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1.
  • Bjornsson, L., et al. (författare)
  • Utilization of a palladium-metal oxide semiconductor (Pd-MOS) sensor for on-line monitoring of dissolved hydrogen in anaerobic digestion
  • 2001
  • Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 73:1, s. 35-43
  • Tidskriftsartikel (refereegranskat)abstract
    • The use of a hydrogen-sensitive palladium-metal oxide semiconductor (Pd-MOS) sensor in combination with a membrane for liquid-to-gas transfer for the detection of dissolved hydrogen was investigated. The system was evaluated with known concentrations of dissolved hydrogen in water. The lowest concentration detected with this set-up was 160 nM. The method was applied to monitoring of a laboratory-scale anaerobic digestion process employing mixed sludge containing mainly food/industrial waste. Pulse loads of glucose were added to the system at different levels of microbial activity, and the microbial status of the culture was reflected in the dissolved hydrogen response. Simultaneous headspace hydrogen measurements were performed, and at the lower levels of dissolved hydrogen no corresponding headspace hydrogen could be detected. When glucose was added to a resting culture the dissolved hydrogen response was rapid and the first response could be detected 9 min after addition of glucose, whereas headspace hydrogen concentrations increased only after 80 to 110 min. This indicates limitations in the liquid-to-gas hydrogen transfer and illustrates the importance of hydrogen monitoring in the liquid. The sensor system developed is flexible, the membrane is easily replaceable, and the probe for liquid-to-gas hydrogen transfer can be adjusted easily to large-scale applications. © 2001 John Wiley & Sons, Inc.
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2.
  • Boer, H., et al. (författare)
  • Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters
  • 2000
  • Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 69:5, s. 486-494
  • Tidskriftsartikel (refereegranskat)abstract
    • Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) pro meter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermenter. Production of Ce17A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K-m values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.
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3.
  • Taherzadeh, Mohammad J, 1965-, et al. (författare)
  • On-line control of fed-batch fermentation of dilute-acid hydrolyzates
  • 2000
  • Ingår i: Biotechnology and Bioengineering. - New York, NY, United States : John Wiley & Sons Inc. - 0006-3592 .- 1097-0290. ; 69:3, s. 330-338
  • Tidskriftsartikel (refereegranskat)abstract
    • Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%). (C) 2000 John Wiley and Sons, Inc.Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).
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4.
  • Bylund, F., et al. (författare)
  • Influence of scale-up on the quality of recombinant human growth hormone
  • 2000
  • Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 69:2, s. 119-128
  • Tidskriftsartikel (refereegranskat)abstract
    • The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer.
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5.
  • Adlercreutz, Dietlind, et al. (författare)
  • Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction.
  • 2002
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 403-411
  • Tidskriftsartikel (refereegranskat)abstract
    • The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.
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6.
  • Adlercreutz, Patrick (författare)
  • Oxygen supply to immobilized cells : 5. Theoretical calculations and experimental data for the oxidation of glycerol by immobilized Gluconobacter oxydans cells with oxygen or p‐benzoquinone as electron acceptor
  • 1986
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 28:2, s. 223-232
  • Tidskriftsartikel (refereegranskat)abstract
    • Theoretical calculations of reaction kinetics were done for one‐step reactions catalyzed by cells immobilized in spherical beads. The reactions catalyzed by free cells were assumed to obey Michaelis–Menten kinetics for a one‐substrate reaction. Both external (outside the beads) and internal (inside the beads) mass transfer of the substrate were considered for the immobilized preparations. The theoretical calculations were compared with experimental data for the oxidation of glycerol to dihydroxyacetone by Gluconobacter oxydans cells immobilized in calcium alginate gel. Glycerol was present in excess so that the reaction rate was limited by oxygen. The correlation between experimental data and theoretical calculations was quite good. The calculations showed how the overall effectiveness factor was influenced by, for example, the particle size and the cell density in the beads. In most cases the reaction rate was mainly limited by internal mass transfer of the substrate (oxygen). As shown previously, p‐benzoquinone can replace oxygen as the electron acceptor in this reaction. The same equations for reaction kinetics and mass transfer were used with p‐benzoquinone as the rate‐limiting substrate. Parameters such as diffusivity, maximal reaction rate, and K were, of course, different. In this case also, the correlation between the model and the experimental results was quite good. Much higher production rates were obtained with p‐benzoquinone as the electron acceptor compared to when oxygen was used. The reasons for this fact were that p‐benzoquinone gave a higher maximal reaction rate for free cells and the solubility of p‐benzoquinone was higher than for oxygen. Different methods of increasing the rate of microbial oxidation reactions are discussed.
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7.
  • Andersson, Jonatan, et al. (författare)
  • Isolation of potato proteins using simulated moving bed technology.
  • 2008
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 101, s. 1256-1263
  • Tidskriftsartikel (refereegranskat)abstract
    • The simulated moving bed (SMB) concept of chromatography was applied to treat potato juice from production of starch. The aim was to harvest proteins. SMB offers possibilities to operate with different process strategies and in this study it was shown possible to harvest up to 80% of the protein in a process utilizing very little extra water besides that already present in the juice. After depleting protein from the juice in the adsorption step, the flow through was used to recondition the column after elution. The present study illustrates a new concept of applying chromatography as a capturing step of bulk products. Biotechnol. Bioeng. (c) 2008 Wiley Periodicals, Inc.
