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Biochemical Engineering Journal (v.34, #2)

Editorial Board (pp. co2).

BEJ Keywords (pp. iv).

Superporous pellicular agarose–glass composite particle for protein adsorption by Xin Zhou; Yan Sun; Zheng Liu (pp. 99-106).

A superporous pellicular agarose–glass (SPAG) composite particle has been prepared by coating 6% agarose gel onto glass beads through water-in-oil emulsification using a mixture of cyclohexane and fine calcium carbonate granules as porogenic agent. After cross-linking, the cyclohexane was removed by proper solvent and the solid granules were removed by dissolving them in hydrochloric acid. Then, the composite particle was modified with diethylaminoethyl (DEAE) groups to create an anion exchanger, DEAE-SPAG for protein adsorption. Both optical microscopy and SEM revealed the superporous pellicular structure of the SPAG composite particle. With the average size (127.8μm) and the mean density (1.7g/ml), the DEAE-SPAG showed favorable an adsorption capacity of 148mg/ml and a diffusivity of D_p, 2.6×10⁻¹¹m²/s for bovine serum albumin (BSA). All the results indicated that the SPAG medium is promising for effective protein adsorption.

Keywords: Superporous pellicular; Composite particle; Protein; Adsorption; Diffusion; Mass transfer

Optimizing bioscouring condition of cotton knitted fabrics with an alkaline pectinase from Bacillus subtilis WSHB04-02 by using response surface methodology by Qiang Wang; Xue-Rong Fan; Zhao-Zhe Hua; Jian Chen (pp. 107-113).

This present study was undertaken to find optimum conditions of pH, temperature, incubation time and enzyme concentration for bioscouring of cotton knitted fabrics with an alkaline pectinase isolated from Bacillus subtilis WSHB04-02 by use of response surface methodology (RSM). A central composite design was used as an experimental design for the analysis of the allocation of treatment combination. A second-order polynomial regression model was fitted and was found adequate with a determination coefficient R² of 0.9844 ( P<0.001). The effect of pH was the most significant factor influencing cotton bioscouring and no significant interactions between different factors were found. Estimated optimum parameters were as follows: pH 9.1, temperature 57°C, incubation time 1.25h and pectinase concentration 1.0g/l. Under these conditions, a desired pectin removal percentage companied with adequate wettability was reached. In addition, a boiling water pretreatment of 30min before enzymatic scouring was found to be useful for subsequent pectinase degradation due to the improvement of the accessibility of pectinase to the pectins in cotton fibers.

Keywords: Optimization; Bioscouring; Cotton; Pectinase; Response surface methodology

Production of MBP–HepA fusion protein in recombinant Escherichia coli by optimization of culture medium by Yin Chen; Xin-Hui Xing; Fengchun Ye; Ying Kuang; Mingfang Luo (pp. 114-121).

Enhanced production of MBP (maltose-binding protein)-heparinase I (HepA) fusion protein in recombinant Escherichia coli was achieved by the optimization of the M9-based culture medium. First, by using the modified expression vector capable of releasing the C-terminus of HepA free to improve the specific activity of MBP–HepA, characteristics of the purified fusion protein were analyzed, which were similar to those of the native HepA except for the decreased affinity towards the substrate. M9-based culture medium was subsequently optimized for the enzyme production by orthogonal experimental design in shake flasks. Three major components were examined, namely glucose, yeast extract and calcium ion. The recombinant E. coli was further cultivated in a fermentor. As a result, total activity of HepA reached 20,650IUl⁻¹ in the optimized medium by a fed-batch mode in the 5-l fermentor . This study indicated that effective production of MBP–HepA by the present system would facilitate the large scale preparation of low molecular weight heparin (LMWH), which is a useful anticoagulant drug.

Keywords: Fusion protein; HepA; MBP; Orthogonal experimental design; Recombinant; Escherichia coli; Vector modification

Thiocyanate degradation during activated sludge treatment of coke-ovens wastewater by Cameron Staib; Paul Lant (pp. 122-130).

