Biochemical Engineering Journal (v.30, #2)

BEJ Keywords (II).

Biosorption thermodynamics of cadmium on coconut copra meal as biosorbent by Yuh-Shan Ho; Augustine E. Ofomaja (117-123).
Coconut copra meal, a waste product of coconut oil production was investigated for its potential use as a biosorbent for cadmium ions from an aqueous solution. A comparison of linear least-squares method and a trial and error non-linear method of three widely used isotherms, Langmuir, Freundlich, and Redlich–Peterson, were examined. Langmuir isotherm parameters obtained from the four Langmuir linear equations by using linear method were not similar, but were the same when non-linear method was used. The best-fitting isotherms were Langmuir and Redlich–Peterson isotherms. Langmuir isotherm is a special case of Redlich–Peterson isotherm when constant g is unity. In addition, various thermodynamic parameters, such as ΔG°, ΔH°, and ΔS°, were calculated. The biosorption process is a spontaneous and exothermic process.
Keywords: Copra meal; Cadmium ions; Langmuir isotherm; Temperature; Adsorption;

Evaluation of the use of volatile electrolyte system produced by ammonia and carbon dioxide in water for the salting-out of proteins: Precipitation of porcine trypsin by Erika Ohta Watanabe; Pedro de Alcântara Pessôa Filho; Everson Alves Miranda; Rahoma Sadeg Mohamed (124-129).
In this work, the precipitation of porcine trypsin in solutions of volatile electrolytes formed by ammonia and carbon dioxide was investigated. The solubility curve of trypsin at 4 °C was determined by measuring the compositions of the precipitate and supernatant phases. Analysis showed that ammonium carbamate is able to induce trypsin salting-out. The verified salting-out property of ammonium carbamate, the easy removal of the salt out of the protein precipitate and mother-solution and the high activity recovery demonstrate the feasibility of using ammonium carbamate as precipitant agent.
Keywords: Downstream processing; Enzyme activity; Protease; Separation; Volatile electrolyte; Trypsin;

Debaryomyces nepalensis was previously isolated, which is capable of producing pectin lyase and pectate lyase using pectin as sole source of carbon. The parameters affecting the activity of these enzymes are categorized into two, viz., chemical (amount of substrate and enzyme) and physical (pH of reaction mixture and temperature of incubation). The effect of these parameters on pectin lyase and pectate lyase was studied using central composite design. The optimal conditions of amount of substrate and enzyme (culture broth) were found to be 545 μl (∼1 g/l pectin) and 123 μl for pectin lyase and 707 μl (∼1.1 g/l polygalacturonic acid) and 96 μl for pectate lyase. The optimum pH and temperature for reaction was found to be 6.4 and 35 °C for pectin lyase and 7.5 and 32 °C for pectate lyase. After optimization, the activity of pectin lyase and pectate lyase was increased by 1.3- and 1.4-fold, respectively.
Keywords: Pectin lyase; Pectate lyase; Pectin; Polygalacturonic acid; Transelimination reaction; Physical parameters; Chemical parameters; Response surface methodology; Central composite design;

Direct recovery of clotting factor IX from unclarified human plasma by expanded bed ion exchange chromatography by Yu-Kaung Chang; Jim-Tong Horng; Ren-Ze Huang; Shiuan-Yaw Lin (138-146).
In this study, STREAMLINE DEAE adsorbent was used for the direct recovery of clotting factor IX (FIX) from unclarified human plasma in expanded bed adsorption (EBA). The equilibrium adsorption studies for general protein showed that the experimental data followed the Langmuir isotherm model. The maximum adsorption capacity for general protein was 35.33 mg/ml with a dissociation constant of 0.18 mg/ml. However, the adsorption of FIX followed the Henry's law with Henry's constant of 59.78. Optimal condition for the elution of FIX with step changes of increasing concentration of NaCl was determined in small packed bed experiments conducted with clarified plasma. The results showed 94.28% of the bound FIX could be eluted with 0.7 M NaCl. The influence of liquid velocities of 100–300 cm/h on the selective adsorption of FIX from clarified plasma was also investigated in packed bed experiments. The results showed that the rate of FIX breakthrough was found to be very similar. In this work, operation at a constant liquid velocity of 200 cm/h in an expanded bed recovery process was chosen for the purification of FIX. The FIX was directly recovered using 0.7 M NaCl from unclarified plasma with a purification factor of 14 and a yield of 90% in a single step.
Keywords: Expanded bed ion exchange chromatography; Adsorption; Human clotting factor IX; STREAMLINE DEAE; Direct recovery;

