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Biochemical Engineering Journal (v.24, #1)
Simulation of biodegradation process of phenolic wastewater at higher concentrations in a fluidized-bed bioreactor by A. Venu Vinod; G. Venkat Reddy (pp. 1-10).
Experiments were carried out to study the biodegradation of phenolic wastewater using microorganisms in a fluidized-bed bioreactor (FBR). Synthetic wastewater has been used in the study. Experiments were conducted at various conditions of wastewater flow rate, dissolved oxygen (DO) concentration and inlet concentration. The wastewater with feed concentration of phenol as high as 1254ppm has been successfully biodegraded to 50ppm in the fluidized-bed bioreactor, whereas feed with concentration up to 1034ppm could be completely biodegraded (to zero ppm).Biokinetic parameters for the growth of the cells were determined in batch experiments and they were found to be μmax=1.436×10−4s−1, Ks=21.92×10−3kg/m3, Ki=522×10−3kg/m3. Model equations describing the simultaneous diffusion and reaction of phenol and oxygen in the biofilm of the solid particles, were solved using the orthogonal collocation technique to calculate the rate of biodegradation of phenol. The rate of biodegradation predicted by the model was compared with that observed experimentally and the difference between the two was found to be about 12%.
Keywords: Biodegradation; Phenol; Biomass; Fluidized-bed bioreactor; Substrate inhibition; Wastewater treatment
Operational stability of subtilisin CLECs in organic solvents in repeated batch and in continuous operation by João F. Amorim Fernandes; Morven McAlpine; Peter J. Halling (pp. 11-15).
The operational stability of cross-linked crystals (CLECs) of subtilisin Carlsberg in organic media is compared between repeated batch and continuous flow operation. The study was designed to create similar conditions in the two modes, although these can never be identical in all respects. The biocatalyst inactivates rapidly during the first two batches of 2h each, losing 50% or more of its activity. The inactivation becomes slower over the following longer batches of 24h. This pattern of rapid and slower phases of inactivation is similar to that observed in continuous reactors. However, in both acetonitrile and tetrahydrofuran, inactivation is somewhat faster over the first 4h of continuous operation than in the equivalent batch mode.
Keywords: Enzyme deactivation; Enzyme bioreactors; Operational stability; Cross-linked enzyme crystals; Protease; Enzyme biocatalysis
Laccase production by Pleurotus ostreatus 1804: Optimization of submerged culture conditions by Taguchi DOE methodology by K. Krishna Prasad; S. Venkata Mohan; R. Sreenivas Rao; Bikas Ranjan Pati; P.N. Sarma (pp. 17-26).
Submerged culture conditions for laccase production by Pleurotus ostreatus 1804 were optimized by Taguchi orthogonal array (OA) experimental design (DOE) methodology. This approach facilitates the study of interaction of a large number of variables spanned by factors and their settings with a small number of experiments leading to considerable saving in time and cost for the process optimization. The proposed Taguchi DOE methodology consists of four phases viz., planning, conducting, analysis and validation, which were connected sequence wise to achieve the overall process optimization. Eight factors viz., carbon, nitrogen source (urea), 2,5-xylidine (inducer), wheat bran (ligninocellulosic material), inoculum size, fermentation broth pH, yeast extract and phosphate source (KH2PO4) at three levels with a OA layout of L18 (21×37) were selected for the proposed experimental design. Laccase yield obtained from the 18 sets of fermentation experiments performed with the selected factors and levels were further processed with Qualitek-4 software at bigger is better as quality character. The optimized conditions showed an enhanced laccase expression of 32.9% (from 538.8 to 803.3U). The optimal combinations of factors obtained from the proposed DOE methodology was further validated by conducting fermentation experiments and the obtained results revealed an enhanced laccase yield of 28.3%. Taguchi approach of DOE resulted in evaluating the main and interaction effects of the factors individually and in combination. This methodology facilitated analysis of the experimental data to establish the optimum conditions for the process, understand the contribution of individual factors and to evaluate the response under optimal conditions. Application of Taguchi approach appears to have potential usage in bioprocess optimization.