The aim of this work was to determine the reaction pathway and kinetics of thiocyanate (SCN⁻) degradation during mixed culture (activated sludge) treatment of coke-ovens wastewater. The effect of phenol and cyanide, both present in coke-ovens wastewater, on thiocyanate degradation was also to be determined. Two 6L perspex sequencing batch reactors (SBRs) were operated continuously for a period of 12 months, fed with coke-ovens wastewater from Port Kembla Steel Works. The results from 18 experimental periods are presented.The degradation of thiocyanate has been quantified by identification of specific rates of removal and oxygen uptake rate. The results indicate that thiocyanate is removed via microbial growth using thiocyanate as a substrate, and that SO₄²⁻, NH₄⁺ and CO₂ are the reaction products. Oxygen uptake rate data and sulphur and nitrogen balances indicate that this reaction was occurring.None of the results obtained showed inhibition of thiocyanate degradation due to phenol. However, phenol was degraded much quicker than thiocyanate, and thus phenol inhibition in coke-ovens wastewater treatment plants, even if it does occur, is not likely to be a significant issue. In contrast, cyanide has been found to have a significant inhibitory effect on the degradation of thiocyanate. This work suggests that thiocyanate removal may be totally inhibited in coke-ovens wastewater at concentrations of CN⁻ in excess of 1mg/L. Thiocyanate degradation is the slowest and most sensitive process, compared with the removal of phenol and cyanide, and will be the determining factor when identifying the hydraulic residence time required for treatment of coke-ovens wastewater (excluding nitrification).

Keywords: Activated sludge; Industrial wastewater; Coke-ovens; Thiocyanate; Cyanide; Phenol

Bioaccumulation of chromium(VI), copper(II) and nickel(II) ions by growing Rhizopus arrhizus by B. Preetha; T. Viruthagiri (pp. 131-135).

The metal resistance capacity and metal ion accumulation capacity during the growth of Rhizopus arrhizus and the inhibition kinetics of heavy metals, namely chromium, copper and nickel were studied in a batch reactor. A maximum percentage uptake yield of 93.84, 95.52 and 61.44% were found for 25mg/l of initial metal ion concentrations of chromium, copper and nickel, respectively. A maximum biomass of 20.17g/l was obtained at 20g/l of initial dextrose concentration and in absence of heavy metals. The inhibition was found to be a competitive inhibition for the bioaccumulation of chromium. Lineweaver–Burk plot, Aiba model and Bazua and Wilke models were used to study the inhibition kinetics of bioaccumulation of heavy metals chromium, copper and nickel using R. arrhizus and the model parameters were evaluated using experimental data.

Keywords: Bioaccumulation; Heavy metals; Rhizopus arrhizus; Growth curve; Inhibition kinetics; Metal–microbe interaction

Equilibrium study of the binary mixture of cadmium–zinc ions biosorption by the Sargassum filipendula species using adsorption isotherms models and neural network by M.R. Fagundes-Klen; P. Ferri; T.D. Martins; C.R.G. Tavares; E.A. Silva (pp. 136-146).

In this work, experiments have been carried out in the batch reactor to obtain equilibrium data of the individual biosorption and the mixture of cadmium and zinc ions by the biomass of the Sargassum filipendula species pre-treated with 0.5mol/L calcium chloride. The experiments have been performed for the chosen temperature of 30°C and operational conditions such as constant agitation and pH 5.0. Six adsorption isotherms models of the Langmuir type have been tested to represent the equilibrium data of the binary system. The artificial neural nets technique was used to fit the equilibrium experimental data. Different types of the net architecture have been tested varying the neurons number of the entrance and the hidden layer. The equilibrium concentrations of the fluid phase were used as input variables and the equilibrium concentrations of the biosorbent were used as output variable. The obtained simulation results have shown that the applied technique of artificial neural network has better adjusted the equilibrium data of the binary system when compared with the conventional biosorption isotherm models.

Keywords: Biosorption; Neural network; Isotherm; Multi-component

A dye–ligand immobilized poly(2-hydroxyethylmethacrylate) membrane used for adsorption and isolation of immunoglobulin G by Gulay Bayramoglu; H. Avni Oktem; M. Yakup Arica (pp. 147-155).