Growth kinetics is one of the most important and critical parameters for studying biofilms as they can be used to model mass transfer and biological reactions. Growth parameters of Phanerochaete chrysosporium were quantified based on the utilisation rate of glucose and biofilm generated in single fibre capillary membrane bioreactors (SFCMBR) operated vertically at 37 °C for 264 h. The growth rate constant value of 0.0348 h−1 was used to model the dry biofilm density profile, which was also classified as equivalent to the specific growth rate. The mass of glucose consumed, increased from 0.051 g/day during the lag phase, to 0.145 g/day in the secondary growth phase. An average glucose consumption rate of 94.0 g/m3  h was determined over the period of 264 h. The average glucose-based growth yield coefficient of approximately 0.202 g biofilm/g glucose was determined. The primary growth phase reached a structural equilibrium (steady state) at an average density of 9.7E05 g/m3 in the period 168–216 h, with the secondary growth phase occurring after 216 h. The glucose maintenance coefficient value of 0.028 h−1, was obtained during the biofilm structural equilibrium phase.
Keywords: Phanerochaete chrysosporium; Growth kinetics; Membrane bioreactor; Biofilms; Filamentous fungi;

Influence of hydrodynamic shear forces on properties of granular biomass in a sequencing batch biofilter reactor by Claudio Di Iaconi; Roberto Ramadori; Antonio Lopez; Roberto Passino (152-157).
The paper reports the results of an experimental investigation aimed at evaluating the role of hydrodynamic conditions on properties (physical, chemical and biochemical) of granular biomass grown under aerobic conditions in a SBBR (i.e. sequencing batch biofilter reactor). The granular biomass was characterised by a high density (i.e. 70–110 g VSS/Lbiomass) that permitted to achieve a biomass concentration such high as 20–38 g VSS/Lbed. The biomass density increased quasi-linearly with the increase of shear forces. The extracellular polymeric substances (EPS) content was rather low (5–6% of the total organic matter) and mostly made up of proteins whereas the cellular protein content, a parameter frequently used as an indicator of microorganisms activity, was high (30–60% of total organic matter).In addition, the EPS content and composition were not affected by hydrodynamic shear forces as well as the biomass hydrophobicity and protein content that, however, both seem to be affected by starvation.
Keywords: Aerobic process; Hydrodynamic shear forces; Biofilter; Granulation; Biomass properties;

Structural stability of glucose oxidase encapsulated in liposomes to inhibition by hydrogen peroxide produced during glucose oxidation by Makoto Yoshimoto; Mitsunobu Sato; Shaoqing Wang; Kimitoshi Fukunaga; Katsumi Nakao (158-163).
The glucose oxidase (GO) consists of two identical subunits each of which contains noncovalently bound flavin adenine dinucleotide (FAD) cofactor. GO is known to be inactivated due to hydrogen peroxide (H2O2) produced in the oxidation of glucose. In our previous paper, the liposomal GO showed a much higher stability to H2O2 than the free enzyme. In this work, to deduce the structure and state of the liposomal GO, the fluorescence properties of the tryptophan residue and FAD cofactor in free GO during the glucose oxidation were measured for its tertiary structure and redox state, respectively. The tryptophan fluorescence data revealed that the initial glucose concentration lower than 0.6 mM resulted in almost no alteration in the tertiary structure, while the higher concentration did in a remarkable change in the structure due to the increase in catalytic turnover. On the other hand, the FAD fluorescence data showed that the reduced FAD was accumulated in the initial stage of the reaction. When glucose was completely consumed, the FAD restored the initial oxidized form for the initial glucose concentrations lower than 0.6 mM, whereas for the higher concentrations the reduced FAD tended to form an inactive complex with H2O2 leading to the deactivated enzyme. In the case of the liposomal GO at even such a high initial glucose concentration as 10 mM, the glucose concentration inside liposome was previously estimated to be lower than 0.2 mM due to its low permeability to glucose. Consequently, the formation of the inactive complex was proved to be effectively depressed in the liposomal GO reaction.
Keywords: Liposomal glucose oxidase; Tryptophan fluorescence; FAD fluorescence; Enzyme tertiary structure; Enzyme inactivation; Glucose permeability;

