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Biochemical Engineering Journal (v.50, #3)
Hydrolysis of liquid pineapple waste by invertase immobilized in PVA–alginate matrix by Nor Azimah Mohd Zain; Mohd Suardi Suhaimi; Ani Idris (pp. 83-89).
A thorough investigation of sucrose hydrolysis by invertase immobilized in recently developed PVA–alginate beads was performed. The developed PVA–alginate bead is distinguished by the treatment with sodium sulfate solution. The performance of invertase immobilized in PVA–alginate matrix beads in terms of leakage, reusability, ideal temperature and pH and kinetic parameters K m and V max was investigated. Results revealed that the ideal temperature and pH for the immobilized invertase are 50°C and 4 respectively. The apparent K m of each bead is relatively lower than that of the free enzyme with the value of 4.7×10−5mM and 2.849×10−5mM for 12PVA–5BA and 12PVA–7BA, respectively. An increase in V max was also observed with the value of 2281.02Umg−1 enzyme and 2281.02Umg−1 enzyme for 12PVA–5BA and 12PVA–7BA, respectively. The reusability of the beads and its storage period were also investigated and were found to be 14 cycles and 60 days, respectively. Finally, the beads with the best performance (12PVA–5BA) were used to hydrolyze liquid pineapple waste. The production yield of glucose was about 745% in 3h in the hydrolysis of the liquid pineapple waste.
Keywords: PVA–alginate beads; Sodium sulfate; Invertase kinetic parameter; Pineapple waste
Physical characterisations of a single-stage Kühni-type aqueous two-phase extraction column by Frank Luechau; Tau Chuan Ling; Andrew Lyddiatt (pp. 90-98).
The main parameters which influence the behaviour of phase separation in a single-stage Kühni-type aqueous two-phase extraction column containing polyethylene (PEG) and di-potassium hydrogen phosphate were characterised. Two aqueous two-phase system (ATPS) composed of 12% (w/w) PEG 1450 and 12% (w/w) di-potassium hydrogen phosphate (designated as 12/12) and 12% (w/w) PEG 1450 and 11% (w/w) di-potassium hydrogen phosphate (designated as 12/11) were chosen in this study. The hold-up ɛD increased with increasing impeller speeds and mobile phase flow rates. Phase separation for the 12/11 system was slower than that for the 12/12 system, which resulted in higher dispersed phase hold-up values for the 12/11 system. For 12/12 system, mass transfer of plasmid DNA (pDNA) from the dispersed mobile phase to the stationary phase increased rapidly with increasing impeller speeds of 130, 160 and 200rpm which was reflected in the decreased values for CT/ CTo. The degree of back-mixing quantified by the axial dispersion coefficient Dax was estimated to be 2.7×10−6m2s−1.
Keywords: Bioseparations; Aqueous two-phase system; Extraction; Precipitation; Physical
Evaluation of mycophenolic acid production by Penicillium brevicompactum MUCL 19011 in batch and continuous submerged cultures by Fatemeh Ardestani; Seyed Safa-ali Fatemi; Bagher Yakhchali; Seyed Morteza Hosseyni; Ghasem Najafpour (pp. 99-103).
Mycophenolic acid (MPA) is a secondary metabolite produced by Penicillium brevicompactum, which has antibiotic and immunosuppressive properties. In this study, production of MPA by P. brevicompactum MUCL 19011 in shake flask and a 2L bench-scale stirred tank bioreactor under batch and continuous culture conditions was evaluated. The maximum concentration of MPA, its yield and rate of production in continuous culture were 1.46gL−1, 29mgg−1 glucose and 6.8mgL−1h−1, respectively. Growth kinetics in batch culture was investigated with five models of Monod, Moser, Verhulst, Contois and Exponential. The compatibility of the experimental data fitted well with the Contois model and the regression value was 0.98. The maximum specific growth rate and saturation constant were 0.07h−1 and 0.61gL−1, respectively. Glucose, biomass and MPA concentration profiles were evaluated during the course of fermentation. Under steady-state conditions, glucose consumption, biomass formation and MPA production fluxes were 9.2, 22.35 and 0.194mmolL−1h−1, respectively. In batch culture, enzymatically hydrolyzed casein and glycine increased MPA production by 165 and 112%, respectively.
