Biochemical Engineering Journal (v.5, #1)

The immobilized cells of five bacterial cultures on different carriers were investigated for the production of cyclodextrin glucosyltransferase (CGTase). The entrapped cells of Bacillus amyloliquefaciens in calcium alginate showed the highest enzyme activity (70.8 U ml−1). The enzyme production with respect to alginate concentration, bead diameter, and maximal cell loading in the immobilization matrix was optimized. In repeated batch fermentation, the immobilized cells retained their ability to produce CGTase consistently over 14 cycles and the activity remain between 70 and 88 U ml−1 throughout the cycles. Continuous culture was investigated in packed-bed and fluidized-bed reactors. In packed-bed reactor, maximal productivity (23 KU l−1  h−1) with enzyme concentration of 48 U ml−1 and specific productivity of 141.8 U g wet cells−1  h−1 was attained at a dilution of 0.48 h−1. Continuous production in fluidized-bed reactor showed maximal productivity (30.4 KU l−1  h−1) with enzyme concentration of 53.0 U ml−1 and specific productivity of 230.9 U g wet cells−1  h−1 at a relatively high dilution rate of 0.57 h−1.
Keywords: Cyclodextrin glucosyltransferase; Production; Immobilized cells; Continuous;

Selective desulfurization of dibenzothiophene by newly isolated Corynebacterium sp. strain P32C1 by S. Maghsoudi; A. Kheirolomoom; M. Vossoughi; E. Tanaka; S. Katoh (11-16).
Corynebacterium sp. strain P32C1 was selected from 30 strains isolated from soil samples of various areas in Iran on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. During 27 h of cultivation in a jar-fermentor, DBT with an initial concentration of 0.25 mM was completely converted to 2-hydroxybip henyl (2HBP). Concentration change of 2′-hydroxybiphenyl-2-sulfinate (HBPS) during cultivation showed the general characteristics of reactions in series, DBT → HBPS → 2HBP. With resting cells prepared in late exponential phase, DBT of 0.5 mM was completely converted to 2HBP during 30 min, and the maximum specific production rate of 2HBP was 37 mmol/(kg dry cells h). Fed-batch addition of DBT in the jar-fermentor resulted in growth continuing up to a high 2HBP concentration of 0.4 mM. Desulfurization ability of P32C1 strain was compared with Rhodococcus sp. IGTS8 as a standard strain, and it was thought that P32C1 strain had a higher desulfurization activity.
Keywords: Biodesulfurization; Dibenzothiophene; Rhodocuccus sp. IGTS8; Corynebacterium sp. P32C1;

In spite of minor limitations due to membrane fouling and catalyst inactivation, one-step hydrolysis of starch with termamyl enzyme in a continuous recycle membrane reactor appears to be a promising way to produce high DE sugar syrups. In addition to the main advantage over batch process to correctly integrating reaction and separation, this type of system opens up to process engineer tunable performance in terms of conversion, capacity and productivity. In this paper the effects of enzyme concentration and space time have been examined. As a whole it appears that by increasing these two parameters the conversion is improved; but the capacity and productivity are reduced leading to higher processing costs. Optimisation of enzyme concentration and space time needs that economic criteria are very carefully taken into account.
Keywords: Membrane bioreactor; Starch; Hydrolysis; Enzyme; Termamyl;

The volumetric mass transfer coefficient k L a is a very important parameter that plays an important role in bioreactor design. Numerous authors have reported that the use of simple models not taking into account the dispersion effects in the gas and/or liquid phase may cause a large error in k L a estimation. We have analysed the effect of another factor on k L a determination, namely, the effect of the pressure profile along the vessel height. We propose a two-phase dispersion model which takes into account the pressure profile. We studied the sensitivity of various parameters to error caused by neglecting the pressure profile. It was found that even when applying an experimental technique that eliminates dispersion effects, the existence of the pressure profile needs to be taken into account, especially in large volume tanks with a non-ideal flow pattern in the liquid phase. We propose a simple correlation describing the error caused by neglecting the pressure profile. The proposed procedure can be used to correct all mass transfer coefficients evaluated by a model not taking into account the pressure profile.
Keywords: Gas–liquid mass transfer; Volumetric mass transfer coefficient k L a; Axial dispersion; Pressure profile; Modelling; Bioreactors;

