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Biochemical Engineering Journal (v.33, #1)
Optimization of serum free medium for cord blood mesenchymal stem cells by Chi-Hsien Liu; Mei-Ling Wu; Shiaw-Min Hwang (pp. 1-9).
Human umbilical cord blood harbors mesenchymal stem cells (MSCs), which can give rise to several mesenchymal lineages. In order to explore their usages in medical applications, the ex vivo expansion of MSCs to sufficient cell numbers is necessary. Additionally, the development of a serum-free medium becomes indispensable for elimination of possible contaminants from the serum-containing medium during expansion. Using fractional factorial designs combined with the steepest ascent approach, we have developed a serum-free medium that could ex vivo expand MSCs over nine passages, resulting in at least 1000-fold increases in cell number within 1-month. Based on Iscove's modified Dulbecco's medium, this medium formulation includes bFGF (17.91ng/mL), human albumin (2.80mg/mL), hydrocortisone (27.65μM) and SITE (1.18%, v/v). The expanded MSCs in the designed medium preserved differentiation potentials into three mesenchymal lineages in vitro, including chondrocytes, adipocytes and osteoblasts. In conclusion, we optimized a serum-free and defined culture medium for cord blood-derived MSCs, which could be applied to cell-based therapy and biomedical research.
Keywords: Biomedical engineering; Cord blood; Fractional factorial designs; Optimization; Stem cell culture; Serum-free medium
The possibility for improvement of ceramic membrane ultrafiltration of an enzyme solution by D.M. Krstić; M.G. Antov; D.M. Peričin; W. Höflinger; M.N. Tekić (pp. 10-15).
Ultrafiltration represents an attractive downstream processing technique for enzymes concentration and their primary purification. However, the process efficiency is often limited by protein fouling and shear-induced enzyme deactivation, resulting in permeate flux decline and loss of enzyme activity. The objective of this work was to investigate the possibility for improvement of ceramic membrane ultrafiltration of endo-pectinase solution. Experimental investigations were performed on a 5nm ceramic membrane with the Kenics static mixer placed inside the membrane in order to improve the process performance. The use of the static mixer resulted in the flux improvement of about 45% at a volume concentration factor (VCF) of 3 leading to the reduction of operation time of 25% and the energy saving of about 40%. Although the rejection of endo-pectinase was higher than 96%, the extensive loss of the enzyme activity during operation indicated that the modification of the feed solution is essential for improved ultrafiltration performance. Addition of pectin to the original endo-pectinase solution led to a significant reduction of the enzyme deactivation: the enzyme activity yield was 90% at a VCF of 1.6 during operation with the static mixer.
Keywords: Ultrafiltration; Ceramic membrane; Enzyme concentration; Enzyme deactivation; Endo-pectinase; Static mixer
Prefermentation to overcome nutrient limitations in food processing wastewater: Comparison of pilot- and bench-scale systems by Zhongda Xu; George Nakhla (pp. 16-25).
A bench- and a pilot-scale anaerobic/aerobic system were evaluated for the treatment of high strength tomato-processing wastewater. The pilot-scale anaerobic tank achieved better prefermentation of organic carbon and nitrogen than the bench-scale system, although overall system performance was comparable with more than 99% SBOD removal and 97% SCOD removal. Hydraulic retention time (HRT) and temperature effects were studied in the bench-scale system. Increase of anaerobic HRT from 0.25 day to 0.5 day favored prefermentation and a better effluent quality was achieved, as demonstrated by reduction in TSS concentrations from 66mg/L to 24mg/L, SCOD from 103mg/L to 78mg/L and SBOD from 8mg/L to 6mg/L, respectively. Specific oxygen uptake rate (SOUR) increased from 0.15–0.23mgO2/mgVSSday at 25°C to 0.67–1.24mgO2/mgVSSday at 32°C. Settling characteristics deteriorated from sludge volume index (SVI) of 24–131mL/g at 25°C to 115–173mL/g at 32°C. Sludge yield decreased from 0.14gVSS/gCOD at 25°C to 0.098gVSS/gCOD at 32°C.
Keywords: Anaerobic/aerobic system; Tomato-processing wastewater; Prefermentation; HRT effect; Temperature effect
Production of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. TS1-1: Optimization of carbon and nitrogen concentration in the feed medium using central composite design by Wan Salwanis Wan Md. Zain; Rosli Md. Illias; Madihah Md. Salleh; Osman Hassan; Roshanida A. Rahman; Aidil Abd. Hamid (pp. 26-33).
Optimisation of nutrient feeding was developed to overcome the limitation in batch fermentation and to increase the CGTase production from Bacillus sp. TS1-1 in fed batch fermentation. Optimisation of the C/N ratio in the feed stream was conducted in a 5l fermenter, where feeding was initiated at constant rate of 0.02h−1. In our initial screening process, the addition of nitrogen source boosted the growth of the microbes, but on the other hand reduced the CGTase production. The amount of tapioca starch and yeast extract was optimised in order to obtain a sufficient growth and thus, increased the CGTase production. Results were analysed using three-dimensional response surface plot, and the optimised values of carbon and nitrogen concentration of 3.30% (w/v) and 0.13% (w/v) were obtained, respectively. CGTase activity increased up to 80.12U/ml, which is 13.94% higher as compared to batch fermentation (70.32U/ml). This also led to 14.54% increment of CGTase production in fed batch culture as compared to the production before the optimisation. The CGTase activity obtained was close to the predicted value, which is 78.05U/ml.
