Biochemical Engineering Journal (v.30, #1)

BEJ Keywords (IV).

Direct recovery of alcohol dehydrogenase from unclarified yeast cell homogenate by IDEBAC using an improved scheme for elution by Yu-Kaung Chang; Jyh-Ping Chen; Jing-Rong Sheu; Po-Jen Cheng; Chia-Hung Su; Shin-Ying Chou (1-10).
Procion Red HE-7B was directly immobilized onto STREAMLINE matrix to obtain a moderately substituted dye-ligand matrix in which the dye concentration was 6.72 μmol/ml adsorbent. The adsorption characteristics of alcohol dehydrogenase (ADH) onto the sticky dyed adsorbent in the absence and/or presence of yeast cells and cell debris were investigated. The Langmuir-type isotherm model was reasonably used to describe the adsorption behavior. The results showed that the adsorption characteristics and uptake rate for ADH did not change significantly even in the presence of cells and cell debris. Measurements of adsorption breakthrough curves in expanded beds have shown that the maximum dynamic binding capacity appeared to be significantly affected by the aspect ratio. The dynamic binding capacity of the dyed adsorbent was found to be 50.75 U/ml as the aspect ratio was up to 16. Optimal conditions for elution of the adsorbed ADH were determined in small scale packed bed experiments conducted with clarified yeast cell homogenates. The one-step elution was carried out by injecting a pulse of NAD+ (15 mM, one settled bed volume) with 0.3 M NaCl as a carrier, resulting in the adsorbed ADH eluting completely from the sticky dyed adsorbent. This case study on the adsorption/desorption characteristics of the sticky dyed adsorbent for ADH showed that this enzyme could be recovered with a purification factor of 4.6 and yield of 89% by immobilized dye-ligand expanded bed affinity chromatography (IDEBAC) in a single step.
Keywords: Adsorption; Elution; Unclarified yeast cell homogenate; Alcohol dehydrogenase; Immobilized dye-ligand; Expanded bed affinity chromatography;

(S)-Mandelic acid [(S)-MA] was prepared in a one-pot fermentation-transformation process by a (R)-MA-degrading bacterium, Pseudomonas putida ECU1009. Using a sequential simplex optimization method, the best results were obtained with growing cells cultivated on a medium composed of 1.0% (RS)-MA, 0.1% NH4NO3, and 0.1% corn steep liquor. The optimal cultivation conditions were 30 °C and pH 6.5. By fed-batch culture under the optimal conditions, (S)-MA was successfully prepared in 1-L Erlenmeyer flask with 41% isolation yield and >99.9% ee, from totally 3% (RS)-MA after 94 h of biotransformation.
Keywords: (S)-Mandelic acid; Pseudomonas putida; Biodegradation; Fed-batch culture; Process optimization;

Low model order approximations of continuously stirred biofilm reactors with Monod kinetics by Torsten Wik; Elin Göransson; Claes Breitholtz (16-25).
Design of controllers and optimization of plants using biofilm reactors require dynamic models and efficient methods of simulation. Continuously stirred biofilm reactors (CSBRs) are useful model units in modeling a variety of different types of biofilm reactors. Often the reaction kinetics in the biofilm is described by a Monod expression. With standard modeling assumptions the equations describing the fast dynamics of a CSBR will then, for each substrate, be one nonlinear partial differential equation coupled with one linear ordinary differential equation. Here, it is shown how a few nonlinear ordinary first order differential equations, which may be solved with standard integration methods, can be used as a close approximation for both dynamic responses and steady state solutions. These low order models can conveniently be used in simulation software and for controller design.
Keywords: Biofilms; Bioreactors; Control; Dynamic modeling; Dynamic simulation; Wastewater treatment;