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8.
  • Andersson, Mats, et al. (författare)
  • Toward an enzyme-based oxygen scavenging laminate. Influence of industrial lamination conditions on the performance of glucose oxidase
  • 2002
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 79:1, s. 37-42
  • Tidskriftsartikel (refereegranskat)abstract
    • The laminate consisted of several polymer layers, aluminium, and one cellulose-based layer containing the active enzymatic system (e.g., glucose oxidase, catalase, glucose, and CaCO3). During the industrial lamination process, the enzyme layer was exposed to three temperature spikes up to 325degreesC without significant enzyme inactivation. Ninety-seven percent of the glucose oxidase activity still remained after the lamination process. The best laminate had an oxygen absorbing capacity of 7.6 +/- 1.0 L/m(2). A reference that was not laminated expressed a corresponding oxygen absorbing capacity of 7.1 +/- 0.8 L/m(2).
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9.
  • Arnling Bååth, Jenny, 1987, et al. (författare)
  • Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica
  • 2022
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 119:2, s. 470-481
  • Tidskriftsartikel (refereegranskat)abstract
    • Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure–function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.
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10.
  • Asadollahi, M. A., et al. (författare)
  • Enhancement of Farnesyl Diphosphate Pool as Direct Precursor of Sesquiterpenes Through Metabolic Engineering of the Mevalonate Pathway in Saccharomyces cerevisiae
  • 2010
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 106:1, s. 86-96
  • Tidskriftsartikel (refereegranskat)abstract
    • The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Overexpression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous overexpression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids harboring cubebol synthase gene. This could be explained by a toxicity effect of cubebol, possibly resulting in higher transcription levels for the genes under control of MET3 promoter, which could lead to accumulation of squalene and ergosterol. Biotechnol. Bioeng. 2010;106: 86-96. (C) 2010 Wiley Periodicals, Inc.
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11.
  • Batstone, Damien, et al. (författare)
  • Kinetics of thermophilic, anaerobic oxidation of straight and branched chain butyrate and valerate
  • 2003
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:2, s. 195-204
  • Tidskriftsartikel (refereegranskat)abstract
    • The degradation kinetics of normal and branched chain butyrate and valerate are important in protein-fed anaerobic systems, as a number of amino acids degrade to these organic acids. Including activated and primary wastewater sludge digesters, the majority of full-scale systems digest feeds with a significant or major fraction of COD as protein. This study assesses the validity of using a common kinetic parameter set and biological catalyst to represent butyrate, n-valerate, and i-valerate degradation in dynamic models. The i-valerate degradation stoichiometry in a continuous, mixed population system is also addressed, extending previous pure-culture and batch studies. A previously published mathematical model was modified to allow competitive uptake of i-valerate, and used to model a thermophilic manure digester operated over 180 days. The digester was periodically pulsed with straight and branched chain butyrate and valerate. Parameters were separately optimized to describe butyrate, i-valerate, and n-valerate degradation, as well as a lumped set optimized for all three substrates, and nonlinear, correlated parameter spaces estimated using an F distribution in the objective function (A Each parameter set occupied mutually exclusive parameter spaces, indicating that all were statistically different from each other. However, qualitatively, the influence on model outputs was similar, and the lumped set would be reasonable for mixed acid digestion. The main characteristic not represented by Monod kinetics was a delay in i-valerate uptake, and was compensated for by a decreased maximum uptake rate (k(m)). Therefore, the kinetics need modification if fed predominantly i-valerate. Butyrate (i- and n-) and n-valerate could be modeled using stoichiometry consistent with beta-oxidation degradation pathways. However, i-valerate produced acetate only, supporting the stoichiometry of a reaction determined by other researchers in pure culture. Therefore, lumping i-valerate stoichiometry with that of n-valerate will not allow good system representation, especially when the feed consists of proteins high in leucine (which produces i-valerate), and the modified model structure and stoichiometry as proposed here should be used. This requires no additional kinetic parameters and one additional dynamic concentration state variable (i-valerate) in addition to the variables in the base model.
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12.
  • Bengtsson, Simon (författare)
  • The utilization of glycogen accumulating organisms for mixed culture production of polyhydroxyalkanoates.
  • 2009
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 104, s. 698-708
  • Tidskriftsartikel (refereegranskat)abstract
    • Production of polyhydroxyalkanoates (PHA) by an open mixed culture enriched in glycogen accumulating organisms (GAOs) under alternating anaerobic-aerobic conditions with acetate as carbon source was investigated. The culture exhibited a stable enrichment performance over the 450 day operating period with regards to phenotypic behavior and microbial community structure. Candidatus Competibacter phosphatis dominated the culture at between 54 and 70 % of the bacterial biomass throughout the study, as determined by fluorescence in situ hybridization. In batch experiments under anaerobic conditions, PHA containing 3-hydroxybutyrate (3HB) and 27 mol-% 3-hydroxyvalerate (3HV) was accumulated up to 49 % of cell dry weight utilizing the glycogen pool stored in the SBR cycle. Under aerobic and ammonia limited conditions, PHA comprising only 3HB was accumulated to 60 % of cell dry weight. Glycogen was consumed during aerobic PHA accumulation as well as under anaerobic conditions, but with different stoichiometry. Under aerobic conditions 0.31 C-mol glycogen was consumed per consumed C-mol acetate compared to 0.99 under anaerobic conditions. Both the PHA biomass content and the specific PHA production rate obtained were similar to what is typically obtained using the more commonly applied aerobic dynamic feeding strategy. (c) 2009 Wiley Periodicals, Inc.