Poly(2-hydroxyethylmethacrylate) (pHEMA) membrane was prepared by photo-polymerization and a dye ligand (i.e., Reactive Green 5) was immobilised on the membrane surface (pHEMA-RG-5). Surface wettability and hydrophilicity of the dye–ligand immobilised membranes were investigated by static contact angle measurements. The chromatographic properties of the dye–ligand immobilized membrane for adsorption and purification of IgG are discussed using bare membrane as a control system. The non-specific adsorption of IgG was low (0.45mgproteinml⁻¹ membrane). The attachment of dye ligand onto the membrane significantly increased the IgG adsorption (33.75mgproteinml⁻¹ membrane). The maximum IgG adsorption was observed at pH 6.0. The reversible IgG adsorption on the membrane obeyed both the Freundlich and the Langmuir–Freundlich isotherm models. To test the efficiency of IgG isolation from human serum with the pHEMA-RG-5 membrane, the purity of the eluted IgG, as determined by HPLC, was 81% with a recovery of 67%.

Keywords: Adsorption; Biomimetics; Microporous membranes; Separation; IgG; Contact angle

Aqueous two phase extraction for purification of C-phycocyanin by Ganapathi Patil; K.S.M.S. Raghavarao (pp. 156-164).

Aqueous two phase extraction is employed for the purification of C-phycocyanin from Spirulina platensis. A systematic approach is suggested to arrive at the optimal process parameters of aqueous two phase extraction by considering a case study of C-phycocyanin. The influence of various process parameters such as type of aqueous two phase systems, phase forming salt, molecular weight of the phase forming polymer, system pH, phase composition, phase volume ratio, and type and concentration of neutral salts on differential partitioning of C-phycocyanin is evaluated. Desirable conditions for the purification of C-phycocyanin are found in polymer–salt systems especially in polyethylene glycol (4000)/potassium phosphate system. Increase in purity of C-phycocyanin to 3.52 from initial purity of 1.18 is achieved at pH 6, tie line length of 35.53% with a phase volume ratio of 0.8 in a single step of aqueous two phase extraction. Multiple extractions resulted in further increase in purity of C-phycocyanin without loosing the yield and a maximum purity of 4.05 is achieved in third aqueous two phase extraction. The integration of ultrafiltration with aqueous two phase extraction facilitated the selective removal of polyethylene glycol from the purified C-phycocyanin. Finally, C-phycocyanin is freeze dried to obtain in powder form.

Keywords: Aqueous two phase extraction; C-phycocyanin; Purification; Differential partitioning; Ultrafiltration

Ethanolysis of a waste material from olive oil distillation catalyzed by three different commercial lipases: A kinetic study by Carlos F. Torres; Ana M. Toré; Tiziana Fornari; F. Javier Señoráns; Guillermo Reglero (pp. 165-171).

Ethanolysis of a raffinate product-obtained after distillation of olive oil-catalyzed by three commercial lipases (from Candida antarctica, Rhizomucor miehei and Thermomyces lanuginosa) was studied. Uni-response model derived from a generalized Michaelis–Menten mechanism was utilized to describe the rates of formation of ethyl esters from the fatty acids present in the precursor oil. The rate constants for the ethanolysis of triacylglycerols and steryl esters under the reaction conditions were compared. Moreover, inactivation of three lipases was evaluated. Because the half-life of the enzyme is comparable to or even shorter than the half-life of the reaction, the intrinsic reaction rate and enzyme deactivation must both be considered in modeling the kinetics.