Keywords: Taguchi method; Design of experiments (DOE); Orthogonal array (OA); Qualitek-4; Optimization; Pleurotus ostreatus; 1804; Laccase activity; Submerged culture conditions
Facile monitoring of baculovirus infection for foreign protein expression under very late polyhedrin promoter using green fluorescent protein reporter under early-to-late promoter by Nimish G. Dalal; William E. Bentley; Hyung Joon Cha (pp. 27-30).
A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) was constructed so that the green fluorescent protein (GFP) was produced via the early-to-late (ETL) promoter. This enabled rapid monitoring of the infection of Sf-9 insect cells. Notably, GFP and its fluorescence appeared ∼18h prior to proteins expressed using very late polyhedrin (Polh) promoter. It is anticipated that the use of GFP under the control of ETL promoter will facilitate vector construction, virus isolation, and titer determination.
Keywords: Baculovirus infection; Early ETL promoter; Green fluorescent protein; Recombinant protein production
Stress-induced physiological responses to starvation periods as well as glucose and lactose pulses in Bacillus licheniformis CCMI 1034 continuous aerobic fermentation processes as measured by multi-parameter flow cytometry by Teresa Lopes da Silva; Alberto Reis; Christopher A. Kent; Maria Kosseva; J. Carlos Roseiro; Christopher J. Hewitt (pp. 31-41).
Multi-parameter flow cytometry (MPFC) coupled with specific fluorescent stains were used to monitor the physiological response of individual cells to starvation periods as well as lactose and glucose pulses during the steady state of a continuous culture of Bacillus licheniformis. Measurements of off-gas (CO2 produced and O2 consumed) and optical density measurements were also made and used to follow bulk biomass proliferation and metabolic activity of the microbial population. It was clear that the physiological response to each perturbation was different in type and extent dependent on the conditions experienced and that these responses could be followed by the techniques employed. The use of MPFC, a rapid, real time analysis of individual microbial cells, is enhancing our knowledge of how different physiological sub-populations develop with time (population dynamics) throughout a bio-process further demonstrating the inherent complex heterogeneity of microbial cultures even during the so called ‘steady-state’ of a continuous culture fermentation. These results are discussed in terms of devising better control systems based on measurements made at the individual cell level for optimisation of bioprocess performance.
Keywords: Bacillus licheniformis; Glucose and Lactose pulses; Starvation; Flow cytometry
Optimization of process conditions to inactivate Bacillus subtilis by high hydrostatic pressure and mild heat using response surface methodology by Yu-Long Gao; Han-Hu Jiang (pp. 43-48).
The results of fractional factorial experiments showed that the significant external factors affecting high pressure processing (HPP) inactivation were pressure, temperature and pressure holding time. Based on these results, response surface methodology (RSM) was employed in the present work and a quadratic equation for HPP inactivation was built. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the experimental values were shown to be significantly in good agreement with predicted values since the adjusted determination coefficient (RAdj2) was 0.9800. The optimum process parameters for six log-cycles reduction of Bacillus subtillis were obtained as: temperature, 46°C; pressure, 479.0MPa and pressure holding time, 14min. The adequacy of the model equation for predicting the optimum response values was verified effectively by the validation data.
Keywords: High pressure processing (HPP); Reduction of; Bacillus subtilis; Response surface methodology (RSM); Optimization
The separation of oil from an oil–water–bacteria mixture using a hydrophobic tubular membrane by M. Konishi; M. Kishimoto; N. Tamesui; T. Omasa; S. Shioya; H. Ohtake (pp. 49-54).
The separation of oil from oil–water–bacteria mixtures is of great importance for the development of whole-cell biocatalytic processes in the presence of organic solvents. We found that a hydrophobic polytetrafluroethylene (PTFE) tubular membrane was effective in separating n-tetradecane, a model oil from 50% (v/v) n-tetradecane in water. When the PTFE tubular membrane was prewetted by n-tetradecane, it was also effective in separating n-tetradecane from 50% (v/v) n-tetradecane in an aqueous medium containing Rhodococcus erythropolis KA2-5-1 as a whole-cell biocatalyst for biodesulfurization. Microscopic observation confirmed that the recovered oil was essentially free of bacterial cells. Though the flux rate fell off with time of filtration, repeated backwashing of the membrane enhanced the flux performance approximately two-fold.