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13.
  • Bergenholm, David, 1987, et al. (författare)
  • Construction of mini-chemostats for high-throughput strain characterization
  • 2019
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 116:5, s. 1029-1038
  • Tidskriftsartikel (refereegranskat)abstract
    • To achieve large-scale, high-throughput experiments for systems biology research of microorganisms, reliable data from robust cultivation systems are needed. Chemostats are such systems, ensuring reproducibility and quality by providing a stable, well-controlled environment for the cells. However, many of the available chemostat systems require large amounts of media and are complex to set up and expensive to purchase and maintain. To address these concerns, we developed a mini-chemostat (MC) system with 16 reactors, each at a working volume of 40 ml. Sensors measure dissolved oxygen in the reactor, while OD600 is measured in the outflow. We further developed a CO 2 and pH sensor array that can be plugged into the outflow of the reactors. The system was used to characterize yeast physiology at four metabolically different conditions: limitations of glucose, both aerobic and anaerobic, nitrogen, and ethanol. The physiology of yeast cells grown at the four different conditions in the MC system was compared with the yeast cells grown in a DASGIP 1 L system using RNAseq analysis. The results show that the MC system provides the same environmental conditions as the DASGIP system and that the MC system is reproducible between different runs. The system is built to be easily scalable with more reactors and to include more sensors, if available. Our study shows that a robust, reproducible chemostat system for high-throughput and large-scale experiments can be built at low costs.
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14.
  • Bergenholm, David, 1987, et al. (författare)
  • Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids
  • 2018
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:4, s. 932-942
  • Tidskriftsartikel (refereegranskat)abstract
    • Chain length and degree of saturation plays an important role for the characteristics of various products derived from fatty acids, such as fuels, cosmetics, and food dditives. The seeds of Theobroma cacao are the source of cocoa butter, a natural lipid of high interest for the food and cosmetics industry. Cocoa butter is rich in saturated fatty acids that are stored in the form of triacylglycerides (TAGs). One of the major TAG species of cocoa butter, consisting of two stearic acid molecules and one oleic acid molecule (stearic acid-oleic acid-stearic acid, sn-SOS), is particularly rare in nature as the saturated fatty acid stearic acid is typically found only in low abundance. Demand for cocoa butter is increasing, yet T. cacao can only be cultivated in some parts of the tropics. Alternative means of production of cocoa butter lipids (CBLs) are, therefore, sought after. Yeasts also store fatty acids in the form of TAGs, but these are typically not rich in saturated fatty acids. To make yeast an attractive host for microbial production of CBLs, its fatty acid composition needs to be optimized. We engineered Saccharomyces cerevisiae yeast strains toward a modified fatty acid synthesis. Analysis of the fatty acid profile of the modified strains showed that the fatty acid content as well as the titers of saturated fatty acids and the titers of TAGs were increased. The relative content of potential CBLs in the TAG pool reached up to 22% in our engineered strains, which is a 5.8-fold increase over the wild-type. SOS content reached a level of 9.8% in our engineered strains, which is a 48-fold increase over the wild type.
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15.
  • Bodin, Aase Katarina, 1977, et al. (författare)
  • Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes
  • 2007
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 97:2, s. 425-434
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacterial cellulose (BC) was deposited in tubular form by fermenting Acetobacter xylinum on top of silicone tubes as an oxygenated support and by blowing different concns. of oxygen, i.e., 21% (air), 35%, 50%, and 100%. Mech. properties such as burst pressure and tensile properties were evaluated for all tubes. The burst pressure of the tubes increased with an increase in oxygen ratio and reached a top value of 880 mmHg at 100% oxygen. The Young's modulus was approx. 5 MPa for all tubes, irresp. of the oxygen ratio. The elongation to break decreased from 30% to 10-20% when the oxygen ratio was increased. The morphol. of the tubes was characterized by SEM (SEM). All tubes had an even inner side and a more porous outer side. The cross section indicated that the tubes are composed of layers and that the amt. of layers and the yield of cellulose increased with an increase in oxygen ratio. We propose that an internal vessel wall with high d. is required for the tube to sustain a certain pressure. An increase in wall thickness by an increase in oxygen ratio might explain the increasing burst pressure with increasing oxygen ratio. The fermn. method used renders it possible to produce branched tubes, tubes with unlimited length and inner diams. Endothelial cells (ECs) were grown onto the lumen of the tubes. The cells formed a confluent layer after 7 days. The tubes potential as a vascular graft is currently under investigation in a large animal model at the Center of Vascular Engineering, Sahlgrenska University Hospital, Gothenburg.
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16.
  • Bramble, J L, et al. (författare)
  • Calcium and Phosphate Effects on Growth and Alkaloid Production in Coffea arabica: Experimental Results and Mathematical Model.
