Biochemical Engineering Journal (v.23, #2)

BEJ Keywords (II).


A miniature (2 ml) bioreactor has been developed for the high throughput cultivation of microorganisms. The microplate bubble column bioreactor (MBCB) consists of a static deep well microtiter plate with polyethylene frits (pore size 20 μm) inserted into the base of each of the 48 wells. Air was supplied at a controlled flow rate to the underside of each well and in this way the air passing through the pores of the frit formed bubbles in the liquid biomedium. A volumetric mass transfer coefficient for oxygen (k L a) of up to 220 h−1 could be achieved in this novel system. Since the system was not shaken, as is usually required with cell cultivations in microtiter plates, its performance was simpler to model and predict. A miniature optical oxygen probe was used to measure the dissolved oxygen concentration and it was found that the derived k L a values were proportional to the superficial gas velocity over the range 0–0.02 m s−1. The usefulness of this new device was demonstrated by carrying out 25 parallel experiments to optimise the growth conditions of Bacillus subtilis ATCC6633, a strict aerobe, over a range of pH values and C/N ratios.
Keywords: Bioreactors; Bubble columns; Gas–liquid mass transfer; Fermentation; Mass transfer;

A 23 factorial design with a central point was utilized to develop a multi-response model of the kinetics of the transesterification reaction between menhaden oil and the ethyl ester of conjugated linoleic acid in the presence of an immobilized lipase from Rhizomucor miehei (Lipozyme L9). The factors considered were the molar ratio of reactants, the enzyme loading, and temperature. Rate expressions were developed to describe both the release of the individual fatty acid residues present in menhaden oil and the incorporation of conjugated linoleic acid (CLA) residues in acylglycerol species. These mathematical models were of the general Michaelis–Menten form characteristic of a ping–pong bi–bi mechanism.
Keywords: Conjugated linoleic acid; Fish oil; Transesterification; Rhyzomucor miehei lipase; Omega-3 fatty acids; Enzyme biocatalysis; Biosynthesis; Immobilized enzyme;

A novel and effective bioreactor with two dynamic changes of air (including air pressure pulsation and internal air circulation) was developed and tested. It could enhance the heat transfer and effectively control the temperature variation in the process of solid-state fermentation (SSF) for cellulase production through changing the pressure range of air pulsation, air pulsation period and rate of internal air circulation and so on. After the optimization of the operating conditions, the maximum temperature gradient in the dynamic system with a bed height of 9.0 cm was 0.12 °C cm−1.
Keywords: Bioreactor; Two dynamic changes of air; Heat transfer; Solid-state fermentation; Optimization;

To investigate the effect of adding large size inert materials into natural organic packing media in waste gas treatment, two biofilters, one packed with buckwheat hulls and inert spheres and the other packed with buckwheat hulls only, were used in parallel experiments to treat toluene gas for 305 continuous days. The toluene removal capacities and pressure drops of the two biofilters were compared during the experiments. The physical properties of the filter beds were also investigated by pulse injection technique combined with a mathematical model. In the late phase of the operation with a sufficient supply of nutrients, the biofilter with inert spheres showed a higher toluene removal capacity than the biofilter without inert spheres. This was due to the fact that the biofilter with inert spheres had less bed compaction and a greater increase in specific surface area. The pressure drop of the biofilter with inert spheres was also significantly lower and more stable than that of the biofilter without inert spheres due to its higher bed void fraction. All the experimental results quantitatively showed that adding inert spheres into the filter beds could improve the performance of the biofilter under long-term operation.
Keywords: Biofilter; Bed compaction; Inert spheres; Pressure drop; Toluene gas;