Supramolecular-mediated thermostabilization of phenylalanine dehydrogenase modified with β-cyclodextrin derivatives by Reynaldo Villalonga; Shinjiro Tachibana; Roberto Cao; Hector L. Ramirez; Yasuhisa Asano (26-32).
Two different monoactivated β-cyclodextrin derivatives, named mono-6-amino-6-deoxy-β-CD (CD1) and mono-6-(5-carboxypentane-1-carboxamidoyl)-6-deoxy-β-CD (CD2) were evaluated as modifying agents for Bacillus badius phenylalanine dehydrogenase. The enzyme glycosidated with CD1 and CD2 contained about 18 mol and 15 mol oligosaccharide per mol of protein and retained 60% and 81% of the initial activity, respectively. The optimum temperature for the catalytic activity of phenylalanine dehydrogenase was increased in 10 °C after attaching the CDs residues. The enzyme thermostability profile was improved, and its resistance to thermal inactivation at different temperatures ranging from 45 °C to 60 °C was noticeably increased after glycosidation. The activation free energy of thermal inactivation was increased by 16.8 kJ/mol and 12.6 kJ/mol for the enzyme modified with CD1 and CD2, respectively. The influence of supramolecular host–guest associations on the improved thermotolerance showed by the modified enzyme forms was demonstrated by fluorescence spectroscopy and enzymatic measurements.
Keywords: Phenylalanine dehydrogenase; Protein denaturation; Enzyme technology; Amino acids; Enzyme activity; Cyclodextrin;

The effect of calcium on the anaerobic digestion treating swine wastewater by Johng-Hwa Ahn; Trong Hoan Do; Sang D. Kim; Seokhwan Hwang (33-38).
A series of experiments with 0, 1, 3, 5, and 7 g Ca2+/l using calcium chloride were performed to evaluate the effect of various calcium concentrations on anaerobic digestion of swine wastewater. The addition of 3 g/l gave the best performance. On the other hand, the calcium concentrations of 5–7 g/l had an inhibitory effect on anaerobiosis. The lag phase durations expected in biogas production were 28–31 days with the addition of 0–3 g/l of calcium, and 43–52 days for calcium concentrations of 5 g/l or more. The concentrations of total volatile fatty acids decreased to less than 100 mg/l with calcium concentrations of 3–7 g/l. On the other hand, propionate and i-valerate concentrations remained over 4 and 0.8 g/l, respectively, when 1 g/l or less of calcium was added.
Keywords: Anaerobic digestion; Batch; Calcium; Lipid; Swine wastewater; VFAs;

Effect of soxR and soxS genes deletion on the central metabolism of Escherichia coli was investigated by combining the information of fermentation characteristics, gene expressions and enzyme activities. Twenty-one different enzyme activities in the central metabolic pathways were assayed in both mutants and its parent E. coli. Semi-quantitative RT-PCR was performed to detect the transcriptional levels of 79 different genes. The deletion of soxR and soxS genes reduced the specific glucose uptake rate and specific growth rate. On the other hand, acetate production rate was increased significantly in the soxS mutant as compared to that in the parent strain, which is partly due to down-regulation of tricarboxylic acid (TCA) cycle. It was found that the pntA (membrane bound transhydrogenase) transcripts, which is involved in NADPH generation were up-regulated in both soxR and soxS mutants by 2.0-fold. The reason was mainly due to the down-regulation of NADPH generating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in pentose phosphate (PP) pathway, and isocitrate dehydrogenase (ICDH) in TCA cycle, since NADPH plays a significant role to reduce oxidative stress. On the other hand, udhA (soluble transhydrogenase) transcripts which is involved in the reoxidation of NADPH were found to be down-regulated by 0.8-fold in the soxR mutant but up-regulated by 1.7-fold in the soxS mutant compared to that in the parent strain. This result was consistent with the measurement of transhydrogenase activity, indicating that soxR mutant suffered from insufficient reoxidation of reducing power. This caused the slow growth for the soxR mutant. It was also found that the expression levels of cyoA and ndh genes were down-regulated in both mutants, demonstrating that soxR and soxS genes deletion adversely affected respiratory system and electron transport chain.
Keywords: soxR; soxS; Batch culture; RT-PCR;

