Biochemical Engineering Journal (v.16, #3)


BEJ Keywords (II).

A hybrid type of computer program was developed for the estimation of intracellular metabolic fluxes based on 13 C -labeling experiment and the measurement of the signals of two-dimensional nuclear magnetic resonance (NMR) spectroscopy of proteinogenic amino acids. The continuous cultures of Saccharomyces cerevisiae were conducted at the dilution rate of 0.1 h−1 with different carbon sources such as glucose, glycerol or acetate labeled by 13 C . The effects of different carbon sources on the intracellular metabolic flux distribution (MFD) were investigated using the program developed. Some of the enzyme activities were also measured to ascertain the results of MFD. It was found that the pyruvate carboxylase instead of PEP carboxylase play the main role as anaplerotic reaction for the case of glucose and glycerol used as carbon sources. It was also found that the pathways via PEP carboxykinase (pck) and malic enzyme as well as glyoxylate shunt were used to a large extent for the assimilation of acetate. The general direction of reaction catalyzed by transketolase 2 reverses and the intermediate metabolite ribulose-5-phosphate and erythrose-4-phosphate were mainly produced by transaldolase and transketolase reactions during growth on acetate.
Keywords: Saccharomyces cerevisiae; 13 C -labeling experiment; Metabolic flux analysis; 2D NMR; Central metabolism; Enzyme assay;

Performance of a perforated rotating disc contactor in the continuous extraction of a protein using the PEG–cashew-nut tree gum aqueous two-phase system by Leonie A Sarubbo; Luciana A Oliveira; Ana Lúcia F Porto; José Luiz Lima-Filho; Galba Maria Campos-takaki; Elias B Tambourgi (221-227).
The characterisation of bovine serum albumin (BSA) mass transfer mechanisms in a perforated rotating disc contactor (PRDC) using an aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and a polysaccharide, the cashew-nut tree gum, was described. The PEG-rich phase was used as the dispersed phase and protein transfer took place from the dispersed to the continuous phase. Studies of the effect of dispersed phase velocity, system composition and disc rotation speed on either protein mass transfer coefficients or column hold-up showed that the dispersed phase hold-up and the volumetric mass transfer coefficient increased with increasing the dispersed phase velocity and disc rotation speed.
Keywords: Liquid–liquid extraction; Aqueous two-phase; Separation; Mass transfer; Perforated rotating disc contactor;

Statistical analysis on some critical parameters affecting the formation of protoplasts from the mycelium of Penicillium griseofulvum by Reddivari Muralidhar; Sathyanarayana N Gummadi; Veeranki Venkata Dasu; Tapobrata Panda (229-235).
The critical parameters, such as slant age, mycelium age and contact time of mycelium with lytic enzymes which affects the formation of protoplast from the Penicillium griseofulvum mycelium were studied using a novel technique called response surface methodology. The design used to optimize these parameters was full factorial central composite design. The number of protoplasts, viability of protoplasts and griseofulvin yield are the three responses studied in this communication. The individual optimum values of slant age, inoculum age and contact time obtained are 187.1, 30.81, 21.4 h, respectively, for number of protoplast/ml 68.92, 33.01, 9.88 h, respectively, for percentage viability of protoplasts and 230.2, 73.6, 31.2 h, respectively, for griseofulvin. In order to obtain a single optimum value for all the three responses, a multi-response analysis was carried out using a generalized distance approach. The simultaneous optimum values of slant age, inoculum age and contact time are 133.78, 37.1 and 15.97 h, respectively, for all the three responses.
Keywords: Penicillium griseofulvum; Protoplasts; Griseofulvin; Slant age; Inoculum age; Contact time;

Fermentative production of bacteriocin, pediocin AcH was studied in a batch fermenter using Pediococcus acidilactici H. An initial pH of 6.5 and a fermentation temperature of 37 °C were found to be favourable for optimum production up to 20 h of fermentation time. Rate of cell growth utilizing glucose as substrate during the exponential growth phase and stationary phase was studied experimentally and the data obtained were simulated into the proposed model. The mathematical model also considered the diffusional mass transfer of glucose into the active mass of the cell. Results showed that the model could adequately account for the transfer mechanism of glucose and the phenomenon of cell growth in the fermenter in exponential growth and stationary phases.
Keywords: Bacteriocin; Bio-preservative; Pediococcus acidilactici H; Pediocin AcH; Monod equation; Mass transfer; Mathematical model; Simulation;