  • 1991
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 37:9, s. 859-868
  • Tidskriftsartikel (refereegranskat)abstract
    • Plant, mammalian, and microbial cells are commonly immobilized in calcium alginate gels for the production of valuable secondary metabolites. However, calcium ions are known to inhibit growth in various type of cells, and calcium is an integral part of such gels. Therefore, an investigation was conducted to evaluate the effect of calcium on the growth and alkaloid production of a model cell-line, Coffea arabica, in suspension culture before attempting to immobilize such cells in alginate. A kinetic model was then developed from the results to describe cell growth and alkaloid production and the mechanism by which calcium influences these variables. In addition, it was observed that there was a characteristic relationship between the concentration of calcium in the external medium and the concentration of extracellular and intracellular phosphate. The intracellular phosphate level was, in turn, related to the production of alkaloids. Using these results, a dynamic mathematical model of cell growth and alkaloid production was developed based on the proposed roles of calcium and phosphate. The model showed satisfactory agreement with three sets of experiments at different calcium concentrations. A possible linkage between the calcium and phosphate results is postulated based on the limited solubility of calcium phosphate. 
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17.
  • Brandberg, T., et al. (författare)
  • Continuous fermentation of wheat-supplemented lignocellulose hydrolysate with different types of cell retention
  • 2007
  • Ingår i: Biotechnology and Bioengineering. - : John Wiley & Sons, Inc.. - 0006-3592 .- 1097-0290. ; 98:1, s. 80-
  • Tidskriftsartikel (refereegranskat)abstract
    • Medium supplementation and process alternatives for fuel ethanol production from dilute acid lignocellulose hydrolysate were investigated. Dilute acid lignocellulose hydrolysate supplemented with enzymatically hydrolysed wheat flour could sustain continuous anaerobic cultivation of Saccharomyces cerevisiae ATCC 96581 if further supplemented with ammonium sulphate and biotin. This medium composition allowed for a hexose utilisation of 73% and an ethanol production of 36 mmol l-1 h-1 in chemostat cultivation at dilution rate 0.10 h-1. Three different methods for cell retention were compared for improved fermentation of supplemented lignocellulose hydrolysate: cell recirculation by filtration, cell recirculation by sedimentation and cell immobilisation in calcium alginate. All three cell retention methods improved the hexose conversion and increased the volumetric ethanol production rate. Recirculation of 75% of the bioreactor outlet flow by filtration improved the hexose utilisation from 76% to 94%. Sedimentation turned out to be an efficient method for cell separation; the cell concentration in the reactor was 32 times higher than in the outflow after 60 h of substrate feeding. However, chemostat and continuous cell recirculation cultures became severely inhibited when the dilution rate was increased to 0.20 h-1. In contrast, an immobilised system kept producing ethanol at a stable level also at dilution rate 0.30 h-1. Biotechnol. Bioeng. 2007; 98: 80-90. © 2007 Wiley Periodicals, Inc.
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18.
  • Brechmann, Nils A., et al. (författare)
  • Antibody capture process based on magnetic beads from very high cell density suspension
  • 2021
  • Ingår i: Biotechnology and Bioengineering. - : John Wiley and Sons Inc. - 0006-3592 .- 1097-0290. ; 118:9, s. 3499-3510
  • Tidskriftsartikel (refereegranskat)abstract
    • Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non-clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed-batch cultures. The performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification. 
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19.
  • Buijs, Nicolaas, 1985, et al. (författare)
  • Long-chain Alkane Production by the Yeast Saccharomyces cerevisiae
  • 2015
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 112:6, s. 1275-1279
  • Tidskriftsartikel (refereegranskat)abstract
    • In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfdl and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFDI together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol.
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20.
  • Cabaneros Lopez, Pau, et al. (författare)
  • Transforming data to information : A parallel hybrid model for real-time state estimation in lignocellulosic ethanol fermentation
  • 2021
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:2, s. 579-591
  • Tidskriftsartikel (refereegranskat)abstract
    • Operating lignocellulosic fermentation processes to produce fuels and chemicals is challenging due to the inherent complexity and variability of the fermentation media. Real-time monitoring is necessary to compensate for these challenges, but the traditional process monitoring methods fail to deliver actionable information that can be used to implement advanced control strategies. In this study, a hybrid-modeling approach is presented to monitor cellulose-to-ethanol (EtOH) fermentations in real-time. The hybrid approach uses a continuous-discrete extended Kalman filter to reconciliate the predictions of a data-driven model and a kinetic model and to estimate the concentration of glucose (Glu), xylose (Xyl), and EtOH. The data-driven model is based on partial least squares (PLS) regression and predicts in real-time the concentration of Glu, Xyl, and EtOH from spectra collected with attenuated total reflectance mid-infrared spectroscopy. The estimations made by the hybrid approach, the data-driven models and the internal model were compared in two validation experiments showing that the hybrid model significantly outperformed the PLS and improved the predictions of the internal model. Furthermore, the hybrid model delivered consistent estimates even when disturbances in the measurements occurred, demonstrating the robustness of the method. The consistency of the proposed hybrid model opens the doors towards the implementation of advanced feedback control schemes.
  •  
21.
  • Cámara, Elena, 1985, et al. (författare)
  • Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
  • 2019
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 3 February:https://doi.org/10.1002/bit.26947
  • Tidskriftsartikel (refereegranskat)abstract
    • The methanol-regulated alcohol oxidase promoter (P AOX1 ) of Pichia pastoris (syn. Komagataella spp.) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures.
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22.