A waste reduction system for the production of useful materials from un-utilized plant biomass was developed for zero emission using a steam explosion, extraction and separation, and various conversion methods. This system utilizes a significant conversion approach without generating pollutants, i.e., waste gas, wastewater, and solid waste materials. The conversion of un-utilized bamboo (Phyllostachys pubescens) into useful materials was studied for the effective utilization of its components such as holocellulose, water-soluble material, methanol-soluble lignin, and Klason lignin. These components were converted into useful materials, i.e., lactic acid, antibacterial violet pigment, methane gas, monosaccharides and oligosaccharides, epoxy resin without estrogenic activity, or activated carbon. The amount of useful materials and products produced from each component was estimated, and the reduction in the amount of pollutants generated in their production process and waste treatment and the detoxification of the lignin resin were confirmed.
Keywords: Bioconversion; Cellulose; Hemicellulose; Lignin; Un-utilized bamboo; Waste treatment;

A general mathematical model has been developed for predicting the performance and simulation of a packed-bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis–Menten kinetics with competitive product (galactose) inhibition. The performance characteristics of a packed-bed immobilized enzyme reactor have been analyzed taking into account the effects of various diffusional phenomena like axial dispersion, internal and external mass transfer limitations. The model design equations are then solved by the method of weighted residuals such as Galerkin's method and orthogonal collocation on finite elements.The effects of intraparticle diffusion resistances, external mass transfer and axial dispersion have been studied and their effects were shown to reduce internal effectiveness factor. The effects of product inhibition have been investigated at different operating conditions correlated at different regimes using dimensionless β xo (St, θ, ϕ). Product inhibition was shown to reduce substrate conversion and to decrease internal effectiveness factor when β s  >  β xo, however it increases internal effectiveness factor when β s  <  β xo. The effectiveness factor is found to be independent of product inhibition at crossover point at which β xo is defined. Effects of St and Pe have been investigated at different kinetic regimes and the results show their effects have a strong dependence on kinetic parameters θγ (i.e. K m/K p ) and β xo.
Keywords: Biocatalysis; Enzyme bioreactors; Immobilisation; Immobilised enzymes; Product inhibition; Internal mass transfer; Effectiveness factor;

Heparinase I (EC is one of the three heparinases purified from Flavobacterium heparinum that cleaves certain sequences of heparin/heparan sulfate specifically. Previous reports have shown that this enzyme expressed in recombinant Escherichia coli was highly prone to aggregate into inactive inclusion bodies even by fusion to cellulose-binding domain (CBD). In this paper, we fused heparinase I to maltose-binding protein (MBP) and expressed the fusion protein in E. coli to develop an expression system of soluble heparinase I. As a result, about 90% of the fusion protein (abbreviated as MBP-hepA) was soluble when expressed in the recombinant E. coli and the fusion protein could reach about 100 mg l−1 with an activity of 88.3 U l−1OD600 −1. To our knowledge, this is the first time to produce soluble heparinase I at such a high yield.
Keywords: Enzyme production; Fusion protein; Heparinase I; Maltose-binding protein (MBP); Protein recovery; Soluble expression;

Influence of alcohol addition on gas hold-up, liquid circulation velocity and mass transfer coefficient in a split-rectangular airlift bioreactor by N. El Azher; B. Gourich; C. Vial; M. Soulami Bellhaj; A. Bouzidi; M. Barkaoui; M. Ziyad (161-167).
In this work, the effect of alcohol addition on gas hold-up, liquid circulation velocity and gas–liquid volumetric mass transfer coefficient was studied in a split-rectangular airlift bioreactor using air-water as a system to which propanol was added in concentrations ranging from 0.01 to 0.1% (v/v). In order to compare the effect of the propanol with other alcohols, methanol and butanol were also used. The experimental results showed that, at high superficial gas velocities, the addition of small amounts of alcohols decreases the difference between the gas hold-up in the riser and the downcomer, which is due to smaller bubble diameter than with the tap water system. This behaviour is enhanced by alcohols with long carbon chain lengths, which has been explained on the basis of surface tension effects. As a result, the addition of alcohol solutions results in a significant decrease in the liquid circulation velocity, which is mainly due to the decrease in the circulation driving force, but also in a strong decrease in the volumetric gas–liquid mass transfer coefficient for superficial gas velocities higher than 0.033 m/s. This surprising result has been explained by a strong decrease in K L values due to the presence of alcohol and by oxygen depletion due to the increasing amount of small recycled bubbles at high superficial gas velocity.
Keywords: Airlift bioreactor; Alcohol addition; Hydrodynamics; Gas–liquid mass transfer;

