Check out our New Publishers' Select for Free Articles

Biochemical Engineering Journal (v.39, #1)

Advert Announcement (pp. i).

BEJ Keywords (pp. v).

Editorial Board (pp. co2).

Influence of hydrodynamics and probe response on oxygen mass transfer measurements in a high aspect ratio bubble column reactor: Effect of the coalescence behaviour of the liquid phase by B. Gourich; Ch. Vial; N. El Azher; M. Belhaj Soulami; M. Ziyad (pp. 1-14).

The usual gassing-in and gassing-out method was applied in a high aspect ratio bubble column using both tap water and coalescence-inhibiting liquid mixtures that represent the coalescence behaviour of biological media. The oxygen concentration curves were analyzed on the basis of different hydrodynamic models available from the literature, taking into account several parameters such as sensor dynamics, mixing in both phases, oxygen depletion in the gas phase or the axial evolution of pressure in the column. The sensitivity of K_L a_L values to the assumptions of each model was examined first in relation to physical considerations, but also as a function of the sensitivity of the optimization procedure. The results showed that the conventional method consisting in cutting the beginning of the oxygen response curves along with an assumption of perfect mixed reactor had to be avoided both in tap water and coalescence-inhibiting mixtures, except at low superficial gas velocity. They showed also that this simple model could not be improved by taking sensor dynamics into account. Conversely, several characteristic times and a dimensionless pressure ratio were established in order to define general quantitative criteria able to determine the best model for reliable K_L a_L estimation from dynamic oxygen response curves with the aim to contribute to design and scale-up of bubble column bioreactors.

Keywords: Bubble column; Hydrodynamics; Mass transfer; Oxygen sensor dynamics; Coalescence behaviour

Isolation and purification of bacterial poly(3-hydroxyalkanoates) by Nicolas Jacquel; Chi-Wei Lo; Yu-Hong Wei; Ho-Shing Wu; Shaw S. Wang (pp. 15-27).

The isolation and the purification of bacterial polyhydroxyalkanoates are the key step of the process profitability in the fermentation system. That is why many scientists have studied this field for the production of this biodegradable polymer. The ideal method should lead to a high purity and recovery level at a low production cost. This paper reviews four isolation methods, i.e. solvent extraction of halosolvent and nonhalosolvent, digestion of non-polyhydroxyalkanoate cell material involving surfactants, sodium hypochlorite or enzyme, mechanical cell disruption methods like using bead mills and high pressure homogenization, and new methods like spontaneous liberation of poly(3-hydroxybutyrate), dissolved air flotation, air classification, or by using supercritical CO₂. The pretreatment of cell disruption and the purification methods and analytical methods of polyhydroxyalkanoates are also presented.

Keywords: Biopolymers; Polyhydroxyalkanoates; Recovery; Isolation; Purification; Bacteria

Inner-profiles of a spherical bio-carrier determined by microelectrodes by Xiao-Hong Zhou; Han-Chang Shi; Yu-Qin Qiu (pp. 28-36).

The microbial activity within bio-carrier is a key factor in the performance of biofilm reactor. In this paper, a spherical bio-carrier comprised of a netlike shell, plastic stuffing, and a perforated hollow duct was introduced. We examined four runs of biofilm processes for simultaneous nitrification and denitrification of the bio-carrier. The heterogeneity of microbial processes and the impact of substrate, oxygen concentrations, as well as the hollow duct on the inner environment of the bio-carrier was determined by microelectrodes for DO, ORP, NH₄⁺ and NO₃⁻. Clear evidence was found that aerobic/anoxic zones could be concurrently in existence at run 2 and run 4. Due to microbial oxygen utilization, the aerobic zone was limited to the surface layer (14–17mm) of the bio-carrier. Although the anoxic zone disappeared at a lower substrate loading and a higher oxygen concentration in the bulk solution, the bio-carrier had great potential for denitrification as long as the quantity of organic substances was not the limiting factor. The contaminant and oxygen concentrations in the bulk solution were correlated with the oxygen uptake rate, which directly influenced the oxygen penetration in the bio-carrier, thus resulting in redox potential changes correspondingly. Different from the DO profiles, the redox potential reflected a comprehensive result of the mixed microbial and chemical activities in the bio-carrier. Moreover, the measured NH₄⁺ and NO₃⁻ profiles showed the obvious nitrification dominated in the surface layer while denitrification occurred in the anoxic zone in the bio-carrier. Based on the kinetics of transport and reaction processes, the calculation of oxygen transport coefficients demonstrated that the intra-carrier convection, which was influenced significantly by the bulk oxygen concentration, prevailed against the molecular diffusion in the bio-carrier.