  • Caspeta-Guadarrama, Luis, 1974 (författare)
  • The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: a scale-down study
  • 2009
  • Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 102:2, s. 468-
  • Tidskriftsartikel (refereegranskat)abstract
    • At the laboratory scale, sudden step increases from 30 to 42°C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this work, the transcriptional and metabolic responses of a recombinant Escherichia coli strain to temperature-induced synthesis of pre-proinsulin in high cell density cultures were examined at different heating rates. Heating rates of 6, 1.7, 0.8, and 0.4°C/min were tested in a scale-down approach to mimic fermentors of 0.1, 5, 20, and 100 m3, respectively. The highest yield and concentration of recombinant protein was obtained for the slowest heating rate. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Compared to 30°C, the mRNA levels of selected heat-shock genes at 38 and 42°C, as quantified by qRT-PCR, increased between 2- to over 42-fold when cultures were induced at 6, 1.7, and 0.8°C/min, but no increase was observed at 0.4°C/min. Only small increases (between 1.5- and 4-fold) in the expression of the stress genes spoT and relA were observed at 42°C for cultures induced at 1.7 and 6°C/min, suggesting that cells subjected to slow temperature increases can adapt to stress. mRNA levels of genes from the transcription–translation machinery (tufB, rpoA, and tig) decreased between 40% and 80% at 6, 1.7 and 0.8°C/min, whereas a transient increase occurred for 0.4°C/min at 42°C. mRNA levels of the gene coding for pre-proinsulin showed a similar profile to transcripts of heat-shock genes, reflecting a probable analogous induction mechanism. Altogether, the results obtained indicate that slow heating rates, such as those likely to occur in conventional large-scale fermentors, favored heterologous protein synthesis by the thermo-inducible expression system used in this report. Knowledge of the effect of heating rate on bacterial physiology and product formation is useful for the rational design of scale-down and scale-up strategies and optimum recombinant protein induction schemes. Biotechnol. Bioeng. 2009;102: 468–482. © 2008 Wiley Periodicals, Inc.
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23.
  • Caspeta-Guadarrama, Luis, 1974, et al. (författare)
  • The yeastGemMap: A process diagram to assist yeast systems-metabolic studies
  • 2021
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:12, s. 4800-4814
  • Tidskriftsartikel (refereegranskat)abstract
    • Visualization is a key aspect of the analysis of omics data. Although many tools can generate pathway maps for visualization of yeast metabolism, they fail in reconstructing genome-scale metabolic diagrams of compartmentalized metabolism. Here we report on the yeastGemMap, a process diagram of whole yeast metabolism created to assist data analysis in systems-metabolic studies. The map was manually reconstructed with reactions from a compartmentalized genome-scale metabolic model, based on biochemical process diagrams typically found in educational and specialized literature. The yeastGemMap consists of 3815 reactions representing 1150 genes, 2742 metabolites, and 14 compartments. Computational functions for adapting the graphical representation of the map are also reported. These functions modify the visual representation of the map to assist in three systems-metabolic tasks: illustrating reaction networks, interpreting metabolic flux data, and visualizing omics data. The versatility of the yeastGemMap and algorithms to assist visualization of systems-metabolic data was demonstrated in various tasks, including for single lethal reaction evaluation, flux balance analysis, and transcriptomic data analysis. For instance, visual interpretation of metabolic transcriptomes of thermally evolved and parental yeast strains allowed to demonstrate that evolved strains activate a preadaptation response at 30 degrees C, which enabled thermotolerance. A quick interpretation of systems-metabolic data is promoted with yeastGemMap visualizations.
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24.
  • Cassimjee, Karim Engelmark, et al. (författare)
  • Silica-immobilized His(6)-tagged enzyme : Alanine racemase in hydrophobic solvent
  • 2008
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 99:3, s. 712-716
  • Tidskriftsartikel (refereegranskat)abstract
    • A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions. The immobilized enzyme showed unaltered kinetic parameters in small-scale stirred reactions and retained its activity after rinsing, drying, freezing or immersion in n-hexane. This practical method is a first step towards a general immobilization method for synthesis applications with any enzyme suitable for His(6)-tagging.
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25.
  • Castan, A., et al. (författare)
  • Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli
  • 2002
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 77:3, s. 324-328
  • Tidskriftsartikel (refereegranskat)abstract
    • Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.
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26.
  • Cavka, Adnan, et al. (författare)
  • Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors
  • 2011
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 108:11, s. 2592-2599
  • Tidskriftsartikel (refereegranskat)abstract
    • Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).
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27.
  • Chen, Yu, 1990, et al. (författare)
  • Genome-scale modeling for Bacillus coagulans to understand the metabolic characteristics
  • 2020
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:11, s. 3545-3558
  • Tidskriftsartikel (refereegranskat)abstract
    • Lactic acid is widely used in many industries, especially in the production of poly-lactic acid. Bacillus coagulans is a promising lactic acid producer in industrial fermentation due to its thermophilic property. In this study, we developed the first genome-scale metabolic model (GEM) of B. coagulans iBag597, together with an enzyme-constrained model ec-iBag597. We measured strain-specific biomass composition and integrated the data into a biomass equation. Then, we validated iBag597 against experimental data generated in this study, including amino acid requirements and carbon source utilization, showing that simulations were generally consistent with the experimental results. Subsequently, we carried out chemostats to investigate the effects of specific growth rate and culture pH on metabolism of B. coagulans. Meanwhile, we used iBag597 to estimate the intracellular metabolic fluxes for those conditions. The results showed that B. coagulans was capable of generating ATP via multiple pathways, and switched among them in response to various conditions. With ec-iBag597, we estimated the protein cost and protein efficiency for each ATP-producing pathway to investigate the switches. Our models pave the way for systems biology of B. coagulans, and our findings suggest that maintaining a proper growth rate and selecting an optimal pH are beneficial for lactate fermentation.