Keywords: Cyclodextrin glucanotransferase; C/N ratio; Fed batch; Central composite design
Effects of post-induction feed strategies on secretory production of recombinant streptokinase in Escherichia coli by Subramanian Ramalingam; Pennathur Gautam; Krishna Jyoti Mukherjee; Guhan Jayaraman (pp. 34-41).
The effects of post-induction nutrient feed rates, on recombinant streptokinase production in fed-batch processes, were investigated using various feed profiles. Recombinant streptokinase was produced using a secretory expression system and was induced by a temperature up-shift, using a temperature-sensitive λPL promoter. The specific growth rates decreased sharply upon induction of recombinant protein expression, thus necessitating a variable feed strategy in the post-induction phase. The various feed profiles employed in the post-induction phase included constant feed rates, linearly increasing feed rate and exponentially varying feed rates. Significant differences were obtained in the specific activity of streptokinase produced in these fed-batch processes. A maximum activity per unit biomass of 4.96×106 and 4.43×106IU/gDCW was achieved for exponentially decreasing feed and linearly increasing feed, respectively. The decrease in specific growth rate during the post-induction phase was also less pronounced in these cases in comparison to other fed-batch experiments. It was observed that streptokinase productivity is governed by the nutrient feed rate per unit biomass at a critical juncture after induction. The highest activity per unit biomass was obtained when the nutrient feed rate per unit biomass was around 0.7–0.8gglucose (gDCW)−1h−1, between 2 and 4h after induction.
Keywords: Secretory expression; Recombinant streptokinase; Fed-batch process; Post-induction feed strategy; Escherichia coli
Stoichiometric analysis and experimental investigation of glycerol–glucose co-fermentation in Klebsiella pneumoniae under microaerobic conditions by Zhi-Long Xiu; Xi Chen; Ya-Qin Sun; Dai-Jia Zhang (pp. 42-52).
The ratio between two substrates is an important parameter in microbial co-fermentation, such as 1,3-propanediol production from glycerol by Klebsiella pneumoniae using glucose as the cosubstrate. In this study, the glycerol–glucose cometabolism by K. pneumoniae is stoichiometrically analyzed according to energy (ATP), reducing equivalent (NADH2) and product balances. The theoretical analysis reveals that the yield of 1,3-propanediol to glycerol under microaerobic conditions depends not only on the ratio of glucose to glycerol initially added, but also on the molar fraction of reducing equivalent oxidized completely by molecular oxygen in tricarboxylic acid (TCA) cycle ( δ) and the molar fraction of TCA cycle in acetyl-CoA metabolism ( γ). The maximum ratio of 0.32mol glucose per mol glycerol is needed to convert glycerol completely to 1,3-propanediol under anaerobic conditions if glycerol neither enters oxidation pathways nor forms biomass. The ratio can be reduced under microaerobic conditions. The experimental results of batch cultures demonstrate that the biomass concentration and yield of 1,3-propanediol on glycerol could be enhanced by using glucose as a co-substrate. The theoretical analysis reveals the relationship between yield of 1,3-propanediol to glycerol, ratio of glucose to glycerol and respiratory quotient (RQ). These results are helpful for the experimental design and control.
Keywords: Stoichiometric analysis; Klebsiella pneumoniae; Glycerol; Glucose; 1,3-Propanediol
Occurrence of virulence genes associated with enterohemorrhagic Escherichia coli in raw municipal sewage by Raheela Awais; Kazuhiko Miyanaga; Hajime Unno; Yasunori Tanji (pp. 53-59).
Municipal sewage influent was screened for the presence of the virulence genes encoding Shiga-like toxins SLT-I and SLT-II ( slt-I and slt-II) and intimin ( eaeA) and those involved in biosynthesis of O157 ( rfbE) and H7 ( fliC) antigens by multiplex PCR to simultaneously identify the enterohemorrhagic Escherichia coli (EHEC) O157:H7 and its virulence factors in a single reaction. The screening was carried out monthly from October 2004 to September 2005. Direct PCR analysis using total DNA from sewage concentrate showed the presence of at least one virulence gene in 100% samples ( n=12). Sixty six percent of these samples were also positive for rfbE (O157) gene and fliC (H7) gene. The PCR amplification of these genes was possible when the concentration was above 20cellsml−1. From the multiplex PCR of the isolates following plating on Cefixime-Tellurite Sorbitol MacConkey (CT-SMAC) agar to detect non-sorbitol fermenting (NSF) colonies ( n=600), one E. coli strain carrying slt-II gene and two strains of E. coli O157:H7 carrying slt-I were detected. The results show that municipal sewage represents a potential reservoir of EHEC. CT-SMAC agar was proved to have limited E. coli O157:H7 selectivity and only 0.005% (3/600) sensitivity for sewage samples due to the high frequency (43%) of NSF strains in sewage. The enrichment of sewage sample in modified E. coli broth (mEC) increased the sensitivity of PCR resulting in the clearer amplification of five genes. Amplification of target cell type in mEC broth implied that EHEC were present in sewage in a culturable and hence potentially infectious state. However, pre-enrichment did not affect the selectivity of CT-SMAC because frequency of NSF colonies remained the same as that obtained without enrichment. The study, therefore, underscores the need for more sensitive screening techniques that can be routinely employed for the regular monitoring of sewage influent.