Preparation and characterization of a temperature-sensitive sulfobetaine polymer–trypsin conjugate by Ming Yan; Jun Ge; Wenguo Dong; Zheng Liu; Pingkai Ouyang (48-54).
A novel temperature-sensitive polymer–trypsin conjugate was prepared by the covalent linking of monodispersed carboxyl-terminated poly(3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate) (PDMAPS) to trypsin. The molar ratio of the polymer to trypsin was 2.4 and its upper critical solution temperature was 14 °C. Fluroscence emission spectroscopy and circular dichroic spectroscopy showed that the conjugated trypsin retained its native conformation. The hydrolysis of N-R-benzoyl-d,l-arginine p-nitroanilide was carried out at different pHs and temperatures using native trypsin and the conjugate, respectively. The optimal pH was 7.8 for native trypsin and 8.0 for the conjugate. Michaelis–Menten kinetics analysis showed that, as compared to the native trypsin, the conjugated trypsin has a smaller K m that decreases with temperature, while the V m of the conjugate was the same. When casein was used to study the catalytic effect of the conjugated trypsin on a high molecular weight substrate, an increase in temperature from 40 to 60 °C gave a 2.6-fold increase in the enzyme activity of the conjugate. The half-life for the enzyme activity at 60 °C was 4.8 min for native trypsin and 315.9 min for the conjugate. The conjugate retained 85% of the initial enzyme activity after 10 cycles of temperature swinging from 4 to 40 °C.
Keywords: Bioconjugate; Trypsin; Temperature-sensitive polymer;

This study proposes an approach to understand biofilm formation in polyphasic anaerobic digestion systems. The objective was to better control biofilm formation in order to reduce the time of colonization during the start-up phase of an anaerobic high-rate biofilm reactor.A three-phase (liquid–gas–solid) inverse turbulent bed reactor (ITBR), with low-density particles used as biomass carrier, was started by stepwise increase of the organic loading rate (OLR) from 0.5 to 20 gCOD  L−1  day−1 while maintaining a COD removal rate of 80%. The reactor was operated at a short and constant hydraulic retention time to favor biofilm accumulation.Biofilm formation and reactor performance have been followed for 90 days. In the initial phase, a COD removal of more than 90% was observed, together with continuous biofilm growth. Thereafter, at a loading rate of 6 gCOD  L−1  day−1, the reactor efficiency suddenly deteriorated, volatile fatty acids (VFA) accumulated in the effluent and the specific methanogenic activity of the biofilm dropped down.This phenomenon was due to a micronutrient limitation, especially cobalt and nickel. After complementation with a mineral solution, optimal methanogenic activity and COD removal were restored. The nutrient supplementation was required to keep on increasing the OLR until the reactor reached a volumetric organic loading rate of 20 gCOD  L−1  day−1 with more than 90% of carbon removal efficiency.
Keywords: Wastewater treatment; Biofilms; Anaerobic process; Fluidization; Nutrient complementation; Start-up;

An improved procedure was developed involving a stirred fluidized bed (SFB) adsorption for direct capture of lysozyme from unclarified preparations of chicken egg whites (CEW). The maximal binding capacity of Streamline SP adsorbent for lysozyme was observed at pH 4. A typical adsorption isotherm curve for lysozyme in crude CEW was well correlated with the Freundlich model. For an initial concentration of 20 mg CEW/ml, adsorption approached equilibrium after 30 min. Determination of dynamic binding capacity for lysozyme in a stirred fluidized bed column (i.d. 2.5 cm) was carried out using frontal analysis. The maximal loading volume of crude CEW feedstock (20 mg/ml) at 1% breakthrough for lysozyme was approximately 5, 22, and 28-fold settled bed volumes, corresponding to 256, 2173, and 4125 ml CEW, respectively. Optimal conditions for elution scheme were determined in small packed bed experiments conducted with clarified CEW. Further experiments were carried out to assess the feasibility of using Streamline SP adsorbent and SFB column for direct recovery of lysozyme from highly crude CEW (20 mg/ml in 20 mM sodium acetate buffer at pH 4). It was found to be successful in achieving purification of lysozyme in a high yield of 98% with a purification factor of 11 in a single step. The specific activity (7.80 × 104  U/mg) was higher than that obtained with a commercially available pure sample of lysozyme (Sigma, 7.30 × 104  U/mg).
Keywords: Stirred fluidized bed column; Adsorption; Lysozyme; Dynamic binding capacity; Crude chicken egg white; Direct recovery;