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28.
  • Clark, Douglas S., et al. (författare)
  • Klaus Mosbach Tribute
  • 2015
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 112:4, s. 645-647
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
  •  
29.
  • Collén, Anna, et al. (författare)
  • Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.
  • 2002
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 385-394
  • Tidskriftsartikel (refereegranskat)abstract
    • Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.
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30.
  • Collén, Anna, et al. (författare)
  • Protein production and induction of the unfolded protein response in Trichoderma reesei strain rut-c30 and its transformant expressing endoglucanase I with a hydrophobic tag
  • 2005
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 89:3, s. 335-344
  • Tidskriftsartikel (refereegranskat)abstract
    • The effect of induction of protein production was studied in bioreactor cultures of T. reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag. The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production. Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations. Induction of the cellulase genes followed a similar temporal pattern in both strains, The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed. Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter. The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions. However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain.
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31.
  • Corominas, Lluis, et al. (författare)
  • Comparison of different modeling approaches to better evaluate greenhouse gas emissions from whole wastewater treatment plants
  • 2012
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 109:11, s. 2854-2863
  • Tidskriftsartikel (refereegranskat)abstract
    • New tools are being developed to estimate greenhouse gas (GHG) emissions from wastewater treatment plants (WWTPs). There is a trend to move from empirical factors to simple comprehensive and more complex process-based models. Thus, the main objective of this study is to demonstrate the importance of using process-based dynamic models to better evaluate GHG emissions. This is tackled by defining a virtual case study based on the whole plant Benchmark Simulation Model Platform No. 2 (BSM2) and estimating GHG emissions using two approaches: (1) a combination of simple comprehensive models based on empirical assumptions and (2) a more sophisticated approach, which describes the mechanistic production of nitrous oxide (N2O) in the biological reactor (ASMN) and the generation of carbon dioxide (CO2) and methane (CH4) from the Anaerobic Digestion Model 1 (ADM1). Models already presented in literature are used, but modifications compared to the previously published ASMN model have been made. Also model interfaces between the ASMN and the ADM1 models have been developed. The results show that the use of the different approaches leads to significant differences in the N2O emissions (a factor of 3) but not in the CH4 emissions (about 4%). Estimations of GHG emissions are also compared for steady-state and dynamic simulations. Averaged values for GHG emissions obtained with steady-state and dynamic simulations are rather similar. However, when looking at the dynamics of N2O emissions, large variability (36?ton?CO2e?day-1) is observed due to changes in the influent wastewater C/N ratio and temperature which would not be captured by a steady-state analysis (4.4?ton?CO2e?day-1). Finally, this study also shows the effect of changing the anaerobic digestion volume on the total GHG emissions. Decreasing the anaerobic digester volume resulted in a slight reduction in CH4 emissions (about 5%), but significantly decreased N2O emissions in the water line (by 14%). Biotechnol. Bioeng. 2012; 109: 28542863. (c) 2012 Wiley Periodicals, Inc.
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32.
  • Corominas, L., et al. (författare)
  • Performance evaluation of fault detection methods for wastewater treatment processes
  • 2011
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 108:2, s. 333-344
  • Tidskriftsartikel (refereegranskat)abstract
    • Several methods to detect faults have been developed in various fields, mainly in chemical and process engineering. However, minimal practical guidelines exist for their selection and application. This work presents an index that allows for evaluating monitoring and diagnosis performance of fault detection methods, which takes into account several characteristics, such as false alarms, false acceptance, and undesirable switching from correct detection to non-detection during a fault event. The usefulness of the index to process engineering is demonstrated first by application to a simple example. Then, it is used to compare five univariate fault detection methods (Shewhart, EWMA, and residuals of EWMA) applied to the simulated results of the Benchmark Simulation Model No. 1 long-term (BSM1_LT). The BSM1_LT, provided by the IWA Task Group on Benchmarking of Control Strategies, is a simulation platform that allows for creating sensor and actuator faults and process disturbances in a wastewater treatment plant. The results from the method comparison using BSM1_LT show better performance to detect a sensor measurement shift for adaptive methods (residuals of EWMA) and when monitoring the actuator signals in a control loop (e.g., airflow). Overall, the proposed index is able to screen fault detection methods. Biotechnol. Bioeng. 2011;108: 333–344. © 2010 Wiley Periodicals, Inc.
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33.
  • Cruys-Bagger, Nicolaj, et al. (författare)
  • An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose
  • 2012
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 109:12, s. 3199-3204
  • Tidskriftsartikel (refereegranskat)abstract
    • An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 mu AmM-1cm-2), low detection limit (25nM), and fast response time (t95%similar to 3s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the beta-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 31993204. (C) 2012 Wiley Periodicals, Inc.
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34.
  •  
35.
  •  
36.
  • de Jongh, Willem A., et al. (författare)
  • The Roles of Galactitol, Galactose-1-Phosphate, and Phosphoglucomutase in Galactose-Induced Toxicity in Saccharomyces cerevisiae
  • 2008
  • Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 101:2, s. 317-326
  • Tidskriftsartikel (refereegranskat)abstract
    • The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combiantions of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furtermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.