Growth kinetics of Pseudomonas fluorescens in sand beds during biodegradation of phenol by I.N. Sgountzos; S. Pavlou; C.A. Paraskeva; A.C. Payatakes (164-173).
The growth kinetics of a bacterium of the strain Pseudomonas fluorescens, which is indigenous in soil, has been investigated in batch cultures and sand packs during biodegradation of phenol. Batch experiments were conducted at 25 °C with four different initial concentrations of phenol for the estimation of the kinetic parameters. Phenol was found to inhibit microbial growth and so Andrews's model was used to describe the growth rate. Growth kinetics in sand packs was studied in a series of eight virtually identical columns, which were operated simultaneously under the same conditions and in a continuous flow mode, with phenol as the sole carbon source. The idea of eight columns allowed monitoring of biomass growth as a function of time. Every 1 or 2 weeks, one column was drawn out of the system and was “sacrificed” to measure the developed biomass in the porous medium. The results indicated that growth of P. fluorescens in sand packs during biodegradation of phenol was an intensely dynamic phenomenon. Detachment of clusters of bacteria from the sand grains was found to be one of the main processes taking place in such a system, caused by a combination of low nutrient supply, oxygen availability and flow velocity.
Keywords: Batch processing; Biofilms; Packed bed bioreactors; Growth kinetics; Detachment; Sand beds;

Biodegradation of high phenol concentration by activated sludge in an immersed membrane bioreactor by B. Marrot; A. Barrios-Martinez; P. Moulin; N. Roche (174-183).
The effect of adaptation of mixed culture in the phenol biodegradation has been studied. The degradation experiments have been conducted at different phenol concentrations from 0.5 to 3 g L−1. Biological treatment has been shown to be economical, practical and it leads to a complete removal of phenol. High concentrations of phenol are inhibitory for growth; so it is for the rates of substrates utilization that are greater at low initial concentrations. Haldane kinetics model for single substrate was used to obtain maximum specific growth rates (μ m  = 0.438 h−1), half saturation (K s  = 29.54 mg L−1) and substrate inhibition constant (K i  = 72.45 mg L−1). Although the concentration in phenol is significant, these results are in agreement with those reported in the literature for phenol removal abilities in different systems and the Haldane model is still acceptable.
Keywords: Activated sludge; Phenol; Biodegradation; Kinetic model; Haldane; Immersed membrane bioreactor;

The nickel(II) ions biosorption process by marine algae Sargassum filipendula in a fixed bed column was investigated for the following experimental conditions: temperature = 30 °C and pH 3.0. The experimental breakthrough curves were obtained for the following chosen flow rates 0.002, 0.004, 0.006, and 0.008 L/min. A mathematical model was developed to describe the nickel ion sorption in a fixed bed column. The model of three partial differential equations (PDE) has considered the hydrodynamics throughout the fixed bed column as well as the sorption process in the liquid and solid phases. The internal and external mass transfer limitations were considered, as well. The nickel ion sorption kinetics has been studied utilizing the Langmuir isotherm. The PDE of the system were discretized in the form of ordinary differential equations (ODE) and were solved for the given initial and boundary conditions using the finite volume method. A new correlation for external mass transfer coefficient was developed. Some of the model parameters were experimentally determined (ɛ, d p) where the others such as (K F, K S) were evaluated on the base of experimental data parameters. The identification procedure was based on the least square statistical method. The robustness and flexibility of the developed model was checked out using four sets of experimental data and the predictive power of the model was evaluated to be good enough for the all studied cases. The developed model can be useful tool for nickel ion removal process optimization and design of fixed bed columns using biomass of S. filipendula as a sorbent.
Keywords: Adsorption; Fixed bed; Nickel; Marine algae; Mass transfer; Modeling;

A vegetable sponge (loofa sponge, LS) from the fibrous interior of dried fruits (Luffa cylindrica) was used as a scaffold in a packed-bed bioreactor for dynamic seeding and perfusion culture of rat hepatocytes. Hepatocytes were immobilized within the highly complex macroscopic architecture of LS by attaching to its rough fiber surface. The cell seeding efficiency was independent of the initial loading cell number up to 2 × 108 hepatocytes (1 × 107 cells/cm3 scaffold) and cell distribution within the bioreactor was uniform. Liver-specific functions of the hepatocytes depended on cell seeding density and medium flow rate with the optimum cell density at 3.5 × 106 cells/cm3 scaffold. Spheroids with diameters around 100 μm and high viability from FDA staining were observed at day 3 and spheroids as large as 400 μm could be found at day 4. Cell damage was enhanced due to direct contacting of cells with the flow stream. Nonetheless, hepatocytes metabolic rates were substantially improved over stationary culture on TCPS dishes with production rates of albumin and urea at day 7 maintained at 90% and 67% of their initial values, respectively.
Keywords: Hepatocytes; Bioartificial liver; Immobilized cells; Tissue cell culture; Bioreactors; Biomedical;