A single continuous-flow fluidized-bed bioreactor system consisting of porous carrier particles for retaining microbes was constructed to simultaneously remove carbonaceous and nitrogenous substances in wastewater under different C/N (mass ratio) values. The suspended microbial concentration in the bioreactor was extremely low compared with that of retained microbes. A TOC removal of >91% and a maximum total nitrogen removal of 85% were achieved under a moderate C/N value.By using a set of simplified reaction kinetics, the multiple microbial reactions involved in the simultaneous removal process of carbonaceous and nitrogenous components were analyzed. The related kinetic parameters in terms of carbonaceous and nitrogenous loadings were obtained.
Keywords: Biokinetics; Bioreactions; Nitrogen removal; Fluidized-bed bioreactor; Organic removal; Wastewater treatment;

Some investigations on bioflocculant producing bacteria by H Salehizadeh; M Vossoughi; I Alemzadeh (39-44).
Bacillus sp. As-101 which produces flocculating substances was isolated from activated sludge, using a proteolytic enzyme. Bioflocculant As-101 was partially purified by cold ethanol and cetylpyridinium chloride. The partially purified flocculating substance was an acidic polysaccharide including uronic, pyruvic and acetic acids, respectively, in an approximate ratio of 11.4%, 6.1%, 0.4%. The biopolymer is also a sugar-protein derivative consisting of glucose, mannose and galactose. The protein, the sugar contents ratio is about 83–17%. Bioflocculant As-101 acted under acidic condition and the maximum activity was observed at optimum pH ∼3.7 and optimum concentration of 30 ppm in kaolin suspension. The flocculating activity was stimulated by the addition of Al3+, Fe3+ and Ca2+ in optimum concentrations of 0.2, 0.25 and 8 mM, respectively. The flocculating activity was linearly decreased on increasing the heating period and was inactivated after 8 min of heating.
Keywords: Bioflocculant; Flocculating activity; Biopolymer; Bacillus;

The selective separation of Penicillin V from phenoxyacetic acid using extraction and transport through liquid membranes consisting of 1,2-dichloroethane and Amberlite LA-2 as carrier was studied. The selective separation is the result of the difference between the acidity and hydrophobicity of the two compounds.The experimental data indicated the decisive influences of pH (difference between the feed and stripping phases), carrier concentration in solvent phase (0–80 g/l Amberlite LA-2) and mixing intensity. Thus, the selectivity factor, defined as the ratio between the final mass flows of Penicillin V and phenoxyacetic acid, was enhancing by reducing the pH gradient between the aqueous solutions and by using low concentrations of carrier inside the liquid membrane, in the conditions of high stirrers rotation speed values. The selectivity factor value varied between 2.5 and 80.4.
Keywords: Liquid membrane; Penicillin V; Phenoxyacetic acid; Amberlite LA-2;

An efficient fermentation process for the production of phytase was developed by statistical optimization from recombinant Escherichia coli BL21. The seed age and inoculum level highly influenced the expression of phytase gene by lactose induction. The optimum of 3.15-h seed culture age instead of an overnight culture and 4.8% (v/v) seed culture yielded high concentrations of cells at short intervals of time for efficient induction. Induction at a lactose concentration of 7.28 mM and at a cell optical density of 0.55 at 550 nm (equivalent to 0.23 g/l dry cell weight) was found to be most suitable in this system. The time of induction and lactose concentration significantly influenced phytase production. The optimized conditions were used to achieve 100% increase in phytase production (174 U/l·h) compared to production before optimizing the conditions.
Keywords: Phytase; Statistical optimization; Fermentation; Lactose; Enzyme production;

Disruption and protein release kinetics by ultrasonication of Acetobacter peroxydans cells by Hamdi Kapucu; Nursel Gülsoy; Ülkü Mehmetoǧlu (57-62).
In this study, the disruption and protein release kinetics of Acetobacter peroxydans by ultrasonication was examined. The effects of acoustic power and the medium pH on the survival percent was investigated. An increase in the cell disruption with increasing acoustic power was observed. The most effective disruption was achieved for 100 W acoustic power. When the pH of ultrasonication medium was increased, the rate of disruption decreased. Within the pH range of 3–10, the most effective value for the cell disruption was determined to be 3. The effects of acoustic power and medium pH on the kinetic constant, β, which determines the pattern of failures according to the equation proposed by Doulah were investigated. The value of rate constant of the protein release was found to be 0.022 min−1 using the first order protein release kinetic equation proposed by Doulah.
Keywords: Cell disruption; Acetobacter; Protein recovery; Kinetic parameters;