Keywords: Bio-carrier; Simultaneous nitrification and denitrification; Microelectrode; Wastewater; Convection; Diffusion

Response surface optimization of medium composition for alkaline protease production by Bacillus clausii by Seyedeh Faranak Ghaemi Oskouie; Fatemeh Tabandeh; Bagher Yakhchali; Fereshteh Eftekhar (pp. 37-42).

A culture medium for production of bacterial alkaline protease was developed following preliminary studies by means of response surface method. Central composite design was applied to optimize the medium constituents and explain the combined effects of three medium constituents, viz., sucrose, yeast extract and potassium nitrate. The optimum values for the tested variables were; sucrose 11gl⁻¹, yeast extract 5gl⁻¹ and KNO₃ 5.2gl⁻¹. A second-order model equation was suggested and then validated experimentally. The model adequacy was very satisfactory as the coefficient of determination was 0.94. The maximum alkaline protease production was 1520±35Uml⁻¹at 42h of incubation which showed about 1.5-fold increase in protease production over the central point and an overall 6-fold enhancement (1520–250Uml⁻¹) over the basal medium.

Keywords: Alkaline protease; Bacillus clausii; Medium optimization; Response surface methodology

Characterization of three-phase partitioned exo-polygalacturonase from Aspergillus sojae with unique properties by Nergiz Dogan; Canan Tari (pp. 43-50).

Exo-polygalacturonase enzyme produced by Aspergillus sojae ATCC 20235 was purified using three-phase partitioning (TPP), an emerging bio-separation technique where a single step as compared to the classical multi-step purification was used. Using this technique, crude enzyme solution (pH 6.6) saturated to 30% (w/v) with ammonium sulphate and with a crude extract to tert-butanol ratio of 1:1 (v/v) at 25°C resulted in 25.5% recovery of exo-polygalacturonase with a 6.7-fold purification. The purified enzyme was characterized with respect to its activity and stability at various pH and temperature ranges. Optimum pH and temperature for maximum activity were determined as pH 4 and 55°C. The enzyme was stable at both acidic and alkaline pH for 2h at 30°C. The thermal stability study showed that the purified enzyme had an inactivation energy of 68.41kcal/mol and a half-life ( t_1/2) value of 3.6h at 75°C presenting a large thermal stability. The kinetic constants K_m and V_max using polygalacturonic acid as substrate were 0.75gl⁻¹ and 1.14μmolmin⁻¹, respectively. SDS-PAGE profiling revealed that the purified exo-polygalacturonase had two bands with the molecular weights of 36 and 53kDa. The enzyme was completely inhibited in the presence of Mn²⁺ and SDS and induced significantly by EDTA, glycerol and β-mercaptoethanol.

Keywords: Exo-polygalacturonase; Three-phase partitioning; Aspergillus sojae; Microbial enzymes; Enzyme characterization

Average shear rate for non-Newtonian fluids in a concentric-tube airlift bioreactor by M.O. Cerri; L. Futiwaki; C.D.F. Jesus; A.J.G. Cruz; A.C. Badino (pp. 51-57).

In the present study, a simple methodology for evaluating the average shear rate (γ˙av) in an internal concentric-tube airlift reactor has been developed for non-Newtonian systems. The volumetric oxygen transfer coefficient ( k_L a) was chosen as the appropriate characteristic parameter to evaluate the average shear rate (γ˙av) because the oxygen transfer occurs through the interfacial area of the air bubbles distributed evenly throughout the bioreactor. The average shear rate (γ˙av) values of up to 9300s⁻¹ were estimated as a function of the gas velocity in the riser (0.0094≤ U_GR≤0.0943ms⁻¹), of the rheological parameters of the fluid (0.180≤ K≤1.833Pasⁿ and 0.234≤ n≤0.461), and of the volumetric oxygen transfer coefficient (0.0056≤ k_L a≤0.0609s⁻¹). Profiles of theγ˙av as a function of the gas velocity in the riser ( U_GR) displayed different behaviors from those of the literature sinceγ˙av increased up to a maximum value and then decreased with an increase in the U_GR. The shear rate estimated by the proposed methodology lay within the range of the correlation values in the literature. Unlike other correlations in the literature, the average shear rate in the present study (γ˙av) is estimated considering the rheological parameters of the fluid, the volumetric oxygen transfer coefficient ( k_L a), and the mass transfer parameter, which is a function of the operating conditions and the geometry of the bioreactor.

Keywords: Airlift; Shear rate; Volumetric oxygen transfer coefficient; Non-Newtonian fluid

A short recursive procedure for evaluating effectiveness factors for immobilized enzymes with reversible Michaelis–Menten kinetics by J.L. Gómez Carrasco; A. Bódalo Santoyo; E. Gómez Gómez; J. Bastida Rodríguez; M.F. Máximo Martín; M. Gómez Gómez (pp. 58-65).