The copper-aided photosterilization of Escherichia coli was examined on TiO2 thin films under a lighting condition with less UV rays from a white light fluorescent lamp. It was found that the coexistence of Cu2+ and H2O2 in liquid phase exerted the synergistic effect on killing E. coli cells though these chemical species were individually at nontoxic levels to the cells (less than 10 mmol/m3). At an incident light intensity of I 0  = 28 W/m2, the addition of Cu2+ (10 mmol/m3) to liquid phase on TiO2 film gave the apparent deactivation rate constant of k′ = 12 × 10−2  min−1, the value of which was about 5 times as large as that on TiO2 film in the absence of Cu2+. This result suggested that in the liquid phase, Cu2+ was reduced to Cu+ by receiving electron from photo-excited TiO2, and then Cu+ reacted with photocatalysis-derived H2O2 to produce •OH via Fenton-type reaction. The deactivation tests were also examined using a modified film preparation made from copper-incorporated TiO2. On this film, the k′ value was 23 × 10−2  min−1 at I 0  = 28 W/m2, which was respectively about two and nine times as large as those on the original TiO2 film with and without Cu2+ addition (10 mmol/m3). The enhancement of biocidal activity on the modified TiO2 film was considered to arise from promoted •OH formation by the aid of copper component anchored in TiO2 solid phase.
Keywords: Titanium dioxide; Photocatalytic sterilization; Escherichia coli; Copper-aided reaction; Reactive oxygen species;

Increased total air pressure versus oxygen limitation for enhanced oxygen transfer and product formation in a Pichia pastoris recombinant protein process by Theppanya Charoenrat; Mariena Ketudat-Cairns; Mehmedalija Jahic; Andres Veide; Sven-Olof Enfors (205-211).
Two strategies to increase the productivity of secreted Thai Rosewood β-glucosidase in Pichia pastoris processes were evaluated. Both methods were based on increasing the oxygen transfer rate (OTR) in the process by simple means. Increasing the driving force for the diffusion from the air bubbles to the medium by elevating the air pressure, from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) by 59% while increasing the driving force by accepting oxygen limitation increased the OUR by 35%. The OTR increased less than in proportion to the increased solubility in the high-pressure process, which indicates that air bubble compression reduces the volumetric oxygen transfer coefficient (K L a). Even though the methanol consumption increased almost in proportion to the OTR in both processes the biomass production did not increase as much. This is explained as a higher maintenance demand for methanol in the oxygen limited (0.027 g g−1  g−1) and high-pressure processes (0.035 g g−1  g−1), compared to 0.022 g g−1  g−1 in the methanol limited reference process. However, in spite of the low effect of increasing OTR on the biomass production the total β-glucosidase yield increased almost in proportion to the increased methanol consumption and reached highest value in the high-pressure process, while the β-glucosidase purity was highest in the oxygen-limited process due to release of less contaminating proteins.
Keywords: Aeration; Oxygen transfer; Dissolved oxygen tension; Enzyme production; β-Glucosidase; Oxygen-limited fed-batch;

The effect of fatty acid concentration and water content on the production of biodiesel by lipase by Sulaiman Al-Zuhair; Kishnu Vaarma Jayaraman; Smita Krishnan; Wai-Hoong Chan (212-217).
The kinetics of the production of biodiesel by esterification of butyric acid with methanol, catalysed by lipase from Mucor miehei, was studied in two types of systems, namely, n-hexane microaqueous and biphasic (n-hexane/water) containing different amounts of water. The experimental results were fit to a Ping-Pong mechanism and the constants found in the rate expression were determined. The results have shown that in a microaqueous media, butyric acid does not inhibit the reaction in the range of initial concentrations considered. The effect of water content on the initial rate of reaction and conversion were illustrated. It was shown that the initial rate of reaction increased as the initial water content increased up to 25% (v/v). However, the conversion was found to be higher at low initial water concentrations.
Keywords: Biodiesel; Lipase; Butyric acid; Esterification; Ping-Pong kinetics;

Flow focusing technology is known to be advantageous for micro- and nanoparticle production. We investigated the applicability of this technology to produce subsieve-size mammalian cell-enclosing capsules of less than 100 μm in diameter. Subsieve-size capsules with a narrow size distribution were obtained via droplets breakup in a water-immiscible co-flowing liquid flow by a flow focusing process. Compared with the production process of non-flow focusing, the capsules could be prepared with less consumption of the water-immiscible fluid of liquid paraffin. No harmful effects resulting from the flow focusing process on the cells enclosed in the capsules were detected in this study. The undamaged percentage of the cells immediately after the enclosing process was 96.6 ± 1.8%, and the cells enclosed in subsieve-size agarose capsules showed mitochondrial activity for over 3 weeks. These results showed that flow focusing is an effective process for producing cell-enclosing subsieve-size capsules.
Keywords: Biomedical; Animal cell culture; Immobilisation; Immobilised cells; Subsieve-size capsules; Cell therapy;

Keywords: Citation error; Pseudo second order kinetic; Sorption;