Keywords: Abbreviations; B; ethanol; E; active form of the enzyme; E; _d; inactive form of the enzyme; FAEEs; fatty acid ethyl esters; G; triacylglycerols; MSS; mean sum of squares; OOP; olive oil pitch; P; lower glycerides; Q; ₁; fatty acid ethyl esters involved in the ethanolysis of triacylglycerols; Q; ₂; fatty acid ethyl esters involved in the ethanolysis of steryl esters; RSS; relative sum of squares; S; sterols; SE; steryl esters; VOOD; vegetable oil deodorizer distillatesDeodorizer distillate; Kinetic parameters; Lipase; Modeling; Steryl esters; Transesterification

Effects of shear stress and mass transfer on chitinase production by Paenibacillus sp. CHE-N1 by Po-Min Kao; Chih-I Chen; Shu-Chen Huang; Yung-Chi Chang; Po-Jen Tsai; Yung-Chuan Liu (pp. 172-178).

Chitinase fermentations with strain Paenibacillus sp. CHE-N1 was carried out in a 5-l stirred-tank bioreactor. The effects of operation variables including agitation rates, aeration rates, and pH values, on cell growth and chitinase activity yield were evaluated. Oxygen transfer limitation was predominant when the fermentation was carried out at low agitation rate (100rpm). At agitation rate of 300rpm, the high shear stress was dominant. Decrease chitinase production and extensive sporulation along with cell degradation were observed. When the pH was controlled at a constant (6.0–9.0) during the cultivation, lower chitinase production was obtained. The operation conditions at an aeration rate of 3vvm, an agitation rate of 200rpm, and without pH control could yield an optimal chitinase activity level of 11.8U/ml.

Keywords: Chitinase; Stirred-tank bioreactor; Paenibacillus; sp.; Shear stress; Mass transfer

Optimization of solid-state medium for the production of inulinase by Kluyveromyces S120 using response surface methodology by Chen Xiong; Wang Jinhua; Li Dongsheng (pp. 179-184).

The optimization of nutrient levels for the production of inulinase by a newly strain Kluyveromyces S120 in solid-state fermentation (SSF) was carried out using response surface methodology (RSM) based on Plackett–Burman design and Box–Behnken design. In the first optimization step, a Plackett–Burman design was used to evaluate the influence of related factors. Inulin, corn steep liquor and (NH₄)₂SO₄ were found to be more compatible supplement with the substrate of wheat bran and positively influenced on inulinase production. In the second step, the concentrations of the three nutrients above were further optimized using a Box–Behnken design. The final concentration of medium optimized with RSM was 12.72% inulin, 10.76% corn steep liquor and 1.61% (NH₄)₂SO₄ by employing wheat bran as the solid substrate. The average inulinase activity (409.8U/g initial dry substrate) in triplicate under optimal medium was obtained.

Keywords: Response surface methodology; Optimization; Kluyveromyces; S120; Inulinase production; Solid-state fermentation

Alkaline protease production by submerged fermentation in stirred tank reactor using Bacillus licheniformis NCIM-2042: Effect of aeration and agitation regimes by Ravichandra Potumarthi; Subhakar Ch.; Annapurna Jetty (pp. 185-192).

The effects of aeration and agitation on alkaline protease production and protease yield were studied by submerged fermentation in a batch STR using Bacillus licheniformis NCIM-2042. The agitation rate varied in the range of 200–400rpm at each airflow rates of 1–3vvm. The maximum protease production was found on third day (72h) and then decreased during fourth and fifth day of operation in all batches of STR operation. The best combination of airflow and agitation rate for the present system was at 3vvm of airflow rate and 200rpm of agitation rate on the basis of maximum specific protease production rate. Maximum specific protease production of 102U/mg DC was observed on third day at 3vvm and 200rpm. The effects of casein concentrations on protease production, viscosity changes and oxygen transfer rate were studied. The increase in casein concentration has resulted in increased viscosity of the fermentation broth with increase in time of bioreactor operation, which in turn resulted in decreased oxygen mass transfer coefficient with reduced oxygen transfer rates.

Keywords: Abbreviations; STR; stirred tank reactor; rpm; revolution per minute; vvm; volume of air per volume of media per minute; DO; dissolved oxygen; OTR; oxygen transfer rate; DCW; dry cell weightStirred tank reactor; Protease production; Yield; Viscosity; Volumetric oxygen mass transfer coefficient; Limiting substrate

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Biochemical Engineering Journal (v.34, #2)