Immunoisolatability, mechanical stability and biocompatibility of cell-enclosing microcapsules are controlled by the formation of external polyelectrolyte complex membranes. The present study aimed to develop cell-enclosing subsieve-size capsules of less than 100 μm in diameter for controlling these properties. We investigated the use of anionic polysaccharide alginate combined with thermosensitive agarose, and prepared particles composed of the two components. For this purpose, agarose in droplets suspended in liquid paraffin was first gelated by cooling, and followed by gelation of alginate in CaCl2 solution. Treatment with a solution containing cationic polysaccharide chitosan inhibited the diffusion of bovine serum albumin into particles compared with non-treated particles. This observation showed that alginate–agarose particles were successfully coated with the polyelectrolyte complex membrane. In addition, cells enclosed in subsieve-size alginate–agarose capsules prepared via the gelation process adapted to the microenvironment and showed mitochondrial activity during 27 days of study. These results showed that alginate–agarose capsules prepared via the droplet breakup method in a water-immiscible co-flowing fluid and the subsequent ordered gelation process were effective vehicles for cell-encapsulation devices for cell therapy.
Keywords: Biomedical; Animal cell culture; Immobilisation; Immobilised cells; Subsieve-size capsule; Cell therapy;

A bifunctional enzyme with chitosanase and carboxymethyl cellulase (CMCase) activity was purified from commercial cellulase, which was produced by trichoderma viride, through sequential steps of DEAE-Sepharose CL-6B ion-exchange chromatography, Phenyl Sepharose CL-4B hydrophobic interaction chromatography and Sephadxe G-75 gel filtration. The purified hydrolase was homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass was 66 kDa. The hydrolase exhibited chitosanase activity for chitosan hydrolysis and cellulase activity for carboxymethyl cellulose (CMC) hydrolysis. For chitosan hydrolysis, the enzyme had an optimum pH of 5.2, temperature of 60 °C and exhibited typical Michaelis-Menten kinetics with K m value and V max of 10 mg/ml and 0.164 U/ml, respectively. For CMC hydrolysis, the pH and temperature optima enzyme were 4.0 and 50 °C. Heavy metal ions such as Hg2+, Ag+ significantly or completely inhibited the enzyme activity. Identification of glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) oligomers as depolymerized products indicated that the enzyme cleaved both GlcN-GlcN and GlcNAc-GlcN linkages. The chitosan hydrolysates were oligomers with one to four glucosamine residues and some oligomers with longer chain length.
Keywords: Cellulase; Chitosanase; Bifunctional; Properties; trichoderma viride;