A short recursive procedure for calculating the effectiveness factor for enzymes immobilized in porous spherical particles is presented. The method is mathematically simple and very precise; it is valid for reversible Michaelis–Menten kinetics including, as particular situations, simple Michaelis–Menten and product competitive inhibition kinetics.The procedure is a modification of the two-parameter model previously published by the authors. The definition of an auxiliary dimensionless concentration leads to a recursive equation which simplifies the resolution of the model by means of elementary numerical methods. The solution algorithm has been transformed into a computer program, whose source code is appended.The exact values of the effectiveness factors for zero and first order kinetics, as a function of the Thiele module, were compared with those obtained with the numerical procedure proposed in this work. The good agreement between both results demonstrates the validity of the method.

Keywords: Effectiveness factor; Immobilized enzymes; Diffusion-reaction; Mathematical model

Optimization of cultivation conditions for extracellular polysaccharide and mycelium biomass by Morchella esculenta As51620 by Hui Xu; Lan-Ping Sun; Ya-Zhong Shi; Ya-Hua Wu; Bin Zhang; Da-Qing Zhao (pp. 66-73).

The quantitative effects of fermentation time, temperature and broth content on mycelial growth in terms of dry cell weight and extracellular polysaccharide content produced by the fungus Morchella esculenta As51620 in submerged fermentation were investigated separately using response surface methodology (RSM). The three factors chosen for the present investigation were based on the results of a previous Plackett–Burman (PB) design. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical methods. By solving the regression equation and also by analyzing the response surface contour plots, the optimal batch fermentation conditions were determined: under conditions of fermentation time 5.31 days, temperature 23.46°C, broth content 59.53ml; the yield of extracellular polysaccharide reached 2427.93μgml⁻¹ of fermentation liquor. When fermentation time, temperature and broth content were 5.6 days, 25.13°C and 67.01ml, respectively, the yield of mycelium biomass reached 6.951mgml⁻¹. In order to obtain simultaneously the maximum yield of extracellular polysaccharide and mycelium biomass, the above conditions would be 4.71 days, 24.82°C and 62.64ml, and under these conditions, the maximum predicted yield of extracellular polysaccharide and mycelium biomass were 2318.52μgml⁻¹ and 6.921mgml⁻¹, respectively. These predicted values were also verified by validation experiments.

Keywords: Morchella esculenta; Extracellular polysaccharide; Optimization; Response surface methodology; Plackett–Burman design; Box–Behnken design

Cellular response to accumulation of recombinant proteins in the E. coli inner membrane: Implications for proteolysis and productivity of the secretory expression system by Balaji Balagurunathan; Guhan Jayaraman (pp. 74-83).

Several literature reports have indicated adverse effects on host cell growth and physiology due to secretory expression of recombinant proteins. In this work, extensive proteolysis of recombinant streptokinase was observed in the secretory expression system, along with severe growth impairment and reduced productivity as compared to intracellular expression in Escherichia coli. These phenomena correlated well with the accumulation of the secretory recombinant protein in the inner membrane and a corresponding up-regulation of an inner-membrane protease (probably ClpX). This protease was found to be activated only in the post-induction phase of the secretory expression system. A reduction in the cultivation temperature led to a significant decrease in the accumulation level of recombinant streptokinase in the inner membrane and a concomitant decrease in the inner-membrane protease activity. This resulted in a considerable enhancement in the overall productivity of the secretory expression system. The results demonstrate that the productivity of the secretory expression system is controlled by the accumulation level of the recombinant protein in the inner membrane. Beyond a particular accumulation level the cellular response triggered in the form impaired growth and enhanced proteolysis significantly affects the productivity of the system. In order to reduce the accumulation in the inner membrane, the synthesis rate needs to be tuned to the protein-specific translocation efficiency of the cell.

Keywords: Escherichia coli; Cellular stress response; Secretory expression; Inner-membrane protease; OmpA signal peptide; Co-translocational proteolysis

Enhancement of stability of recombinant streptokinase by intracellular expression and single step purification by hydrophobic interaction chromatography by Balaji Balagurunathan; Narkhede Sachin Ramchandra; Guhan Jayaraman (pp. 84-90).