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37.
  • Diano, A., et al. (författare)
  • Physiology of Aspergillus niger in Oxygen-Limited Continuous Cultures: Influence of Aeration, Carbon Source Concentration and Dilution Rate
  • 2009
  • Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 103:5, s. 956-965
  • Tidskriftsartikel (refereegranskat)abstract
    • In industrial production of enzymes using the filamentous fungus Aspergilhis niger supply of sufficient oxygen is often a limitation, resulting in the formation of by-products such as polyols. In order to identify the mechanisms behind formation of the different by-products we studied the effect of low oxygen availability, at different carbon source concentrations and at different specific growth rates, on the metabolism of A. niger, using continuous cultures. The results show that there is an increase in the production of tricarboxylic acid (TCA) cycle intermediates at low oxygen concentrations. Indeed, at these conditions, a decrease in the mitochondrial respiratory chain activity leads to an accumulation of NADH and to a decreased ATP production which uncouples catabolism and anabolism, influences the intracellular pH and leads to production and excretion of organic acids. Moreover, mannitol is being produced in order to ensure reoxidation of NADH, and this is the main cellular response to balance the ratio NADH/NAD at low oxygen availability. Mannitol production is also coupled to low specific growth rate, which suggests a control of carbon catabolite repression on the mannitol pathway. The roles of two other polyols, erythritol and glycerol, were also investigated. Both compounds are known to accumulate intracellularly, at high osmotic pressure, in order to restore the osmotic balance, but we show that the efficiency of this system is affected by a leakage of polyols through the membrane. Biotechnol. Bioeng. 2009;103: 956-965. (C) 2009 Wiley Periodicals, Inc.
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38.
  • Dopson, Mark, et al. (författare)
  • Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms.
  • 2007
  • Ingår i: Biotechnol Bioeng. - : Wiley. - 0006-3592 .- 1097-0290. ; 97:5, s. 1205-15
  • Tidskriftsartikel (refereegranskat)abstract
    • An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C. (c) 2006 Wiley Periodicals, Inc.
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39.
  • Dopson, Mark, et al. (författare)
  • Silicate mineral dissolution during heap bioleaching
  • 2008
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 99:4, s. 811-820
  • Tidskriftsartikel (refereegranskat)abstract
    • Silicate minerals are present in association with metal sulfides in ores and their dissolution occurs when the sulfide minerals are bioleached in heaps for metal recovery. It has previously been suggested that silicate mineral dissolution can affect mineral bioleaching by acid consumption, release of trace elements, and increasing the viscosity of the teach solution. In this study, the effect of silicates present in three separate samples in conjunction with chalcopyrite and a complex multi-metal sulfide ore on heap bioleaching was evaluated in column bioreactors. Fe2+ oxidation was inhibited in columns containing chalcopyrite samples A and C that leached 1.79 and 1.11 mM fluoride, respectively but not in sample B that contained 0.14 mM fluoride. Microbial Fe2+ oxidation inhibition experiments containing elevated fluoride concentrations and measurements of fluoride release from the chalcopyrite ores supported that inhibition of Fe2+ oxidation during column leaching of two of the chalcopyrite ores was due to fluoride toxicity. Column bioleaching of the complex sulfide ore was carried out at various temperatures (7-50 degrees C) and pH values (1.5-3.0). Column leaching at pH 1.5 and 2.0 resulted in increased acid consumption rates and silicate dissolution such that it became difficult to filter the leach solutions and for the leach liquor to percolate through the column. However, column temperature (at pH 2.5) only had a minor effect on the acid consumption and silicate dissolution rates. This study demonstrates the potential negative impact of silicate mineral dissolution on heap bioleaching by microbial inhibition and liquid flow.
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40.
  • Du, Liping, et al. (författare)
  • Multigene expression in vivo : Supremacy of large versus small terminators for T7 RNA polymerase
  • 2012
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 109:4, s. 1043-1050
  • Tidskriftsartikel (refereegranskat)abstract
    • Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TF large (class I) terminator with adjacent copies of the vesicular stomatitis virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within Escherichia coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e., more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TF terminators for BioBrick synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.
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41.
  • Feldman, Hannah, et al. (författare)
  • Model-based analysis and optimization of a full-scale industrial high-rate anaerobic bioreactor
  • 2018
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; , s. 2726-2739
  • Tidskriftsartikel (refereegranskat)abstract
    • The objective of this paper is to present the model-based optimization results of an anaerobic granular sludge internal circulation reactor. The International Water Association Anaerobic Digestion Model No. 1 extended with phosphorus (P), sulfur (S), and ethanol is used to describe the main biological and physico-chemical processes. The high-rate conditions within the reactor are simulated using a flow + reactor model comprised of a series of continuous stirred tank reactors followed by an ideal total suspended solids separation unit. Following parameter estimation by least squares on the measured data, the model had a relative mean error of 13 and 15% for data set #1 and data set #2, respectively. Response surfaces show that the reactor performance index (a metric combining energy recovery in the form of heat and electricity, as well as chemicals needed for pH control) could be improved by 45% when reactor pH is reduced down to 6.8. Model-based results reveal that influent S does not impose sufficient negative impacts on energy recovery (+5.7%, in MWh/day,+0.20 M€/year when influent S is removed) to warrant the cost of its removal (3.58 M€/year). In fact, the process could handle even higher S loads (ensuring the same degree of conversion) as long as the pH is maintained above 6.8. Nevertheless, a higher S load substantially increases the amount of added NaOH to maintain the desired operational pH (>25%) due to the acidic behavior of HS −. CO 2 stripping decreases the buffer capacity of the system and hence use of chemicals for pH control. Finally, the paper discusses the possibilities and limitations of the proposed approach, and how the results of this study will be put into practice.