Butyl isobutyrate is used mainly as a fixative and modifier in flavour industry. The present work focuses on the synthesis of butyl isobutyrate by esterification of isobutyric acid with n-butanol by using lipases. Effects of various parameters were studied to deduce the kinetics and mechanism of the reaction. Novozym SP 435 was found to be the most efficient catalyst and heptane was the best solvent. With equimolar quantities of the reactants in heptane, Novozym SP 435 offered a conversion of 56% at 30 °C in 6 h. Initial rate data and progress curve data were used to arrive at a suitable model. The initial rate studies showed that the Michaelis constant for n-butanol was very low indicating lower affinity between the enzyme and the reactant. The kinetics was found to obey the Ping-Pong bi-bi mechanism with n-butanol substrate inhibition.
Keywords: Immobilised lipase; Enzyme catalysis; Butyl isobutyrate; Lineweaver–Burk plots; Ping-Pong bi-bi mechanism; Substrate inhibition; Kinetics; Novozym SP 435;

Model description of dibenzothiophene mass transfer in oil/water dispersions with respect to biodesulfurization by C.L.M Marcelis; M van Leeuwen; H.G Polderman; A.J.H Janssen; G Lettinga (253-264).
A mathematical model was developed in order to describe the mass transfer rate of dibenzothiophene within the oil droplet to the oil/water interface of droplets created in a stirred tank reactor. The mass transfer rate of dibenzothiophene was calculated for various complex hydrocarbon distillates and model solvents in the temperature range of 20–60 °C, at volume fractions of oil of 10 and 25% (v/v). The viscosity of the various oil phases used appeared to be the most critical physical parameter governing the dibenzothiophene mass transfer rate, while density and interfacial tension were found to be of minor importance. Based on the model calculations, we estimated that the mass transfer rate of dibenzothiophene within the oil droplet to the oil/water interface is at least a factor 10 up to 104 higher compared to experimentally determined specific dibenzothiophene conversion rates. Due to the prevailing mass transfer resistance from the oil/water interface to the bacterium it is essential to maximize the specific surface area to enhance the surface contact between the bacteria and the oil droplets. The microbial desulfurization rate is the overall rate-limiting process step.
Keywords: Bacteria; Biodesulfurization; Bioreactor; Dibenzothiophene; Mass transfer; Mixing; Modeling; Viscosity;

A mathematical model was established to describe protein adsorption behavior in expanded-bed chromatography by taking into account the axial size classification of adsorbent within the bed. This model has been used to simulate the expanded-bed adsorption (EBA) of bovine serum albumin (BSA) to Streamline DEAE under various operating conditions, such as different feed protein concentrations, liquid velocities and liquid viscosities. The parameters involved in the model are determined by the independent experiments or calculated from correlation to ensure a reliable comparison between the experimental and simulated results. Using these independently determined parameters, the model prediction agreed reasonably well to the experimental data. Moreover, the model was found more precise than that without considering the axial particle size distribution.
Keywords: Expanded-bed chromatography; Streamline DEAE; Bovine serum albumin; Modeling; Particle size distribution; Simulation;

Effect of medium compositions on biosensing of benzene derivatives using recombinant Escherichia coli by Shinya Ikeno; Chiaki Ogino; Takeo Ito; Yasuhiro Sugino; Nobuaki Shimizu (273-278).
TOL plasmid pWW0 of Pseudomonas putida mt-2 encodes a series of enzymes for the degradation of benzene derivatives. In the presence of benzene derivatives, the complex formed from the regulating protein XylR and Ps promoter controls the expression of these enzymes in P. putida. For detecting of benzene derivatives, the plasmid called as pTS301-GFP has been constructed by introducing a gene encoding green fluorescent protein (GFP) into the downstream of the Ps promoter gene. GFP functioned as a reporter protein is transcribed by activation of the XylR complex. In the present study, the effects of the composition of culture medium and ATP addition on fluorescence of transformant were investigated. The optimal culture medium was found to NZM, and presence of casamino acid decreases the sensitivity for detection of chemicals. Furthermore, it was observed that the addition of 200 μM ATP was effective for detecting of fluorescence of transformants exposed to the low concentration (0.05 mM) of benzene derivatives for 4 h. Applying these results for the detection of the benzene derivatives, it will be expected that transformant with pTS301-GFP can be useful for environmental water evaluation.
Keywords: XylR; Benzene derivative; GFP; Casamino acid; ATP;