Maintenance of stability, high biological activity and purity are essential requirements for recombinant therapeutic products and processes must be designed to meet these needs. This work addresses issues related to obtaining a high amount of stable recombinant streptokinase in its soluble form and a method to purify it in a single step with high levels of recovery and purity. The streptokinase gene without its native signal peptide was cloned and expressed in Escherichia coli, using the pRSETB expression vector. A significant amount of the expressed streptokinase was found in the soluble form in the cytoplasm. With an inducer concentration of 0.1mM IPTG, recombinant streptokinase accumulated to about 20% of the total soluble protein in the cell. The biomass-specific activity of recombinant streptokinase obtained in the intracellular expression system (∼20,000IU/mg DCW) was two-fold higher than the maximum reported in the literature. Furthermore, the expressed soluble protein was found to be highly stable. A higher concentration of the recombinant protein obtained from the intracellular system enabled single-step purification by hydrophobic interaction chromatography, with nearly 100% purity and 68% recovery. The whole process of expression and purification involved a minimal set of operations and would be very useful for economic production on a large scale of proteins like streptokinase which have therapeutic significance.

Keywords: Recombinant streptokinase; Escherichia coli; Intracellular expression; Single-step purification; Hydrophobic interaction chromatography

Application of response surface methodology in the permeabilization of yeast cells for lactose hydrolysis by Parmjit S. Panesar (pp. 91-96).

Enzymatic hydrolysis of lactose is one of the most promising biotechnological applications in the food industry, which can be used to solve the problems of lactose intolerant individuals. However, the industrial applications of enzymatic hydrolysis processes are being hampered, due to intracellular location of the yeast enzyme, which makes its extraction difficult and expensive However, the problem of enzyme extraction and poor permeability of cell membrane to lactose can be overcome using permeabilization technique. Response surface methodology was used to investigate the effect of different parameters (concentration, temperature and treatment time) on the permeabilization of Kluyveromyces marxianus cells. The experimentation was aimed to find the values of process variables to achieve maximal β-galactosidase activity by numerical optimization technique. The optimum operating conditions for permeabilization process were 49.6% (v/v) ethanol concentration, 23°C temperature and process duration of 18min. At these conditions of process variables, the predicted value of enzyme activity was found to be 1573IU/g. These permeabilized yeast cells displayed 89.7% lactose hydrolysis in skim milk after 150min of incubation time.

Keywords: Yeast; β-Galactosidase; Permeabilization; Response surface methodology; Lactose hydrolysis

Stress of pH and acetate on product formation of fermenting polysaccharide-rich organic waste by Fan Lü; Pin-Jing He; Li-Ming Shao; Duu-Jong Lee (pp. 97-104).

Effects of stress of pH and/or acetate on the fermentation product formation of polysaccharide-rich organic waste were examined in this work, with externally dosed acetate (up to 333mmoll⁻¹) and adjusted pH (5–9). Ethanol, acetate, and lactate were the main metabolites. Concentrations and dissociation states of acetate affected the productions of ethanol and lactate. With dosed acetate, ethanol was favorably produced at pH 6, but was completely inhibited at pH 5. On the other hand, lactate was less favorably produced with dosed acetate, but was also completely inhibited at pH 5. With no externally dosed acetate, the metabolism gradually shifted from acetate to lactate and ethanol-forming pathway with fermentation. With dosed acetate, except for at pH 5, the pathway to ethanol was strengthened with time. The acetate stress on product formation was stronger than from pH stress.

Keywords: Anaerobic processes; Fermentation; Acetic acid; Ethanol; Product inhibition; Anaerobic Digestion Model No. 1 (ADM1)

Elaboration of a biased random walk model with a high spatial resolution for the simulation of the microorganisms exposure to gradient stress in scale-down reactors by F. Delvigne; J. Destain; P. Thonart (pp. 105-114).

The success of a bioprocess scaling-up operation is dependent of the mixing operation. Indeed, mixing is important in order to provide favourable extracellular conditions for microorganisms growth or metabolites production. However, the increase of the volume of the bioreactor leads to mixing efficiency drop and thus to fluctuations at the level of the extracellular conditions. In the present work, this phenomenon has been studied by a biased random walk model (BRWM) able to reproduce the displacement of microorganisms inside the non-mixed part of a scale-down reactor (SDR). Considering its stochastic nature and the possibility to increase to a great extent its spatial resolution, the model is able to simulate the great diversity of circulation paths taken by the individual cells of a microbial population crossing the reactor. The model has been validated and is able to reproduce experimental residence time distributions (RTD). In a second time, the simulated circulation paths have been superimposed to dye mixing experimental data in order to obtain the concentration profile experienced by the microorganisms. This method of superimposition has been previously used to characterize the dissolved oxygen and substrate gradients experienced by a single microbial cell in large-scale reactor [D. Vlaev, R. Mann, V. Lossev, S.D. Vlaev, J. Zahradnik, P. Seichter, Macro-mixing and Streptomyces fradiae: modelling oxygen and nutrient segregation in an industrial bioreactor, Trans. IChemE 78 (2000) 354–362]. The chemical engineering and physiological constraints have been discussed in order to fix an optimal spatial resolution for the model. Spectral analysis has highlighted two supplemental periodic components at the level of the simulated concentration profile when the spatial resolution of the model increases. However, it has been shown that there are a lot of biological implications that need to be considered in a future work.

