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1.	Bjornsson, L., et al. (author) Utilization of a palladium-metal oxide semiconductor (Pd-MOS) sensor for on-line monitoring of dissolved hydrogen in anaerobic digestion 2001 In: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 73:1, s. 35-43 Journal article (peer-reviewed)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.
2.	Boer, H., et al. (author) Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters 2000 In: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 69:5, s. 486-494 Journal article (peer-reviewed)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.
3.	Taherzadeh, Mohammad J, 1965-, et al. (author) On-line control of fed-batch fermentation of dilute-acid hydrolyzates 2000 In: Biotechnology and Bioengineering. - New York, NY, United States : John Wiley & Sons Inc. - 0006-3592 .- 1097-0290. ; 69:3, s. 330-338 Journal article (peer-reviewed)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%).
4.	Bylund, F., et al. (author) Influence of scale-up on the quality of recombinant human growth hormone 2000 In: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 69:2, s. 119-128 Journal article (peer-reviewed)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.
5.	Adlercreutz, Dietlind, et al. (author) Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction. 2002 In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 403-411 Journal article (peer-reviewed)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.
6.	Adlercreutz, Patrick (author) 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 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 28:2, s. 223-232 Journal article (peer-reviewed)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.
7.	Andersson, Jonatan, et al. (author) Isolation of potato proteins using simulated moving bed technology. 2008 In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 101, s. 1256-1263 Journal article (peer-reviewed)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.
8.	Andersson, Mats, et al. (author) Toward an enzyme-based oxygen scavenging laminate. Influence of industrial lamination conditions on the performance of glucose oxidase 2002 In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 79:1, s. 37-42 Journal article (peer-reviewed)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).
9.	Arnling Bååth, Jenny, 1987, et al. (author) Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica 2022 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 119:2, s. 470-481 Journal article (peer-reviewed)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.
10.	Asadollahi, M. A., et al. (author) Enhancement of Farnesyl Diphosphate Pool as Direct Precursor of Sesquiterpenes Through Metabolic Engineering of the Mevalonate Pathway in Saccharomyces cerevisiae 2010 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 106:1, s. 86-96 Journal article (peer-reviewed)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.
11.	Batstone, Damien, et al. (author) Kinetics of thermophilic, anaerobic oxidation of straight and branched chain butyrate and valerate 2003 In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:2, s. 195-204 Journal article (peer-reviewed)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.
12.	Bengtsson, Simon (author) The utilization of glycogen accumulating organisms for mixed culture production of polyhydroxyalkanoates. 2009 In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 104, s. 698-708 Journal article (peer-reviewed)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.
13.	Bergenholm, David, 1987, et al. (author) Construction of mini-chemostats for high-throughput strain characterization 2019 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 116:5, s. 1029-1038 Journal article (peer-reviewed)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.
14.	Bergenholm, David, 1987, et al. (author) Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids 2018 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:4, s. 932-942 Journal article (peer-reviewed)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.
15.	Bodin, Aase Katarina, 1977, et al. (author) Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes 2007 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 97:2, s. 425-434 Journal article (peer-reviewed)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.
16.	Bramble, J L, et al. (author) Calcium and Phosphate Effects on Growth and Alkaloid Production in Coffea arabica: Experimental Results and Mathematical Model. 1991 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 37:9, s. 859-868 Journal article (peer-reviewed)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.
17.	Brandberg, T., et al. (author) Continuous fermentation of wheat-supplemented lignocellulose hydrolysate with different types of cell retention 2007 In: Biotechnology and Bioengineering. - : John Wiley & Sons, Inc.. - 0006-3592 .- 1097-0290. ; 98:1, s. 80- Journal article (peer-reviewed)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.
18.	Brechmann, Nils A., et al. (author) Antibody capture process based on magnetic beads from very high cell density suspension 2021 In: Biotechnology and Bioengineering. - : John Wiley and Sons Inc. - 0006-3592 .- 1097-0290. ; 118:9, s. 3499-3510 Journal article (peer-reviewed)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.
19.	Buijs, Nicolaas, 1985, et al. (author) Long-chain Alkane Production by the Yeast Saccharomyces cerevisiae 2015 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 112:6, s. 1275-1279 Journal article (peer-reviewed)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.
20.	Cabaneros Lopez, Pau, et al. (author) Transforming data to information : A parallel hybrid model for real-time state estimation in lignocellulosic ethanol fermentation 2021 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:2, s. 579-591 Journal article (peer-reviewed)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. (author) Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter 2019 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 3 February:https://doi.org/10.1002/bit.26947 Journal article (peer-reviewed)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.
22.	Caspeta-Guadarrama, Luis, 1974 (author) 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 In: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 102:2, s. 468- Journal article (peer-reviewed)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.
23.	Caspeta-Guadarrama, Luis, 1974, et al. (author) The yeastGemMap: A process diagram to assist yeast systems-metabolic studies 2021 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:12, s. 4800-4814 Journal article (peer-reviewed)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.
24.	Cassimjee, Karim Engelmark, et al. (author) Silica-immobilized His(6)-tagged enzyme : Alanine racemase in hydrophobic solvent 2008 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 99:3, s. 712-716 Journal article (peer-reviewed)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.
25.	Castan, A., et al. (author) Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli 2002 In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 77:3, s. 324-328 Journal article (peer-reviewed)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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