Keywords: Enterohemorrhagic; E. coli; (EHEC); E. coli; O157:H7; Municipal sewage; Sorbitol MacConkey agar; Modified; E. coli; broth
An improved method of lipase preparation incorporating both solvent treatment and immobilization onto matrix by Md. Mahabubur Rahman Talukder; Sriappareddy Tamalampudy; Chong Jia Li; Le Yanglin; Jinchuan Wu; Akihiko Kondo; Hideki Fukuda (pp. 60-65).
A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol–buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9mmolh−1g−1, which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222nm suggests that the α-helical content of lipase increase after pretreatment.
Keywords: Lipase; Immobilization; Isopropanol treatment; Enzyme activity; Adsorption; Macroporous resin
Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme by Li-Li Shen; Xue-Jun Cao (pp. 66-71).
The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28°C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05M NaClO4. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30μmolg−1 polymer. The adsorption capacity of lysozyme on the polymer was 3.4mgg−1polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.
Keywords: Thermo-sensitive; N; -Alkyl substituted acrylamide polymer; Lower critical solution temperature; Affinity precipitation; Cibacron Blue F3GA
Analysis of the transient response of a CSTR containing immobilized enzyme particles by Arturo Horta; José R. Álvarez; Susana Luque (pp. 72-87).
The transient response of the bulk substrate concentration in a CSTR containing immobilized enzyme (IMEs) in porous solid supports and the possibilities of exploiting the minimum behavior for parameter estimation purposes are studied in this work. For that purpose, mathematical models have been developed for several kinetic expressions with parallel deactivation mechanisms and for two different particle shapes (cylindrical and spherical). The influence of a number of system variables and non-dimensional parameters, i.e., pellet radius, mass of particles, flow rate, effective diffusivity, mass transfer coefficient, Thiele modulus, Michaelis constant, and enzyme deactivation constant, is also reported in a systematic way. Simulations, using realistic data, show that the minimum bulk substrate concentration is sufficiently pronounced and the time scale for the minimum to occur is sufficiently practical, so that it can be used to extract several parameters of the immobilized enzyme system.
Keywords: Transient response; Simulation; Bioreactors; Multiphase reactors; Parameter identification; Immobilized enzyme particles
Improved accumulation of phenylethanoid glycosides by precursor feeding to suspension culture of Cistanche salsa by Jin-Yan Liu; Zhi-Gang Guo; Zhao-Lin Zeng (pp. 88-93).
Effects of some precursors on phenylethanoid glycosides (PeGs) accumulation in Cistanche salsa cell suspension cultures were investigated. Precursors such as tyrosine, phenylalanine, caffeic acid and cucumber juice at proper concentrations could increase the total accumulation of PeGs (echinacoside, acteoside, 2′-acetylacteoside) by 50%, 12%, 12% and 23%, respectively. Under the combined feeding of precursors at proper concentrations, the total production of PeGs in bio-staged culture reached the highest amount of 1358.1mgl−1 (640.8mg echinacosidel−1, 689.4mg acteosidel−1 and 54.9mg 2′-acetylacteosidel−1), which was about two-fold of that in the control. This study showed promise for obtaining large-scale production of active ingredients in plant cells by the solid–liquid two step culture (SLTSC) technique and also provided for the first time an example for producing PeGs by C. salsa cell culture. The improved production of PeGs was higher than that in previous reports on PeG production by Cistanche deserticola cell culture fed with precursors.
Keywords: Cistanche salsa; Phenylethanoid glycosides; Plant cell culture; Biosynthesis; Large scale cultivation; Precursor
Thermolysis of recombinant Escherichia coli for recovering a thermostable enzyme by Xiaodong Ren; Dawei Yu; Siping Han; Yan Feng (pp. 94-98).
The development of recombinant DNA has made it feasible to produce a wide range of valuable protein products in the bacterium Escherichia coli. Extraction of intracellular protein from E. coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, thermolysis, which differs from the traditional ones, is presented for disruption of E. coli cells to release recombinant thermostable enzyme. Heat treatment of E. coli at 80°C is highly effective to destroy the integrity of the bacterial cell wall and release the recombinant thermostable enzyme. At the same time of disruption, the recombinant thermostable enzyme was partially purified. Moreover, thermolysis was carried out in fermentation broth in situ, which may make it a more applicable approach for industrial-scale processes.
Keywords: Abbreviations; rpm; round per minute; IPTG; isopropyl-β-; d; -thiogalactopyranoside; SDS; -; PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresisBioseparations; Cell risruption; Downstream processing; Protein recovery; Thermolysis; Escherichia coli