batch fermentations with Escherichia coli by Maria C. Cuellar; Tiemen W. Zijlmans; Adrie J.J. Straathof; Joseph J. Heijnen; Luuk A.M. van der Wielen (280-288).
Fermentations with cell retention by membrane technology offer a means to increase fermentation productivity. In these systems, fermentation and membrane filtration interact with each other, and consequently, these two operations should be evaluated simultaneously. In this work we performed glucose-limited fermentations with Escherichia coli using an ultrafiltration membrane for cell retention. Models were derived to evaluate both fermentation and membrane performance. Concerning the fermentation, a model derived from fermentations without cell retention could describe the system with cell retention properly. This model had correctly assumed that the cell retention system would not affect the fermentation performance. The membrane performance, on the other hand, was well described in short-term experiments, but the long-term effects could not be predicted and the calculated cake mass and particle diameter were unrealistic. Further evaluation of the model and experimental results suggest that other fouling sources besides the bacterial cells – such as medium components and/or intracellular products released to the medium due to cell lysis – might play a more important role in flux decline.
Keywords: Fermentation; Ultrafiltration; Modeling; Escherichia coli; Cell retention; Membrane fouling; Separation;

Refolding of single-chain Fv by use of an antigen-coupled column by Kazutaka Ikeda; Yoichi Kumada; Tomohisa Katsuda; Hideki Yamaji; Shigeo Katoh (289-291).
A single-chain variable fragment (scFv) antibody against bisphenol A was refolded using an antigen (bisphenol A)-coupled column. The refolding efficiency was compared with that used in dialysis. The refolding efficiency of the antigen-coupled column was about 50–60%, which was much higher than with dialysis, due to a decrease in the concentration of the refolding molecules and to the suppression of the aggregate formation.
Keywords: Single-chain Fv; Refolding; Antigen-coupled column; Aggregate;

Development of a practical small-scale circulation bioreactor and application to a drug metabolism simulator by Hiroyuki Ijima; Yasuo Kakeya; Takahiro Ogata; Takanori Sakai (292-296).
We developed an easy-to-use, small-scale circulation-type bioreactor system that enables the simultaneous evaluation of many specimens. Medium flow was generated by a magnetic stirrer in this system. Primary rat hepatocytes formed a monolayer, and there were no morphological differences between cells in circulation and stationary cultures. The mitochondrial activity of hepatocytes in the circulation culture was 23% lower than that in the stationary culture after 2 days of culture. On the other hand, albumin production activity in the circulation culture after 2 days of culture was 1.4 times higher than that in the stationary culture. Albumin production activity per cell in the circulation culture was 1.9 times higher than that in the stationary culture after 2 days of culture. In addition, lidocaine metabolism rate per cell in the circulation culture was 1.3 times higher than that in the stationary culture. The lidocaine clearance of the circulation culture in our circulation-type bioreactor was 1.3 times higher than that of the stationary culture. It was shown that this bioreactor is suitable for the expression of the liver-specific functions of primary rat hepatocytes. Therefore, we can expect that this circulation-type bioreactor system will be a practical drug metabolism simulator.
Keywords: Bioreactor systems; Hepatocyte; Drug metabolism simulator; Immobilized cell; Animal and insect cell culture engineering; Cell engineering;

Deep desulfurization of diesel by integrating adsorption and microbial method by Wangliang Li; Huang Tang; Qingfen Liu; Jianmin Xing; Qiang Li; Dan Wang; Maohua Yang; Xin Li; Huizhou Liu (297-301).
Aiming for the deep desulfurization of diesel, a novel adsorption–bioregeneration system was constructed by combining adsorption and biodesulfurization processes. The sequence of adsorption capacity of DBT (dibenzothiophene) is AC (activated carbon) > NiY > AgY > alumina > 13X. The sequence of selectivity of DBT toward naphthalene is NiY > AgY > alumina ≈ 13X > AC. For hydrotreated diesel, MAS (mesoporous aluminosilicates) showed high adsorption capacity, while MCM-41 and NaY showed low adsorption capacity. The bioregeneration process of these adsorbents was also carried out with P. delafieldii R-8 cells. Adding P. delafieldii R-8 cells can improve DBT desorption from adsorbents. The desorption of DBT from adsorbents by bioregeneration follows the sequence: 13X > alumina > AgY > NiY > AC. Ag-MAS can be completely regenerated in in situ adsorption–bioregeneration system.
Keywords: Desulfurization; Adsorption; P. delafieldii R-8; Bioregeneration; Hydrotreated diesel; Integrating;