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1.
  • Anasontzis, George E, 1980, et al. (författare)
  • Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris
  • 2014
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 30:3, s. 728-735
  • Tidskriftsartikel (refereegranskat)abstract
    • Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. (c) 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728-735, 2014
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2.
  • Andersson, Christian, et al. (författare)
  • Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli
  • 2007
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 23:2, s. 381-388
  • Tidskriftsartikel (refereegranskat)abstract
    • Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.
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3.
  • Andersson, Christian, et al. (författare)
  • Inhibition of succinic acid production in metabolically engineered Escherichia Coli by neutralizing agent, organic acids, and osmolarity
  • 2009
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 25:1, s. 116-123
  • Tidskriftsartikel (refereegranskat)abstract
    • The economical viability of biochemical succinic acid production is a result of many processing parameters including final succinic acid concentration, recovery of succinate, and the volumetric productivity. Maintaining volumetric productivities >2.5 g L-1 h(-1) is important if production of succinic acid from. renewable resources should be competitive. In this work, the effects of organic acids, osmolarity, and neutralizing agent (NH4OH, KOH, NaOH, K2CO3, and Na2CO3) on the fermentative succinic acid production by Escherichia coli AFP184 were investigated. The highest concentration of succinic acid, 77 g L-1. was obtained with Na2O3. In general, irrespective of the base used, succinic acid productivity per viable cell was significantly reduced as the concentration of the produced acid increased. Increased osmolarity resulting from base addition during succinate production only marginally affected the productivity per viable cell. Addition of the osmoprotectant glycine betaine to cultures resulted in an increased aerobic growth rate and anaerobic glucose consumption rate, but decreased succinic acid yield. When using NH4OH productivity completely ceased at a succinic acid concentration of similar to 40 g L-1. Volumetric productivities remained at 2.5 g L-1 h(-1) for tip to 10 h longer when K- or Na-bases where used instead of NH4OH. The decrease in cellular succinic acid productivity observed during the anaerobic phase was found to be due to increased organic acid concentrations rather than medium osmolarity.
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4.
  • Andersson, Niklas, et al. (författare)
  • Design and control of integrated chromatography column sequences
  • 2017
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 00:00
  • Tidskriftsartikel (refereegranskat)abstract
    • To increase the productivity in biopharmaceutical production, a natural step is to introduce integrated continuous biomanufacturing which leads to fewer buffer and storage tanks, smaller sizes of integrated unit operations, and full automation of the operation. The main contribution of this work is to illustrate a methodology for design and control of a downstream process based on integrated column sequences. For small scale production, for example, pre-clinical studies, integrated column sequences can be implemented on a single chromatography system. This makes for a very efficient drug development platform. The proposed methodology is composed of four steps and is governed by a set of tools, that is presented, that makes the transition from batch separations to a complete integrated separation sequence as easy as possible. This methodology, its associated tools and the physical implementation is presented and illustrated on a case study where the target protein is separated from impurities through an integrated four column sequence. This article shows that the design and control of an integrated column sequence was successfully implemented for a tertiary protein separation problem.
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5.
  • Bijmans, MFM, et al. (författare)
  • Sulfate reduction at pH 4.0 for treatment of process and wastewaters
  • 2010
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 26:4, s. 1029-1037
  • Tidskriftsartikel (refereegranskat)abstract
    • Acidic industrial process and wastewaters often contain high sulfate and metal concentrations and their direct biological treatment is thus far not possible as biological processes at pH < 5 have been neglected. Sulfate-reducing bacteria convert sulfate to sulfide that can subsequently be used to recover metals as metal-sulfides precipitate. This study reports on high-rate sulfate reduction with a mixed microbial community at pH 4.0 and 4.5 with hydrogen and/or formate as electron donors. The maximum sulfate reducing activity at pH 4.0 was sustained for over 40 days with a specific activity 500-fold greater than previously reported values: 151 mmol sulfate reduced/L reactor liquid per day with a maximum specific activity of 84 mmol sulfate per gram of volatile suspended solids per day. The biomass yield gradually decreased from 38 to 0.4 g volatile suspended solids per kilogram of sulfate when decreasing the reactor pH from pH 6 to 4. The microorganisms had a high maintenance requirement probably due maintaining pH homeostasis and the toxicity of sulfide at low pH. The microbial community diversity in the pH 4.0 membrane bioreactor decreased over time, while the diversity of the sulfate reducing community increased. Thus, a specialized microbial community containing a lower proportion of microorganisms capable of activity at pH 4 developed in the reactor compared with those present at the start of the experiment. The 16S rRNA genes identified from the pH 4.0 grown mixed culture were most similar to those of Desulfovibrio species and Desulfosporosinus sp. M1. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1029-1037, 2010
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6.