Keywords: Biocatalysis; Biodesulfurization; Downstream processing; Hydrophobic tubular membrane; Rhodococcus erythropolis; Separation
The mechanism of PNIPAAm-assisted refolding of lysozyme denatured by urea by Diannan Lu; Zhixia Liu; Minlian Zhang; Zheng Liu; Haimeng Zhou (pp. 55-64).
The molecular interaction of a temperature stimuli-responsive polymer, poly- N-isopropyl acrylamide (PNIPAAm), with lysozyme of different status was studied with an emphasis on the application of PNIPAAm for protein refolding. The refolding of lysozyme was performed by directly diluting denatured lysozyme into a refolding buffer containing PNIPAAm, in which PNIPAAm with the weight average molecular weight of 22,000, denoted as M-PNI, gave the best refolding yield in terms of the recovery of lysozyme activity. The interaction between M-PNI and lysozyme was investigated using non-reductive SDS–PAGE, circular dichroism (CD), fluorescence emission spectroscopy, and reverse phase HPLC. It was shown that the use of M-PNI increased the secondary structures of lysozyme and reduced the formation of protein aggregate. The correctly folded lysozyme has a weaker hydrophobicity compared to the denatured lysozyme. The PNIPAAm-lysozyme complex dissociates once lysozyme is correctly folded. The increase in the operational temperature leads to increases in both the refolding yield and the apparent rate of refolding. Based on above experimental results, a kinetic model of the refolding, both with and without PNIPAAm, was determined and a molecular view of lysozyme refolding using PNIPAAm was presented.
Keywords: Poly(; N; -isopropyl acylamide); Lysozyme; Refolding in vitro; Folding kinetics; Protein downstream process
Adsorption of urea nitrogen onto chitosan coated dialdehyde cellulose under biocatalysis of immobilized urease: Equilibrium and kinetic by Zu Pei Liang; Ya Qing Feng; Zhi Yan Liang; Shu Xian Meng (pp. 65-72).
Chitosan coated dialdehyde cellulose (CDAC) and immobilized urease in gelatin membrane (IE) were prepared. The adsorption of urea nitrogen onto CDAC under biocatalysis of IE was studied in batch system. The equilibrium isotherm of urea nitrogen adsorption onto CDAC with different degrees of oxidation (DO) and the kinetics of adsorption with respect to the DO of CDAC (53, 78 and 95%), CDAC/IE weight ratio (50:4, 50:5 and 50:6), the initial urea nitrogen concentration (395.2, 648.8 and 767.3mg/L) and temperature (37, 42 and 47°C) were investigated. Langmuir and Freundlich adsorption models were applied to describe the experimental isotherm and isotherm constants. Equilibrium data fitted very well to the Langmuir model in the entire saturation concentration range (128.2–708.2mg/L). The maximum monolayer adsorption capacities obtained from Langmuir model are 70.18, 79.56 and 90.42mg/g for the CDACs with DO 53, 78 and 95%, respectively, at CDAC/IE weight ratio 10:1 and 37°C. The pseudo first-order and pseudo second-order kinetic models were used to describe the kinetic data, and the rate constants were evaluated. The results showed that adsorption process of urea nitrogen onto CDAC followed the pseudo second-order kinetic model. The DO of CDAC, CDAC/IE weight ratio, initial urea nitrogen concentration and temperature significantly affected the adsorption capacity. The apparent activation energy is 8.934kJmol−1 for the adsorption of the urea nitrogen onto CDAC under catalysis of IE at DO 95% of CDAC, CDAC/IE weight ratio 10:1 and initial urea nitrogen concentration 648.8mg/L.
Keywords: Adsorption; Equilibrium; Kinetic; Urea nitrogen; Chitosan coated dialdehyde cellulose; Immobilized urease; Biocatalysis
Mathematical modelling of cyclodextrin-glucanotransferase production by batch cultivation by N. Burhan; Ts. Sapundzhiev; V. Beschkov (pp. 73-77).