Keywords: Scale-down; Bioreactors; Mixing; Modelling; Stochastic; Extracellular environment

Peptide synthesis in aqueous–organic media catalyzed by proteases from latex of Araujia hortorum ( Asclepiadaceae) fruits by Evelina Quiroga; Nora Priolo; David Obregón; José Marchese; Sonia Barberis (pp. 115-120).

An enzymatic preparation containing cysteine phytoproteases belonging to the papain family and isolated from the latex of Araujia hortorum Fourn. ( Asclepiadaceae) fruits was used as catalyst of the synthesis of the bitter dipeptide precursor Z-Ala-Phe-OMe in aqueous–organic media. Considering the good stability and activity of araujiain in different media, N, N-dimethylformamide (with low water content) and mixtures of the Tris–HCl buffer (0.1M, pH 8.5) and hexane, ethyl acetate or propanone in 50:50 ratio were selected to perform the synthesis of that peptide. The araujiain-catalyzed synthesis was carried out at 40°C using 2-mercaptoethanol as activator, TEA as neutralizing agent of the amino component (Phe-OMe·HCl) and different carboxylic components (Z-Ala, Z-Ala-OMe and Z-Ala- pNo). Under these conditions, araujiain only was able to form peptide bonds in biphasic media. The highest dipeptide conversion was obtained in the kinetically controlled synthesis using p-nitrophenyl ester derivative as carboxylic component in 50% (v/v) ethyl acetate. It is noteworthy that araujiain was unable to form oligopeptides derivatives, showing a good selectivity towards the synthesis of Z-Ala-Phe-OMe. For this reason and in general terms, araujiain constituted a new catalyst for the synthesis of small peptides.

Keywords: Enzyme biocatalysis; Dipeptide synthesis; Cysteine protease; Aqueous–organic media; Araujiain; Araujia hortorum; (; Asclepiadaceae; )

Bioelectricity generation from chemical wastewater treatment in mediatorless (anode) microbial fuel cell (MFC) using selectively enriched hydrogen producing mixed culture under acidophilic microenvironment by S. Venkata Mohan; G. Mohanakrishna; B. Purushotham Reddy; R. Saravanan; P.N. Sarma (pp. 121-130).

Bioelectricity generation from composite chemical wastewater treatment was evaluated in a dual chambered microbial fuel cell (MFC) [anode chamber (mediatorless; perforated plain graphite electrode); cathode chamber (50mM potassium ferricyanide [K₃Fe(CN)₆] in phosphate buffer; pH 7.5; plain graphite electrode)] inoculated with selectively enriched hydrogen (H₂) producing mixed culture under acidophilic microenvironment (pH 5.5). Anode chamber, which resembles anaerobic suspended contact reactor was fed with wastewater and operated in absence of artificial mediator at acidic environment to proliferate H₂ producing bacteria. Experimental data showed the feasibility of producing bioelectricity from wastewater treatment, though power production was found to be dependent on the substrate loading rate. Maximum voltage of 271.5mV (5.43mA) and 304mV (6.08mA) was recorded at operating organic loading rates (OLR) of 1.165kgCOD/(m³day) and 1.404kgCOD/(m³day), respectively when measured at 50Ω external resistors at stable operating conditions. COD removal efficiency of 35.4% (substrate degradation rate (SDR) of 0.412kgCOD/(m³day)) and 62.9% (SDR, 0.88kgCOD/(m³day)) was observed at OLRs 1.165kgCOD/(m³day) and 1.404kgCOD/(m³day), respectively. Maximum specific power production of 0.163W/kgCOD_R (1.165kgCOD/(m³day); 50Ω) and 0.198W/kgCOD_R (1.404kgCOD/(m³day); 100Ω) was observed during stable phase of fuel cell operation. Current density of 747.96mA/m² (1.165kgCOD/(m³day)) and 862.85mA/m² (1.404kgCOD/(m³day)) was documented at 10Ω. Utilizing chemical wastewater for the production of renewable energy (bioelectricity) from anaerobic treatment is considered as a feasible, economical and sustainable process.

Keywords: Bioelectricity; Microbial fuel cell (MFC); Chemical wastewater treatment; Acidophilic; Anaerobic; Biohydrogen; Mixed culture; Graphite

Clavulanic acid production processes in a tower bioreactor with immobilised cells by Susana C.S. Lavarda; Carlos O. Hokka; Maria Lucia G.C. Araujo (pp. 131-136).