  • Bornadel, Amin, et al. (författare)
  • Kinetic modeling of lipase-catalyzed esterification reaction between oleic acid and trimethylolpropane: A simplified model for multi-substrate multi-product ping-pong mechanisms.
  • 2013
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 29:6, s. 1422-1429
  • Tidskriftsartikel (refereegranskat)abstract
    • Kinetic models are among the tools that can be used for optimization of biocatalytic reactions as well as for facilitating process design and upscaling in order to improve productivity and economy of these processes. Mechanism pathways for multi-substrate multi-product enzyme-catalyzed reactions can become very complex and lead to kinetic models comprising several tens of terms. Hence the models comprise too many parameters, which are in general highly correlated and their estimations are often prone to huge errors. In this study, Novozym® 435 catalyzed esterification reaction between oleic acid (OA) and trimethylolpropane (TMP) with continuous removal of side-product (water) was carried out as an example for reactions that follow multi-substrate multi-product ping-pong mechanisms. A kinetic model was developed based on a simplified ping-pong mechanism proposed for the reaction. The model considered both enzymatic and spontaneous reactions involved and also the effect of product removal during the reaction. The kinetic model parameters were estimated using nonlinear curve fitting through unconstrained optimization methodology and the model was verified by using empirical data from different experiments and showed good predictability of the reaction under different conditions. This approach can be applied to similar biocatalytic processes to facilitate their optimization and design.
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7.
  • Bornadel, Amin, et al. (författare)
  • Optimization of a two-step process comprising lipase catalysis and thermal cyclizationimproves the efficiency of synthesis of six-membered cyclic carbonate from trimethylolpropane and dimethylcarbonate.
  • 2013
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 29:1, s. 66-73
  • Tidskriftsartikel (refereegranskat)abstract
    • Six-membered cyclic carbonates are potential monomers for phosgene and/or isocyanate free polycarbonates and polyurethanes via ring-opening polymerization. A two-step process for their synthesis comprising lipase-catalyzed transesterificationofa polyol, trimethylolpropane(TMP) with dimethylcarbonate(DMC)in a solvent-free system followed by thermal cyclization was optimized to improve process efficiency and selectivity. Using full factorial designed experiments and partial least squares (PLS) modeling for thereaction catalyzed by Novozym®435 (N435; immobilized Candida antarctica lipase B), the optimum conditions for obtaining either high proportion of mono-carbonated TMP and TMP-cyclic-carbonate (3 and 4), or di-carbonated TMP and monocarbonated TMP-cyclic-carbonate (5 and 6) were found. The PLS model predicted that the reactions using 15-20% (w/w) N435 at DMC:TMP molar ratio of 10-30 can reach about 65% total yield of 3 and 4 within 10 h, and 65-70% total yield of 5 and 6 within 32-37 h, respectively. High consistency between the predictedresults and empirical data was shown with 66.1% yield of 3 and 4 at 7 h and 67.4% yield of 5 and 6 at 35 h, using 18% (w/w) biocatalyst and DMC:TMPmolar ratio of20. Thermal cyclization of the product from 7 h reaction, at 110 °C in the presence of acetonitrile increased the overall yield of cyclic carbonate 4 from about 2% to more than 75%within 24 h.N435 was reused for 5 consecutive batches, 10 h each, to give 3+4 with a yield of about 65% in each run. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012.
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8.
  • Bramble, J L, et al. (författare)
  • Plant Cell Culture using a Novel Bioreactor: The Magnetically Stabilized Fluidized Bed
  • 1990
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 6:6, s. 452-457
  • Tidskriftsartikel (refereegranskat)abstract
    • A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared to the results obtained in shake flask culture. 
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9.
  • Brechmann, Nils Arnold, et al. (författare)
  • Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture
  • 2019
  • Ingår i: Biotechnology progress (Print). - : AIChE. - 8756-7938 .- 1520-6033.
  • Tidskriftsartikel (refereegranskat)abstract
    • High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the developmentof a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarifiedCHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum bindingcapacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for anIgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture stepfrom 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions wereobtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scalepurification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single proteinA capture step, the mAb purity was similar to the one obtained by column chromatography, whilethe host cell protein content was very low, <10 ppm. Our results showed that this magnetic beadmAb purification process, using a dedicated pilot-scale separation device, was a highly efficientsingle step, which directly connected the culture to the downstream process without cell clarification.Purification of mAb directly from non-clarified cell broth without cell separation can providesignificant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.