Sequencing batch reactor (SBR) of biofilm formed on fibrous carriers was effective for phosphorus removal from wastewater when conditions included a 30% packing ratio for the carriers and 9 h hydraulic retention time (3 h for the anaerobic reaction phase and 6 h for the aerobic reaction phase). After 6 months of operation at chemical oxygen demand (COD) loading of 1.00 kg COD/m3 per day, the biomass concentration in the biofilm reached a high level of 8855 mg/l in terms of MLSS and 5532 mg/l in terms of MLVSS, while as the suspended biomass concentration in the liquid phase of the reactor, it was almost 60 mg/l in terms of MLSS. In every cycle, phosphorus removal was achieved by removing the biomass detached from the biofilm (excess sludge) after 20 min sedimentation. When the influent COD loading rate increased from 0.27 to 1.32 kg COD/m3 per day, the phosphorus removal efficiency surpassed 90%, and this process showed a strong tolerance for the increase of COD loading. The dominant bacterial species were found to be Pseudomonas, Aeromonas and Bacillus. The excess sludge detached from the biofilm had a high phosphorus content of 5.67% (w/w) in dry biomass and good settling performance with an SVI of 99.5–103.8 ml/g.
Keywords: Sequencing batch reactor; Biofilm process; Biological phosphorus removal; Wastewater treatment;

Shear stress tolerance and biochemical characterization of Phaeodactylum tricornutum in quasi steady-state continuous culture in outdoor photobioreactors by Asterio Sánchez Mirón; M.Carmen Cerón Garcı́a; Antonio Contreras Gómez; Francisco Garcı́a Camacho; Emilio Molina Grima; Yusuf Chisti (287-297).
A bubble column and two airlift photobioreactors (a draft-tube sparged vessel and a split-cylinder device) of the same general design (0.19 m column diameter, 2 m tall, 0.06 m3 working volume) were evaluated for outdoor continuous culture of the microalga Phaeodactylum tricornutum at a dilution rate of 0.03 h−1. At a daily averaged irradiance (photosynthetically active) value of 900 μE m−2  s−1, all bioreactors attained a quasi steady-state biomass concentration of ∼1 kg m−3 and a biomass productivity of ∼0.3 kg m−3 per day when the aeration velocity was 0.01 m s−1. The microalgal cells were susceptible to aeration-associated hydrodynamic stress if the superficial aeration velocity exceeded 0.01 m s−1. Supplementing the culture medium with 0.02% or more carboxymethyl cellulose (CMC), allowed stable culture under conditions that had previously damaged the cells.The average elemental composition of the biomass was: 49.2% C, 6.3% H, 0.8% N, and 1.3% S. The chlorophylls, carotenoids, and pigments content of the biomass changed with irradiance within a given day. Low irradiance favored accumulation of the light capture pigments. Increasing daily irradiance led to accumulation of carbohydrates. Some of the carbohydrate accumulated during the day was consumed at night and partly converted to proteins. Eicosapentaenoic acid (EPA, 20:5n3) constituted between 27 and 30% of the total fatty acids present, or 2.6–3.1% of the dry biomass. The other main fatty acids present were palmetic acid (16:0), palmoleic acid (16:1n7), and myristic acid (14:0). On average, these three fatty acids constituted 16.9% (16:0), 14.0% (16:1n7) and 9.4% (14:0) of the total fatty acids present.
Keywords: Phaeodactylum tricornutum; Microalgal culture; Photobioreactors; Airlift reactors;

The creation of microscale fermentation procedures could have significant benefits at all stages of fermentation process development from discovery through to process optimisation. For both microbial and mammalian fermentations, pH is a vital process parameter as it has a marked affect on cell growth rate, viability and product synthesis. In this work, we describe the influence of various pH control strategies on growth and erythromycin synthesis by Saccharopolyspora erythraea CA340 at the 7 l scale and show that the effects can be reproduced in pH-controlled microscale fermentations (thousandfold scale translation). At the 7 l scale the implementation of base only or full pH control (NaOH and H3PO4 additions) significantly increased both the maximum growth rate and biomass concentrations attained compared to fermentations without pH control. There was over a twofold increase in erythromycin biosynthesis and the ratio of erythromycin A (EA) to erythromycin C (EC) increased from 2:1 to 6:1 (base only pH control) to 11:1 (full pH control). In order to measure pH during microscale fermentations, a specially designed microtitre plate was built that allowed the insertion of a micro-pH probe into each well (total well volume 7 ml). This, allowed manual base only pH control to be implemented in microwell fermentations which enhanced both the maximum specific growth rate and the maximum biomass concentration. Total erythromycin synthesis and the ratio of EA:EC were also significantly enhanced. This work has demonstrated the benefits of implementing pH control in microscale fermentations and now allows the specification of an automated pH control system.
Keywords: Microscale fermentation; pH control; Saccharopolyspora erythraea CA340; Erythromycin biosynthesis;