For the purpose of a large-scale production of native cyclodextrin-glucanotransferase (CGTase, EC 2.4.1.19), a reliable kinetic model of this microbial process is required. The goal of the present paper is to propose a mathematical model based on experimental data, and suitable for description and prediction of the process of microbial synthesis of CGTase.The model consists of a set of ordinary differential equations taking into account the microbial growth, the substrate inhibition, and the decay of the microbial culture with time.The model is used to evaluate kinetic constants from experimental data for the microbial production of CGTase by the strain Bacillus circulans ATCC 21783 in a bubble-column bioreactor. The model adequacy was proved statistically.It was found that the model shows the sensitivity of the strain towards the initial substrate concentration, thus, predicting maximum values for the rate constants of CGTase production.It was shown that the Andrews’ equation for substrate inhibition of microbial growth is more appropriate for the considered case than the Aiba's one.The validity of the model is demonstrated treating of other data published in the literature.
Keywords: Cyclodextrin-glucanotransferase; Enzyme production; Bubble column; Kinetic parameters; Modelling
Hydrogen sulphide adsorption on a waste material used in bioreactors by A. Barona; A. Elías; A. Amurrio; I. Cano; R. Arias (pp. 79-86).
The biofilter packing, support or bed material, is considered to be the “heart� of the biofiltration system, especially when it also provides active biomass. Furthermore, when biodegradation performance is not effective, the ability of the packing material surface to retain or adsorb the contaminant is a desirable property. In this paper, a pelletized organic material has been researched in order to quantify and to model its adsorption capacity when used as bed material in biofilters. For comparison purposes, adsorption tests have been carried out using sterilized organic material and activated carbon. Adsorption equilibrium isotherms have been established for both materials in a H2S concentration range from 40 to 330ppmv at room temperature. A type I adsorption isotherm for the activated carbon and type III for the organic material have been obtained. The experimental adsorption data for the activated carbon can equally be represented by both Langmuir and Freunlich models. However, only the Freundlich approach is adequate for the organic bed with KF=9.61mg1− nL ng−1 of dry bed and n=1.55. When the sterilized organic material is not dry, the contribution of absorption to the retention of the contaminant in the organic bed is negligible.
Keywords: Adsorption; Bioreactors; Biofilters; Air pollution; Packing material; Hydrogen sulphide
Reversed micellar extraction of an extracellular protease from Nocardiopsis sp. fermentation broth by T.I.R.C. Monteiro; T.S. Porto; A.M.A. Carneiro-Leão; M.P.C. Silva; M.G. Carneiro-da-Cunha (pp. 87-90).
Extraction of an extracellular alkaline protease from Nocardiopsis sp. fermentation broth using reversed micelles of sodium di(2-ethylhexyl) sulfosuccinate (AOT) in isooctane was performed with equal phase volume ratio. This work describes the effects of pH, ionic strength and surfactant concentration on the enzyme transfer process from the aqueous to the organic phase by direct contact (5min) between the two phases. The best conditions for extraction (38.4% of protein content with about 83.5% of activity) were obtained using 200mM AOT, at pH 4.0 with 50mM KCl. For back extraction, sodium carbonate buffer, at pH 5.0 with 100mM of KCl allowed for the best conditions (47.2% of protein content with about 14.0% of activity). The low protease activity yield of 11.69% obtained for the total process, extraction and back-extraction, suggests enzyme denaturation or its allocation near the AOT hydrophobic tail.
Keywords: Protease; AOT; Protein recovery; Reversed micelles; Liquid–liquid extraction
Immobilization of surfactant–lipase complexes and their high heat resistance in organic media by Muneharu Goto; Chiaki Hatanaka; Masahiro Goto (pp. 91-94).
Surfactant–lipase complexes were immobilized in an n-vinyl-2-pyrrolidone gel matrix. Features of a native lipase and gel-immobilized surfactant–lipase complexes were measured by esterification reaction between lauric acid and benzyl alcohol in isooctane. Optimal gel-immobilized surfactant–lipase complex activity was 37.2molh−1kg−1-lipase. Gel-immobilized lipase complexes showed a 51-fold increase in activity and exhibited superior heat resistance compared to native lipases. The optimum temperature for the immobilized lipase complex was 60°C, as compared to 37°C for native lipase activity. Gel-immobilized lipase complexes could be readily recovered, and their high activity was completely preserved, even after 10 reuses.
Keywords: Enzyme activity; Esterification reaction; Immobilized enzymes; Lipase; Surfactant–enzyme complex