The feasibility of using Streptomyces clavuligerus ATCC 27064 bioparticles supported on alginate gel containing alumina to produce clavulanic acid (CA) was investigated. To this end, effectiveness factors for spherical bioparticles, relating respiration rates of immobilised and free cells, were experimentally determined for various dissolved oxygen (DO) levels and bioparticle radii. Monod kinetics was assumed as representative of the oxygen consuming reaction, while internal oxygen diffusion was considered the limiting step. A comparison was made of the results from a tower bioreactor operating under batch, repeated-batch and continuous conditions with immobilised bioparticles. The theoretical curve of the effectiveness factor for the zero-order reaction model, considering an inert nucleus – the dead core model – was very well fitted to the experimental data. The results of the bioprocess indicated that the batch operation was the most efficient and productive, requiring a DO concentration in the reactor above 60% of the saturation value.

Keywords: Clavulanic acid; Immobilised Streptomyces; Tower bioreactor; Oxygen transfer; Diffusion–reaction; Effectiveness factor

A fuzzy logic approach for regulation in flux balance analysis by Aris Tepeli; Amable Hortaçsu (pp. 137-148).

Technological advances in experimental observations provide the opportunity to examine complex biological systems and the chance to forecast bacterial behaviour in silico. The optimal growth of bacteria on defined carbon sources is now easily predicted from the solutions of constraint-based metabolic models. However, these methods are unable to predict the sequence of carbon utilization and changes in cellular behaviour for growth in mixed substrates. In this work, a regulatory structure describing transcriptional regulation of catabolic genes or operons expressed in fuzzy logic formalism is combined with dynamic flux balance analysis (dFBA). The fuzzy logic formalism is a good alternative to differential equation models that require kinetic parameter values and superior to Boolean Formalism which automatically sets regulation as “on” or “off” rules. The dFBA/fuzzy logic combination was successfully used to simulate aerobic growth of Escherichia coli in mixed double (glucose–lactose, glucose–sorbitol) or triple (glucose–lactose–galactose) substrates and for anaerobic growth of Lactococcus lactis in the triple substrate (glucose–lactose–galactose). When well-defined data are available, the computed results are in good agreement with the data. The method also allows for the prediction of growth lag periods upon substrate substitution and changes in growth pattern, and the dynamics of substrate substitution upon pulse injection of substrate in existing cultures.

Keywords: Batch processing; Dynamic simulation; Fuzzy logic; Lac operon; Microbial growth; Regulatory flux balance analysis

Calorimetric on-line monitoring of proteolytic activity of P. aeruginosa cultivated in a bench-scale biocalorimeter by Senthilkumar Sivaprakasam; Surianarayanan Mahadevan; Sudharshan Sekar (pp. 149-156).

The aim of this work is to generate thermokinetic data for the production of proteases which would be immensely useful not only for successful scale up of a ferementor for protease production but also to monitor and control of the process by following the heat signals. The biocalorimetric studies prove that the protease production and heat generation profiles are comparable. Oxygen uptake rate was found to correlate well with both biomass growth and heat production rate at all lag, exponential and stationary growth phases. Heat yield due to biomass growth (anabolic) related reactions and substrate uptake (catabolic) related reactions were estimated from the calorimetric experiments and the results were analyzed. Crude protease secreted in calorimeter was extracted separately and characterized by Zymogram and SDS-PAGE analytical techniques. The protease was found to be an alkaline protease. This study reveals that the protease secretion by P. aeruginosa could be monitored effectively in a biological reaction calorimeter. Metabolic heat generated is correlated to protease secretion (non-growth process) for the first time in this study. The heat yields due to biomass growth, substrate uptake and oxygen uptake could help to understand the metabolic process in realistic manner and the results would be useful for bioreactor design and scale up.

Keywords: Alkaline protease; Bio-RC1; P. aeruginosa; Heat generation; SDS-PAGE

Design a chemically defined/medically approved medium for cell transplantation according to the metabolic characteristics of microencapsulated cells and the process of encapsulation by Guojun Lv; Zhijie Sun; Na Li; Shuangyue Li; Ying Zhang; Yubing Xie; Weiting Yu; Wei Wang; Xiaojun Ma (pp. 157-163).

Cell microencapsulation is a promising therapeutic strategy to many diseases; however, the metabolic characteristics of microencapsulated cells and also the optimum microcapsule additives that meet the criteria for long-term functional immunoisolated transplantation have rarely been taken into consideration hitherto. In this work, the optimized amino acid supplements and several chemically defined compounds were applied to cultivation of the microencapsulated CHO cells. The results show that the optimized medium significantly enhances cell survival and, particularly, endostatin secretion of encapsulated CHO cells over a time period of 8 weeks. Furthermore, long-term in vitro stability of alginate–poly-l-lysine–alginate (APA) microcapsules underwent no changes, which indicated that this optimized medium might be feasible to maintain high viability of microencapsulated cell for short periods of time before implantation.