Keywords: Mycophenolic acid; Penicillium brevicompactum; Continuous culture; Growth kinetics; Submerged; Stirred tank
Biodesulfurization of dibenzothiophene, its alkylated derivatives and crude oil by a newly isolated strain Pantoea agglomerans D23W3 by Sumedha Bhatia; D.K. Sharma (pp. 104-109).
Biocatalytic desulfurization (BDS) of fuels has been shown to be a potential alternative to the conventional hydrodesulfurization (HDS) process used in refineries, since HDS cannot remove the heterocyclic organo-sulfur compounds such as dibenzothiophene (DBT). Herein the isolation of a DBT desulfurizing mesophilic bacterium, characterized as Pantoea agglomerans D23W3, from contaminated soils collected from refinery has been reported. HPLC analysis revealed that P. agglomerans D23W3 could convert DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway and that it could degrade 93% of the 100ppm DBT within 24h of culture. In addition P. agglomerans D23W3 could also desulfurize 4,6-dimethyl DBT and benzothiophene which are among the most difficult DBT derivatives to be removed by HDS. Further, adapted cells of P. agglomerans D23W3 were found to remove 26.38–71.42% of sulfur from different petroleum oils with highest sulfur removal from light crude oil. Therefore, P. agglomerans D23W3 has a potential for the BDS of the petroleum oils.
Keywords: Abbreviations; HDS; hydrodesulfurization; BDS; biocatalytic desufurization; ATF; aviation turbine fuel; BT; benzothiophene; DBT; dibenzothiophene; 2-HBP; 2-hydroxybiphenyl; DEDBT; diethyldibenzothiophene; DMDBT; dimethyldibenzothiophene; SFM; sulfur free mediumBiodesulfurization; Dibenzothiophene; Crude oil; Pantoea agglomerans
Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato by Büşra Özer; Emine Akardere; Evran Bıçak Çelem; Seçil Önal (pp. 110-115).
Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato ( Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.
Keywords: Bioseparation; t-Butanol; Invertase; Protein purification; Three-phase partitioning; Tomato
Investigation of medium perfusion through scaffold-free tissue constructs using endothelial cell-covered spheroids in vitro by Masakazu Inamori; Hiroshi Mizumoto; Toshihisa Kajiwara (pp. 116-121).
A scaffold-free tissue construct was formed by assembling endothelial cell-covered spheroids, and medium perfusion through the tissue construct was investigated using hydrostatic pressure-driven culture circuit. Primary rat hepatocyte spheroids covered by human umbilical vein endothelial cells (HUVECs) were assembled in culture chambers with a cylindrical culture space of 2mm in diameter, and then medium was perfused through the assembled spheroids for 48h. The medium flow rate through the culture chamber was measured over the perfusion culture time, which decreased during the first several hours, then increased or remained low depending on the amount of spheroids in the culture chamber. Histochemical analyses showed single tissue construct formation by spheroid fusion when cultured from 2×105nuclei spheroids, with the loss of boundaries between the spheroids. Moreover, a viable cell region was found at the center of the tissue construct in several locations. Poor adhesion was found between spheroids cultured from 4×105nuclei spheroids. The total nuclei density in cultured tissue constructs was estimated to be about half of that in HUVEC-covered hepatocyte spheroids.This study demonstrated the possibility of medium perfusion through scaffold-free tissue constructs by assembling endothelial cell-covered spheroids, promising for a large tissue construct culture in vitro.
Keywords: Spheroid; Perfusion culture; Hepatocyte; Endothelial cell; Scaffold-free
A descriptive model and methods for up-scaled process routes for interfacial partition of bioparticles in aqueous two-phase systems by Frank Luechau; Tau Chuan Ling; Andrew Lyddiatt (pp. 122-130).
In this paper, we have developed a descriptive model and methods for the up-scaled interfacial partition of bioparticles in polymer–salt aqueous two-phase systems (ATPS). The model is only valid for ideal spherical particles. The model might shed some light on the mechanism of interfacial partition, which allows one to draft some process routes for its potential operation on a larger scale.