Sorption of mono-carboxylic acids by an anion-exchange membrane by Hiroshi Takahashi; Kazuya Ohba; Ken-ichi Kikuchi (311-315).
Sorption characteristics of eight mono-carboxylic acids by an anion-exchange membrane were studied. The experiments were carried out as batch determinations of the equilibrated concentrations of chloride and carboxylic acid in the membrane and the solution. The sorption of a carboxylic acid by the ion-exchange membrane rapidly increased with increase in the solution concentration of the carboxylic acid, and was considerably different at different pH. However, these pH-dependent sorption equilibria were successfully represented as one curve in terms of the concentration of the dissociated carboxylic acid ion in the solution. This means that the sorption behavior of carboxylic acids can be explained by the ion exchange between a dissociated carboxylic acid ion and a counter ion on the fixed charge in the membrane. On the basis of the experimental results, the selectivity coefficients of mono-carboxylic acids were determined by a model which considered with dissociation equilibrium of carboxylic acids, material balance in the solution, and electrical neutrality in the membrane. The model successfully explained the observed sorption characteristics for carboxylic acids.
Keywords: Anion-exchange membrane; Carboxylic acid; Sorption; Selectivity;

Chitosan was used for the surface coating of porous silica beads and copper(II) ions were immobilized to the chitosan layer to prepare an immobilized metal affinity (IMA) absorbent for protein adsorption. The amount of chitosan adsorbed on silica bead was about 8.5 mg/g of wet beads. Nonspecific adsorption of chitosan-coated silica beads decreased significantly from 0.23 to less than 0.08 mmol/l after the surface coating. A proper cross-linking ratio of glutaraldehyde was determined by investigating the adsorption of Cu2+ and then BSA. Copper(II) ions were loaded to the chitosan-coated silica at 48 mmol/l. BSA adsorption to this IMA adsorbent showed a maximum (0.91 mmol/l) at liquid phase pH of 5.0, close to its isoelectric point. The adsorbed protein could be dissociated by increasing liquid phase pH 8.0, indicating the reversibility of the adsorption. Most importantly, the chitosan-coated silica showed significantly enhanced stability in alkaline solutions, as demonstrated by the long-term treatment in 0.1 mol/l sodium hydroxide solution and repeated uses for protein adsorption.
Keywords: Protein; Adsorption; Affinity; Chromatography; Chitosan; Surface coating;

The condensation of octyl β-d-glucoside and octanoic acid and the hydrolysis of 6-O-octanoyl octyl β-d-glucoside using an immobilized lipase from Candida antarctica were carried out in acetonitrile under various conditions. The kinetics for these reactions could be expressed based on the ping-pong bi-bi mechanism, and all the kinetic parameters in the expression were evaluated. The activity of the lipase decreased at water concentrations lower than 0.05 mol/l. The dependence of the activity on the water concentration was expressed by an empirical equation. These equations were useful for predicting the transient changes in the concentrations of the substrates and the products under any conditions even when molecular sieves were added to the reaction system.
Keywords: Condensation; Hydrolysis; Kinetics; Lipase; Organic solvent;

Evaluation of a smart bioconjugate of pectinase for chitin hydrolysis by Ipsita Roy; Meryam Sardar; Munishwar N Gupta (329-335).
Pectinex™ Ultra SP-L was immobilized non-covalently on alginate. The smart (reversibly soluble–insoluble) bioconjugate showed 56% activity as compared to the equivalent free enzyme for hydrolysis of chitin. The bioconjugate showed a broader pH optimum while the temperature optimum remained unchanged, as compared to the free enzyme. The bioconjugate also showed enhanced thermal stability at 55 and 65 °C. The K m and V max values of the bioconjugate were 6 mg ml−1 and 3.4 nmol min−1, respectively. There was 55% reduction in the bioconjugate activity after first cycle. The reason behind this reduction was investigated and is briefly discussed.
Keywords: Bioconversion; Chitin hydrolysis; Enzyme technology; Immobilized enzymes; Pectinase; Waste treatment;