An on-line adaptive control based on DO/pH measurements and ANN pattern recognition model for fed-batch cultivation by Shengbing Duan; Zhongping Shi; Haojie Feng; Zuoying Duan; Zhonggui Mao (88-96).
An on-line adaptive control strategy based on DO/pH measurements and artificial neural network pattern recognition (ANNPR) model for fed-batch cultivation processes was proposed. Various changing patterns of pH and dissolved oxygen concentration (DO) under pH-Stat, DO-Stat, and the conditions of substrate in excess were collected and used to train the ANNPR models. Based on the on-line measured pH and DO data, the recognition results on current physiological state was deduced by the ANNPR models, and then the on-line adaptive control of nutrient feeding rate was implemented. Compared with the traditional pH-Stat control, the proposed control strategy increased cell productivity about 150% without byproduct accumulation. The control strategy is potentially useful for high cell density cultivation of recombinant microorganisms to efficiently express value-added foreign proteins or enzyme with the most traditional pH and DO sensors.
Keywords: Artificial neural network; Fed-batch culture; On-line adaptive control; Pattern recognition; Traditional pH and DO sensors;

Production of xylose from oil palm empty fruit bunch fiber using sulfuric acid by S.H.A. Rahman; J.P. Choudhury; A.L. Ahmad (97-103).
Oil palm empty fruit bunch fiber is a lignocellulosic waste from palm oil mills. It is a potential source of xylose which can be used as a raw material for production of xylitol, a high value product. The increasing interest on use of lignocellulosic waste for bioconversion to fuels and chemicals is justifiable as these materials are low cost, renewable and widespread sources of sugars. Batch hydrolysis of oil palm empty fruit bunch fiber was performed at operating temperature 120 °C using various concentration of sulfuric acid (2–6%) and reaction time (0–90 min). Concentration of xylose, glucose, furfural and acetic acid in the resulting hydrolysate were determined. Kinetic parameters of mathematical models were obtained in order to predict concentration of xylose, glucose, furfural, acetic acid in the hydrolysate and to optimize the process. Optimum H2SO4 concentration and reaction time obtained under operating temperature of 120 °C was 6% and 15 min, respectively. Optimum concentration of xylose, glucose, furfural and acetic acid found in the hydrolysate were 29.4, 2.34, 0.87 and 1.25 (g/l), respectively.
Keywords: Oil palm empty fruit bunch; Hydrolysis; Xylose; Kinetic parameters; Optimisation; Cellulose; Glucose;

This study evaluates the influence of pH and temperature on the partition coefficient of β-glucosidase and total protein in order to determine the best conditions for the purification of the enzyme in poly(ethylene glycol) (PEG) 4000 (8%, w/w) and potassium phosphate salt (13%, w/w) aqueous two-phase systems. The partition coefficient increased with pH and temperature for the ranges investigated (i.e., pH: 6.0–7.5; temperature: 25–55 °C). Yield and selectivity were key criteria for the selection of the optimum separation condition for enzyme purification. The highest selectivity was achieved at a pH of 6.5 and a temperature of 25 °C. A β-glucosidase yield greater than 92% was obtained in the bottom phase for pH values less than 7.0 and temperatures below 35 °C.
Keywords: Aqueous two-phase system; Bioseparations; β-d-Glucosidase; Protein; Purification; Partition coefficient;

Development of optimal culture method of Sparassis crispa mycelia and a new extraction method of antineoplastic constituent by Akihiro Kurosumi; Fumihisa Kobayasi; Godliving Mtui; Yoshitoshi Nakamura (109-113).
The optimal culture condition of basidiomycete Sparassis crispa mycelia was determined for producing a large amount of antineoplastic constituent, i.e. β-1,3-glucan. Furthermore, a new extraction method with steam explosion as a pretreatment was attempted to increase the amount of antineoplastic constituent extracted from S. crispa mycelia. The shake-flask liquid culture of fungal mycelia could shorten a culture time up to about 1/3 compared with the conventional stationary liquid culture. The productivity of S. crispa mycelia reached its maximum at an initial glucose concentration of 30 g/L, a culture temperature of 25–30 °C, and pH 5. Steam explosion at a steam temperature of 225 °C for 5 min not only enhanced the amount of antineoplastic constituent extracted from the mycelia but also shortened the extraction time significantly.
Keywords: Bioconversion; Filamentous fungi; β-1,3-Glucan; Microbial growth; Plant cell culture; Sparassis crispa;