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42.
  • Fernandes, Rita Lencastre, et al. (författare)
  • Cell mass and cell cycle dynamics of an asynchronous budding yeast population: Experimental observations, flow cytometry data analysis, and multi-scale modeling
  • 2013
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 110:3, s. 812-826
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development in experimental single-cell studies has taken place in the last decades. It has however not been fully accompanied by similar contributions within data analysis and mathematical modeling. Indeed, literature reporting, for example, quantitative analyses of experimental single-cell observations and validation of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model is a suitable tool for describing the microbial population dynamics in a bioreactor. This study therefore contributes towards the understanding of the development of heterogeneous populations during microbial cultivations. More generally, it consists of a step towards a paradigm change in the study and description of cell cultivations, where average cell behaviors observed experimentally now are interpreted as a potential joint result of various co-existing single-cell behaviors, rather than a unique response common to all cells in the cultivation. Biotechnol. Bioeng. 2013; 110: 812826. (c) 2012 Wiley Periodicals, Inc.
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43.
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44.
  • Fernandes, Sheryl, et al. (författare)
  • Selective recovery of lactate dehydrogenase using affinity foam.
  • 2002
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 79:4, s. 472-480
  • Tidskriftsartikel (refereegranskat)abstract
    • Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.
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45.
  • Ferreira, Sofia, et al. (författare)
  • Metabolic engineering strategies for butanol production in Escherichia coli
  • 2020
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:8, s. 2571-2587
  • Forskningsöversikt (refereegranskat)abstract
    • The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.
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46.
  • Fletcher, Eugene, 1986, et al. (författare)
  • Industrial systems biology and its impact on synthetic biology of yeast cell factories
  • 2016
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:6, s. 1164-1170
  • Forskningsöversikt (refereegranskat)abstract
    • Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal of developing improved yeast cell factories. (C) 2015 Wiley Periodicals, Inc.
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47.
  • Gahan, Chandra Sekhar, et al. (författare)
  • Effect of chloride on ferrous iron oxidation by a leptospirillum ferriphilum-dominated chemostat culture
  • 2010
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 106:3, s. 422-431
  • Tidskriftsartikel (refereegranskat)abstract
    • Biomining is the use of microorganisms to catalyze metal extraction from sulfide ores. However, the available water in some biomining environments has high chloride concentrations and therefore, chloride toxicity to ferrous oxidizing microorganisms has been investigated. Batch biooxidation of Fe2+ by a Leptospirillum ferriphilum dominated culture was completely inhibited by 12gL(-1) chloride. In addition, the effects of chloride on oxidation kinetics in a Fe2+ limited chemostat were studied. Results from the chemostat modeling suggest that the chloride toxicity was attributed to affects on the Fe2+ oxidation system, pH homeostasis, and lowering of the proton motive force. Modeling showed a decrease in the maximum specific growth rate (mu(max)) and an increase in the substrate constant (K-s) with increasing chloride concentrations, indicating an effect on the Fe2+ oxidation system. The model proposes a lowered maintenance activity when the media was fed with 2-3 g L-1 chloride with a concomitant drastic decrease in the true yield (Y-true). This model helps to understand the influence of chloride on Fe2+ biooxidation kinetics. Biotechnol. Bioeng. 2010;106: 422-431. (C) 2010 Wiley Periodicals, Inc.
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48.
  • Gárdonyi, Márk, et al. (författare)
  • Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae
  • 2003
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 7:82, s. 818-824
  • Tidskriftsartikel (refereegranskat)abstract
    • Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization. We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C, for the xylose transport was calculated from the response and elasticity coefficients. The value of C for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L-1. However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L-1, while C stayed below 0.2 for strain TMB3001 irrespective of xylose concentration. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 818-824, 2003.
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49.
  • Glahder, J, et al. (författare)
  • Transfection of HeLa-cells with pEGFP plasmid power-assisted by impedance electroporation
  • 2005
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 92:3, s. 267-276
  • Tidskriftsartikel (refereegranskat)abstract
    • Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 mu g DNA in 90 mu l low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value R-infinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance R-M decay exponentially (r(2) = 0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, R-M,(O), and the value R-M,R-N after each pulse makes an index of the effect of electroporation on the cells. The ratio R-M,R-N/R-M,(O) as well as the relative change of the dissipation factor, tan delta, on the "Loss Change Index" (LCI) fits well a dose-response model (r(2) = 0.98) with the number of applied pulses. The changes in the model parameters membrane resistance Delta R-M = [1- R-M,(N)/R-M,o] and loss factor [1- tan delta(O)/tan Omega(N)] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.
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50.
  • Grabherr, R., et al. (författare)
  • Stabilizing plasmid copy number to improve recombinant protein production
  • 2002
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 77:2, s. 142-147
  • Tidskriftsartikel (refereegranskat)abstract
    • The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization. © 2002 John Wiley & Sons, Inc.
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