The aim of this work was to determine whether or not the variable maintenance equation proposed by Neijssel and Tempest [Arch. Microbiol. 107 (1976) 215] and modified by Pirt [Arch. Microbiol. 133 (1982) 300] could explain the differences in the experimentally determined and predicted yield and maintenance coefficients of the ferrous-iron oxidising bacteria used in bioleaching operations determined under different growth conditions. The results obtained indicated that the maintenance requirement of energy sufficient cultures is a combination of the variable and constant maintenance energy requirements. For this reason, modelling the bioenergetics of energy sufficient cultures assuming a constant maintenance energy requirement results in a significant overestimation of the maximum bacterial yield. However, the variable maintenance equation proposed by Pirt [Arch. Microbiol. 133 (1982) 300] may be used to determine the maximum bacterial yields and the maintenance coefficients from both energy limited and energy sufficient experiments. This is because this model is able to account for variations in the growth rate as a result of changes in the maintenance requirement due to the micro-organisms being limited by more than one factor. It is thus suggested that the variable maintenance equation be used instead of the constant maintenance equation when quantifying the bioenergetics of micro-organisms unless the energy source is known to be the limiting substrate.
Keywords: Batch processing; Bioreactors; Chemoautotrophs; Kinetic parameters; Maintenance requirements; Modelling;

In this paper immobilisation effectivity of α-amylase, β-amylase and glucoamylase on copolymers of butyl acrylate and pentaerhitrite triacrylate or ethylene glycol dimethacrylate was evaluated. Special attention was focused on copolymers having three kinds of anchor groups: OH, COOH, and NH2. The anchor groups were generated on polymer matrices by aminolysis with ethylenediamine. Particular carrier with the same structure was activated by glutaraldehyde, divinyl sulfone or carbodiimide procedure. The first method was found to give enzyme-carrier preparations of the highest activities. From seven investigated carriers the copolymer of butyl acrylate and ethylene glycol dimethacrylate was found as the best for three amylolytic enzymes immobilisation. The activities of selected immobilised enzyme preparations and thermal and pH stability were determined. These efforts resulted in selection of preparation based on one polymer matrix to form dual enzyme systems: α-amylase and β-amylase, or α-amylase and glucoamylase.
Keywords: Immobilisation; Glucoamylase; α-Amylase; β-Amylase; Acrylic carriers; Copolymers;

Production of gellan gum by Sphingomonas paucimobilis NK2000 with soybean pomace by Hyuck Jin; Nam-Kyu Lee; Myung-Kyo Shin; Sung-Koo Kim; David L Kaplan; Jin-Woo Lee (357-360).
The ammonium nitrate as a nitrogen source for the production of gellan gum by Sphingomonas paucimobilis NK2000 enhanced the cell growth but its depletion was essential for higher production of gellan gum. The highest production of gellan gum was 3.27 g l−1 when the concentrations of glucose and bacto-peptone were 2.0% (w/v) and 0.05% (w/v). The production of gellan gum by S. paucimobilis NK2000 increased with increased concentration of soybean pomace substituted for bacto-peptone up to 2.0% (w/v). The highest production of gellan gum was 7.33 g l−1 in a flask scale when the concentrations of glucose and soybean pomace without ammonium nitrate were 2.0% and 2.0% (w/v). The highest production of gellan gum with the same concentrations of glucose and soybean pomace was 7.50 g l−1 in a 7 l bioreactor whereas that with 2.0% (w/v) glucose and 0.05% (w/v) bacto-peptone was 4.95 g l−1.
Keywords: Bioreactors; Biosynthesis; Fed-batch culture; Fermentation; Gellan gum; Soybean pomace;

This paper reports the biosorption study of Cu2+, Mn4+, and Zn2+ by Rhizopus Oligosporus biomass cultured in Tempe. The maximum adsorption rate occurred at pH 8 and initial concentration of 200 mg/l. The Cu2+ uptake was in equilibrium at 8 mg/g after 15 min of contact time. The Mn4+ uptake was at 9 mg/g after 60 min of contact time. The equilibrium adsorption for Zn2+ was at 12 mg/g after 30 min of contact time.
Keywords: Tempe; Biomass; Organisms; Biosorption; Rhizopus Oligosporus;