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10.
  • Calles, Karin, et al. (författare)
  • Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.
  • 2006
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 22:3, s. 653-659
  • Tidskriftsartikel (refereegranskat)abstract
    • The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.
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11.
  • Calles, Karin, et al. (författare)
  • Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity
  • 2006
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 22:2, s. 394-400
  • Tidskriftsartikel (refereegranskat)abstract
    • The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Low passage (LP) cells at passages 20-45 were compared to high passage (HP) cells at passages > 100. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. LP cells passed a switch in growth kinetics, characterized by a shorter lag phase and a higher growth rate, after 30-40 passages. After this point, CM lost its stimulating effect on proliferation. HP cells displayed a still shorter lag phase and reached the maximum cell density 24-48 earlier than LP cells. HP cells also exhibited higher specific productivity of recombinant protein compared to LP cells, when infected with baculovirus during the initial 48 h of culture. The specific productivity of LP cells was decreased by 30-50% by addition of 20% CM or 10 kDa filtrated CM, whereas addition of CM to cells having passed the switch in growth kinetics had no negative effect on productivity. Cell cycle analysis showed that a large proportion of HP cells, >60%, was transiently arrested in G2/M after inoculation. In LP cultures this proportion was lower, 40-45%, and addition of CM decreased the arrested population further. This correlated to the cell size, the HP cells being the largest: HP cells > LP > LP + 20% CM > LP + 20% 10 kDa filtrated CM. Since the degree of synchronization in G2/M correlated to the productivity, yeastolate limitation was used to achieve 85% G2/M synchronized cells. In this culture the specific productivity was maintained during a prolonged production phase and a 69% higher volumetric yield was obtained. The results suggest that a decreasing degree of synchronization during the course of culture partly explains the cell-density-dependent drop in productivity in Sf9 cells.
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12.
  • Cimander, C., et al. (författare)
  • Assessment of the performance of a fed-batch cultivation from the preculture quality using an electronic nose
  • 2002
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 18:2, s. 380-386
  • Tidskriftsartikel (refereegranskat)abstract
    • An electronic nose, a gas-phase multisensor system, was used to monitor precultivations of a recombinant tryptophan-producing Escherichia coli strain. The electronic nose signals showed a high correlation toward the main stages of the precultivations, namely, exponential growth, oxygen-limited growth, and glucose depletion. Principal component analysis (PCA) of the electronic nose signals was performed and shown to be useful for monitoring preculture progression. More importantly, PCA also allowed a qualitative assessment of the preculture performance during subsequent fed-batch cultivations. The electronic nose signals from the precultures showed, furthermore, a high correlation to the time of phosphate limitation and the tryptophan yield coefficient of the subsequent fed-batch cultivations, which allowed an accurate prediction of these process variables using partial least squares (PLS). The results demonstrate on data from 12 cultivations how the electronic nose can be a useful tool for the assessment of inoculum quality, thereby providing means of reducing batch-to-batch variation and increasing the productivity of bioprocesses.
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13.
  • Clincke, Marie-Francoise, et al. (författare)
  • Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactorpart II : Applications for antibody production and cryopreservation
  • 2013
  • Ingår i: Biotechnology progress (Print). - : Wiley-Blackwell. - 8756-7938 .- 1520-6033. ; 29:3, s. 768-777
  • Tidskriftsartikel (refereegranskat)abstract
    • A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed-batch were compared. Cell densities higher than 108 cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell-specific productivity was comparable at cell densities up to 1.3 x 108 cells/mL in perfusion and was comparable or lower in fed-batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed-batch and 28x more in a 1-month perfusion at 108 cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 x 108 and 108 cells/mL was performed using cells from a perfusion run at 108 cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing.
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14.
  • Clincke, Marie-Francoise, et al. (författare)
  • Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I. Effect of the cell density on the process
  • 2013
  • Ingår i: Biotechnology progress (Print). - : Wiley-Blackwell. - 8756-7938 .- 1520-6033. ; 29:3, s. 754-767
  • Tidskriftsartikel (refereegranskat)abstract
    • High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor using external hollow fiber filter as cell separation device. Both classical tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF- and ATF-based cultures was shown at 20-35 x 106 cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by-product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9-1.3 x 108 cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 x 108 cells/mL, achieved for the first time in a wave-induced bioreactor, and 1.32 x 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 x 108 cell/mL based on the analysis of the theoretical distance between the cells for the present cell line.