Keywords: Cell transplantation; Microencapsulation; Recombinant CHO cell; Chemically defined medium; Amino acid

Design and characterisation of a miniature stirred bioreactor system for parallel microbial fermentations by N.K. Gill; M. Appleton; F. Baganz; G.J. Lye (pp. 164-176).

The establishment of a high productivity microbial fermentation process requires the experimental investigation of many interacting variables. In order to speed up this procedure a novel miniature stirred bioreactor system is described which enables parallel operation of 4–16 independently controlled fermentations. Each miniature bioreactor is of standard geometry (100mL maximum working volume) and is fitted with a magnetically driven six-blade miniature turbine impeller ( d_i=20mm, d_i/ d_T=1/3) operating in the range 100–2000rpm. Aeration is achieved via a sintered sparger at flow rates in the range of 0–2vvm. Continuous on-line monitoring of each bioreactor is possible using miniature pH, dissolved oxygen and temperature probes, while PC-based software enables independent bioreactor control and real-time visualisation of parameters monitored on-line. In addition, a new optical density probe is described that enables on-line estimation of biomass growth kinetics without the need for repeated sampling of individual bioreactors. Initial characterisation of the bioreactor involved quantification of the volumetric oxygen mass transfer coefficient as a function of agitation and aeration rates. The maximum k_L a value obtained was 0.11s⁻¹. The reproducibility of E. coli TOP10 pQR239 and B. subtilis ATCC6633 fermentations was shown in four parallel fermentations of each organism. For E. coli (1000rpm, 1vvm) the maximum specific growth rate, μ_max, was 0.68±0.01h⁻¹ and the final biomass concentration obtained, X_final, was 3.8±0.05gL⁻¹. Similarly for B. subtilis (1500rpm, 1vmm) μ_max was 0.45±0.01h⁻¹ and X_final was 9.0±0.06gL⁻¹. Biomass growth kinetics increased with increases in agitation and aeration rates and the oxygen enrichment for control of DOT levels enabled μ_max and X_final as high as 0.93h⁻¹ and 8.1gL⁻¹ respectively to be achieved. Preliminary, scale-up studies with E. coli in the miniature bioreactor (100mL working volume) and a laboratory scale 2L bioreactor (1.5L working volume) were performed at matched k_L a values. Very similar growth kinetics were observed at both scales giving μ_max values of 0.94 and 0.97h⁻¹, and X_final values of 5.3 and 5.5gL⁻¹ respectively. The miniature bioreactor system described here thus provides a useful tool for the parallel evaluation and optimisation of microbial fermentation processes.

Keywords: Miniature bioreactor; Parallel operation; Fermentation; On-line monitoring

Effect of organic carbon sources and Fe²⁺ ions on growth and β-carotene accumulation by Dunaliella salina by M. Mojaat; J. Pruvost; A. Foucault; J. Legrand (pp. 177-184).

Effects of adding Fe²⁺ ions in photoheterotrophic cultivation of Dunaliella salina on growth and β-carotene synthesis were investigated. Different concentrations of Fe²⁺ ions and organic carbon source (acetate and malonate) were studied. A significant increase of cellular β-carotene content was observed, with a maximum value of 70pg/cell in a culture enriched with 67.5mM of acetate and 450μM of FeSO₄ and 33pg/cell in the case of 67.5mM of malonate and 450μM of FeSO₄. For comparison, the well-known nitrate starvation protocol was applied, and 35pg/cell of β-carotene were obtained. By inducing an oxidative stress, Fe²⁺ ions revealed to stimulate β-carotene synthesis, especially when acting in the presence of the carbon source. Furthermore for some concentration of Fe²⁺ ions and organic carbon source, the loss in cells division was reduced compared to nitrate starvation.

Keywords: β-carotene; Dunaliella salina; Oxidative stress; Iron; Acetate; Malonate

Enzymatic production of biodiesel from Jatropha oil: A comparative study of immobilized-whole cell and commercial lipases as a biocatalyst by Sriappareddy Tamalampudi; Mahabubur Rahman Talukder; Shinji Hama; Takao Numata; Akihiko Kondo; Hideki Fukuda (pp. 185-189).