Keywords: Bioseparations; Aqueous two-phase system; Integrated processing; Protein recovery; Purification; Downstream processing
Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture by Lefki-Maria Papaspyridi; Petros Katapodis; Zacharoula Gonou-Zagou; Evangelia Kapsanaki-Gotsi; Paul Christakopoulos (pp. 131-138).
This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57gL−1 xylose and 37gL−1 corn steep liquor, high yields (39.2gL−1) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140±4 and 625±9mgg−1 mycelium dry weight, respectively.
Keywords: Pleurotus ostreatus; FF Biolog MicroPlate; Response surface methodology; Bioreactor; Dietary fibres; Glucan
Evaluation of anthrax vaccine production by Bacillus anthracis Sterne 34F2 in stirred suspension culture using a miniature bioreactor: A useful scale-down tool for studies on fermentations at high containment by Tarit K. Mukhopadhyay; Nigel Allison; Susan Charlton; Michael J. Hudson; Bassam Hallis; Annemarie King; Rebecca Baker; Sara Noonan; Joanne McGlashan; Katie West; M. Susana Levy; John M. Ward; Gary J. Lye (pp. 139-144).
The licensed UK anthrax vaccine is produced by static cultures of Bacillus anthracis Sterne 34F2 in glass Thompson bottles, each batch consisting of multiple bottles grown for 24–28h. In this work, a novel miniature bioreactor was used as a scale-down tool to investigate the possible transfer of anthrax vaccine production from static culture to stirred tank operation and to explore the effects of this change in culture conditions on process performance. It is shown that the change to stirred culture conditions is possible and that the concentration of the two main vaccine components, Protective Antigen (PA) and Lethal Factor (LF), are reached in less than half the time compared to standard Thompson bottle methods. Furthermore, because higher cell densities were attained in the miniature bioreactor, a 74% increase in antigen concentration was achieved. More detailed analysis of the stirred bioreactor results operated with and without aeration showed antigen degradation in the presence of aeration. Overall this work demonstrates the usefulness of miniaturisation techniques to perform process characterisation studies safely and without significant capital investment for large-scale containment.
Keywords: Bacillus anthracis; Sterne 34F; 2; Miniature bioreactor; Anthrax vaccine; Protective Antigen (PA); Lethal Factor (LF)
Three-phase partitioning of protease from Calotropis procera latex by Saroat Rawdkuen; Phanuphong Chaiwut; Punyawatt Pintathong; Soottawat Benjakul (pp. 145-149).
Three-phase partitioning (TPP) was used to partially purify protease from the latex of Calotropis procera ( C. procera). To optimize the TPP for protease isolation a ratio of crude extract to t-butanol, percent saturation of (NH4)2SO4, and the cycle of TPP was required. The highest proteolytic recovery (first cycle) of 182% with a purification of 0.95 folds was obtained at the interphase of the system comprising the ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 50% (NH4)2SO4. The second cycle of TPP was prepared by adding of (NH4)2SO4 up to 65% (w/v) to the bottom phase obtained from 30% (NH4)2SO4–1.0:0.5 system of the first TPP. A purification of 6.92-fold was achieved with about 132% activity recovery. SDS-PAGE and zymography profiles revealed the substantial isolation of protease from C. procera latex by the TPP. The molecular weight of major protease was found to be around 28kDa. The present study shows high interesting outcomes and could be used as a primary purification process in comparison with existing literature's values.
Keywords: Calotropis procera; Latex; Protease; Three-phase partitioning; t; -Butanol
Technical comments on “Oxic-settling-anoxic (OSA) process combined with 3,3′,4′,5-tetrachlorosalicylanilide (TCS) to reduce excess sludge production in the activated sludge system” by Fenxia Ye and Ying Li, Biochem. Eng. J. 49 (2010) 229–234 (doi:10.1016/j.bej.2010.01.001) by Gaurav Saini (pp. 150-151).
M; etabolic uncoupling; OSA; TCS; E; nergy wastage