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15.
  • Dainiak, Maria, et al. (författare)
  • Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.
  • 2008
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 24:6, s. 1373-1383
  • Tidskriftsartikel (refereegranskat)abstract
    • Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests.
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16.
  • Dainiak, Maria, et al. (författare)
  • Cell chromatography: Separation of different microbial cells using IMAC supermacroporous monolithic columns
  • 2005
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:2, s. 644-649
  • Tidskriftsartikel (refereegranskat)abstract
    • Supermacroporous monolithic columns with CU2+-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10- 100 mu m) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the CU2+-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.
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17.
  • Dainiak, Maria, et al. (författare)
  • Improved methods for prepurification and detection of Staphylococcal Enterotoxin B from cell-free culture filtrate
  • 2005
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:4, s. 1347-1351
  • Tidskriftsartikel (refereegranskat)abstract
    • An improved ELISA method for the detection of Staphylococcal. Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.
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18.
  • Dainiak, Maria, et al. (författare)
  • Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption
  • 2002
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 18:4, s. 815-820
  • Tidskriftsartikel (refereegranskat)abstract
    • Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths. However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads. To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA). The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions. PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent. Thus, the shielding layer of PAA was adsorbed, only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study. PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which other-wise bound,strongly to the native adsorbent at neutral conditions. Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells. The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles.
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19.
  • Derelöv, Micael, 1973-, et al. (författare)
  • Engineering Design Methodology for Bio-Mechatronic Products
  • 2008
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 4:1, s. 232-244
  • Tidskriftsartikel (refereegranskat)abstract
    • Four complex biotechnology products/product systems (a protein purification system, a bioreactor system, a surface plasmon resonance biosensor, and an enzymatic glucose analyzer) are analyzed using conceptual design principles. A design model well-known in mechanical system design, the Hubka-Eder (HE) model, is adapted to biotechnology products that exemplify combined technical systems of mechanical, electronic, and biological components, here referred to as bio-mechatronic systems. The analysis concludes that an extension of the previous HE model with a separate biological systems entity significantly contributes to facilitating the functional and systematic analyses of bio-mechatronic systems.
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20.
  • Digaitis, Ramunas, PhD, et al. (författare)
  • Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis : Part II
  • 2021
  • Ingår i: Biotechnology progress (Print). - : John Wiley & Sons. - 8756-7938 .- 1520-6033. ; 37:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Lignocellulose breakdown in biorefineries is facilitated by enzymes and physical forces. Enzymes degrade and solubilize accessible lignocellulosic polymers, primarily on fiber surfaces, and make fibers physically weaker. Meanwhile physical forces acting during mechanical agitation induce tearing and cause rupture and attrition of the fibers, leading to liquefaction, that is, a less viscous hydrolysate that can be further processed in industrial settings. This study aims at understanding how mechanical agitation during enzymatic saccharification can be used to promote fiber attrition. The effects of reaction conditions, such as substrate and enzyme concentration on fiber attrition rate and hydrolysis yield were investigated. To gain insight into the fiber attrition mechanism, enzymatic hydrolysis was compared to hydrolysis by use of hydrochloric acid. Results show that fiber attrition depends on several factors concerning reactor design and operation including drum diameter, rotational speed, mixing schedule, and concentrations of fibers and enzymes. Surprisingly, different fiber attrition patterns during enzymatic and acid hydrolysis were found for similar mixing schedules. Specifically, for tumbling mixing, slow continuous mixing appears to function better than faster, intermittent mixing even for the same total number of drum revolutions. The findings indicate that reactor design and operation as well as hydrolysis conditions are key to process optimization and that detailed insights are needed to obtain fast liquefaction without sacrificing saccharification yields.
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21.
  • Doverskog, Magnus, et al. (författare)
  • Cell cycle progression in serum-free cultures of Sf9 insect cells : Modulation by conditioned medium factors and implications for proliferation and productivity
  • 2000
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 16:5, s. 837-846
  • Tidskriftsartikel (refereegranskat)abstract
    • Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (mu(N,max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining mu(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase mu(N,max), decrease the time to reach mu(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean;population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.
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22.