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha by Jin Seol Kim; Bong Hee Lee; Beom Soo Kim (169-174).
Ralstonia eutropha ATCC 17699 was grown to produce poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from fructose and γ-butyrolactone. Through flask cultures, it was found that R. eutropha cells were inhibited by high concentrations of fructose (more than 20 g/L) and γ-butyrolactone (more than 6 g/L). The 4HB fraction could be increased from 37.9 to 53.7 mol% by adding small amount (2 g/L) of acetate or propionate as a stimulator. Fed-batch cultures (DO-stat and constant feeding) were carried out to improve cell and PHA concentration. In the first step, cells were grown to 38–48 g/L by DO-stat feeding of fructose. Nitrogen limitation was applied and the fructose feed was replaced with the mixture of fructose and γ-butyrolactone. The effects of γ-butyrolactone to fructose ratio in the second step feeding solution were examined. As the ratio increased, 4HB fraction increased, while cell and PHA concentration, PHA content, and productivity decreased. The results obtained were in the range of 33.6–49.1 g/L of cell concentration, 13.8–24.4 g/L of PHA concentration, 38.8–50.2% of PHA content, 1.64–25.2 mol% of 4HB fraction, and 0.32–0.55 g/L/h of productivity.
Keywords: Poly(3-hydroxybutyrate-co-4-hydroxybutyrate); Ralstonia eutropha; Fed-batch culture; Fermentation; Microbial growth;

In this study, the central composite design and the response surface methodology (RSM) were applied to determine the optimum physiological conditions for the production of volatile fatty acids (VFA) and H2 from sucrose-rich wastewater by mixed anaerobic culture. The experimental results show the acidogenesis was affected by pH, temperature, and substrate concentration individually and interactively. The interaction between temperature and pH on the VFA and H2 production was significant, whereas there was no interaction between pH and sucrose concentration, temperature and sucrose concentration. The optimum conditions were pH 5.6, temperature 33.5 °C and sucrose concentration 24.2 g l−1 for total VFA yield; pH 5.5, temperature 33.1 °C and sucrose concentration 24.4 g l−1 for butyrate yield; pH 5.5, temperature 35.1 °C and sucrose concentration 26.0 g l−1 for H2 yield. The RSM was demonstrated as an appropriate approach to the optimization of acidogenesis.
Keywords: Acidogenesis; pH; Response surface methodology (RSM); Substrate concentration; Sucrose; Temperature;

Study on biosorption of Cr(VI) by Mucor hiemalis by Neetu Tewari; P. Vasudevan; B.K. Guha (185-192).
Many investigations have been carried on metal binding capacity of different groups of microorganisms. However, the reports on the kinetic, thermodynamic and desorption study of biosorption process are quite limited. The present study was carried out in a batch system using Mucor hiemalis for its sorption and desorption study of Cr(VI). M. hiemalis exhibited the highest Cr(VI) uptake of 53.5 mg/g at an initial pH of 2.0. Equilibrium data fitted well to Langmuir isotherm model. Biosorption showed pseudo-second order rate kinetics at different initial concentration of Cr(VI) and different dose of M. hiemalis. The activation energy of the biosorption (E a) was estimated as 4.0 kJ/mol using Arrhenius equation. Using the equilibrium constant value obtained at different temperature, the thermodynamics properties of the biosorption (ΔG°, ΔH° and ΔS°) were also determined. The biosorption of Cr(VI) onto M. hiemalis was found to be endothermic. Desorption data showed that nearly 99% of the Cr(VI) adsorbed on M. hiemalis could be desorbed using 0.1N NaOH. Study with the cyclic use of a batch of M. hiemalis repeatedly after desorption, showed that it retain its activity up to five sorption and desorption cycles.
Keywords: Adsorption; Biosorption; Bioremediation; Mucor hiemalis; Wastewater treatment; Chromium; Desorption;