The large percentage of biodiesel fuel (BDF) cost associated with feedstock oil and enzyme. In order to reduce the cost of BDF production, the lipase producing whole cells of Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) was used for the production of BDF from relatively low cost non-edible oil from the seeds of Jatropha curcas. The activity of ROL was compared with that of commercially available most effective lipase (Novozym 435). Different alcohols as a hydroxyl donor are tested, and methanolysis of Jatropha oil progresses faster than other alcoholysis regardless of lipases used. The maximum methyl esters content in the reaction mixture reaches 80wt.% after 60h using ROL, whereas it is 76% after 90h using Novozym 435. Both the lipases can be used for repeated batches and both lipases exhibit more than 90% of their initial activities after five cycles. Our results suggest that whole-cell ROL immobilized on BSP is a promising biocatalyst for producing BDF from oil.

Keywords: Lipase; Transesterification; Filamentous fungi; Immobilized cells

Bioprocess hybrid parametric/nonparametric modelling based on the concept of mixture of experts by J. Peres; R. Oliveira; S. Feyo de Azevedo (pp. 190-206).

This paper presents a novel method for bioprocess hybrid parametric/nonparametric modelling based on mixture of experts (ME) and the expectation maximisation (EM) algorithm. The bioreactor system is described by material balance equations whereas the cell population subsystem is described by an adjustable mixture of parametric/nonparametric sub-models inspired in the ME architecture. This idea was motivated by the fact that cellular metabolism has an inherent “modular” structure, organised in metabolic pathways, with complex interactions. This study was supported by simulations using models of different levels of complexity. The proposed method was compared with the conventional hybrid technique employing the multi-layer perceptron (MLP) and the radial basis function (RBF) networks. As main conclusions it can be stated that MEs trained with the EM algorithm are able to systematically detect metabolic shifts with the individual experts developing expertise in describing the individual pathways. The hybrid ME model with thin-plate spline RBF network as experts outperforms both the hybrid MLP model and the hybrid RBF model in its ability to describe metabolic switches.

Keywords: Bioprocesses; Bioreactors; Hybrid modelling; Mixture of experts; Expectation maximisation; Artificial intelligence; Waste-water treatment; Control

Optimization of lignocellulolytic enzyme production by the white-rot fungus Trametes trogii in solid-state fermentation using response surface methodology by Laura Levin; Claudia Herrmann; Victor L. Papinutti (pp. 207-214).

The white-rot basidiomicete Trametes trogii (MYA 28-11) is an outstanding producer of laccase. A Doehlert experimental design was applied to optimize its lignocellulolytic enzyme production in solid-state fermentation. The impact on enzyme production of three quantitative variables, namely pH, copper and nitrogen concentrations, was investigated by using a wood-based solid medium supplemented with malt extract. Optimization was aimed at simultaneously minimizing cellulase activity and maximizing ligninolytic enzyme production. Such conditions were: pH 4.5, peptone 12.5gl⁻¹, and CuSO₄ 11mM. Highest activities achieved were: laccase 901 and Mn-peroxidase 20Ug⁻¹. There was not a direct correlation between the levels of enzymatic activities and the extent of losses of wood weight or components. The factorial approach also allowed us to quantify the enzyme activity in any part of the experimental domain, consequently, it was also possible to determine a culture medium to obtain crude extracellular extracts with high laccase (510Ug⁻¹) and endoxylanase (780Ug⁻¹) yields. These enzymes have gained renewed interest mainly due to their applications in paper industries for pulp treatment, improving the effectiveness of conventional bleaching. Considering the results obtained, T. trogii could be an attractive source of both enzymes.

Keywords: Response surface methodology; Lignocellulolytic enzymes; White-rot fungi; Solid-state fermentation

Simultaneous biological removal of ammonia, iron and manganese from potable water using a trickling filter by A.G. Tekerlekopoulou; D.V. Vayenas (pp. 215-220).

A pilot-scale trickling filter with dual layer support material was constructed and tested for simultaneous biological removal of ammonia, iron and manganese from potable water. The performance of the trickling filter was tested at constant hydraulic loading of 226m³/m²d while feed concentrations of iron, ammonia and manganese were varied between 0.5 and 4.0, 0.5 and 3.0, and 0.5 and 1.3mg/l, respectively. The system was inoculated with a mixed culture and a series of experiments was performed to investigate the interactions among ammonia, iron and manganese removal when simultaneously present in the trickling filter. The oxidation reduction potential increased along the filter depth from about 150 to 600mV, depending on the feed concentrations, thus enabling one-stage simultaneous removal of the three pollutants. Ammonia and iron drastically affected manganese oxidation and manganese was found to be the rate-limiting pollutant. The results are presented using an operating diagram of the system, that determines the range of operating conditions resulting in optimal operation, keeping iron, ammonia and manganese concentration under the maximum permitted limits in potable water.

Keywords: Trickling filter; Biological oxidation; Manganese; Iron; Ammonia; Potable water

Sections

Personal tools

Biochemical Engineering Journal (v.39, #1)

Sections

Personal tools

Local resources

Biochemical Engineering Journal (v.39, #1)