Microbial lipase from Candida rugosa was immobilised by physical adsorption onto styrene–divinylbenzene (STYDVB) copolymer. Two different coupling media were evaluated: aqueous solution (sodium phosphate buffer) and organic solvent (heptane). The choice of the immobilisation medium had a large effect on the hydrolytic and synthetic lipase activities. Better results were found when the coupling procedure was performed in the presence of heptane. The effects of enzyme loading and support capacity regeneration were also studied. Both, free and immobilised lipases were characterised by determining the activity profile as a function of pH, temperature and thermal stability. For the immobilised lipase, operational and storage stabilities were also determined. Operational stability tests indicated that a small enzyme deactivation occurs after 12 consecutive batches of 24 h each. Such results revealed good potential for recycling under non-aqueous system.
Keywords: Lipase; Styrene–divinylbenzene copolymer; Aqueous and non-aqueous environment; Hydrolytic and esterification activities;

In this paper we report on the structural changes to the milk protein β-lactoglobulin induced by the combined effects of pressure and temperature. A series of treatment combinations were studied using pressures up to 100 MPa, temperatures up to 75°C and pH values of 5.6 and 7.0 as being appropriate to milk. Examination of the pressure-temperature treated protein by circular dichroism, differential scanning calorimetry (DSC) and spectrofluorometry indicated that sufficient energy had been applied to disrupt irreversibly the molecular structure at both the secondary and tertiary level caused by both pressure and temperature. Pressure and temperature effects were observed most clearly to changes in the tertiary structure using circular dichroism and DSC for which complete loss of structure was observed following treatment at 100 MPa and 75°C at pH 7.0 and 72% loss at pH 5.6. Combined pressure and temperature effects appeared to have less effect to the secondary structure and on the temperature of denaturation. The mechanism by which pressure and temperature function in combination, however, is not clear.
Keywords: Denaturation; Protein structure; Food processing; High pressure;

The adsorption behaviour of immobilised tannin towards 14 different proteins including albumins, globulins, schleroproteins, phosphoproteins, chromoproteins, glycoproteins and some enzymes has been studied. pH and protein concentration have been varied in the range of 4–7 and 0.05–1.0 mg/ml respectively. For most of the proteins under present investigation bell-shaped curves are obtained when the adsorption selectivity of immobilised tannin at a protein concentration of 0.25 mg/ml is plotted against pH. A paraboloc equation is proposed to explain this behaviour. For two proteins, namely, concanavalin A (a globulin) and gelatin (a schleroprotein) the combined effect of bulk phase protein concentration and pH on adsorption selectivity have been mathematically described by coupling a parabolic function with the conventional Langmuir isotherm. The simulated results have been compared with the experimental observations.
Keywords: Immobilised tannin;

A membrane bioreactor with novel modules for effective biodegradation of toluene by Zeyi Xiao; M.Hatta Dahlan; X.-H Xing; Yumi Yoshikawa; K Matsumoto (83-88).
Two novel membrane modules, tubular and spiral type with numerous hollow fibers of silicone rubber were incorporated into a bioreactor system for effective degradation of volatile organic chemicals (VOC) in wastewater. Biodegradation and transfer of toluene as a model VOC were studied in the membrane bioreactors using Pesudmonas putida mt-2 as the degrader. The overall mass-transfer coefficient of toluene across the membranes was estimated to be 4.8×10−7  m/s for the tubular type module and 4.6×10−7  m/s for the spiral one. Both bacterial growth and toluene degradation in the two types of membrane bioreactors were the same as those obtained from the culture under an ideal condition. The mass transfer of toluene across the membranes was not a rate-limiting factor for the bacterial degradation of toluene.
Keywords: Biodegradation; Mass transfer; Membrane bioreactor; Volatile organic chemicals; Wastewater treatment;