  • Falco, Cigdem Yucel, et al. (författare)
  • Hybrid coating of alginate microbeads based on protein-biopolymer multilayers for encapsulation of probiotics
  • 2019
  • Ingår i: Biotechnology progress (Print). - : John Wiley & Sons. - 8756-7938 .- 1520-6033. ; 35:3
  • Tidskriftsartikel (refereegranskat)abstract
    • A hybrid coating based on multilayers of proteins and biopolymers was developed to enhance the protection performance of alginate microbeads against acidic conditions for delivery of probiotics (Lactobacillus rhamnosus GG). Zeta potential measurements and quartz crystal microbalance with dissipation confirmed layer-by-layer deposition of protein-polymer layers. The stability of protein-based coatings during simulated gastric fluid (SGF) treatment was monitored by microscopy. Protein-coated microbeads were partially dismantled, whereas polymer-coated microbeads were intact after a sequential treatment in simulated gastric and intestinal fluids. This suggests that hybrid formulation offers an advantage over the coatings based on biopolymer multilayers in terms of better release of bacteria. Uncoated alginate microbeads completely dissolved and could not protect bacteria after SGF treatment whereas microbeads with hybrid coating showed increased physical stability and a modest decrease of culturability of 3.8 log units. Therefore, this work provides a concept for future protein-based hybrid coatings for bacterial delivery systems.
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23.
  • Fernandes, Sheryl, et al. (författare)
  • Recovery of recombinant cutinase using detergent foam
  • 2002
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 18:1, s. 116-123
  • Tidskriftsartikel (refereegranskat)abstract
    • Foam generated by vigorous stirring of a nonionic detergent, Triton X-114, was used for the recovery of recombinant cutinase expressed by Saccharomyces cerevisiae. The enzyme with a hydrophobic fusion tag, (Trp-Pro)(4), was recovered with a higher yield as compared to the wild-type cutinase, indicating the involvement of hydrophobic interactions in protein isolation with the foam. The influence of various factors including volume, dilution, pH, different additives, and cell concentration in the medium on enzyme recovery was investigated. Interaction of the enzyme with detergent was monitored using fluorescence spectroscopy. No significant changes in protein conformation after the isolation procedure were observed using circular dichroism.
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24.
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25.
  • Fexby, Sara, et al. (författare)
  • Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems
  • 2004
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 20:3, s. 793-798
  • Tidskriftsartikel (refereegranskat)abstract
    • The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase.
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26.
  • Garcia-Aparicio, Maria, et al. (författare)
  • Evaluation of Steam-Treated Giant Bamboo for Production of Fermentable Sugars
  • 2011
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 27:3, s. 641-649
  • Tidskriftsartikel (refereegranskat)abstract
    • Giant bamboo plantations are currently being established in the Southern Africa region and can be considered as potential lignocellulosic feedstock for the production of second generation bioethanol. In this study, giant bamboo internodal material was subjected to sulphur dioxide (SO2) impregnated steam pretreatment prior to enzymatic hydrolysis. The effect of temperature, residence time, and acidity on the overall sugar recovery and byproduct formation was studied using response surface response technology according to a central composite experimental design (CCD) at a fixed SO2 concentration of 2.5% (w/w liquid) after impregnation. The results showed that pretreatment conditions with combined severity factor (CSF) values and enzyme dosages greater than 1.72 and 30 FPU/g water insoluble solid, respectively, were required to obtain an efficient glucan digestibility and a good overall glucose recovery. Up to 81.2% of the sugar in the raw material was recovered for a CSF of 2.25. However, considering overall sugar yield and byproducts concentration, the pretreated material obtained with a CSF of 1.62 can be considered as the most appropriate for SSF experiments using a xylose-utilizing yeast. At these conditions, it could be possible to obtain up to 247 L of ethanol per dry ton of giant bamboo considering hexose and pentose sugars fermentation. This amount could be increased up to 292 L of ethanol per dry ton of giant bamboo with the maximum sugar yield obtained (CSF 2.25) if the microorganism possesses robust fermentative characteristics as well as a high resistance to pretreatment by-products. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 641-649, 2011
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27.
  • Gomis-Fons, Joaquín, et al. (författare)
  • Model-based design and control of a small-scale integrated continuous end-to-end mAb platform
  • 2020
  • Ingår i: Biotechnology progress (Print). - : John Wiley and Sons Inc.. - 8756-7938 .- 1520-6033.
  • Tidskriftsartikel (refereegranskat)abstract
    • A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained. 
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28.
  • Gowtham, Yogender Kumar, et al. (författare)
  • Novel two-stage fermentation process for bioethanol production using Saccharomyces pastorianus
  • 2014
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 30:2, s. 300-310
  • Tidskriftsartikel (refereegranskat)abstract
    • Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose-metabolizing pathways. In this study, a novel two-stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non-Saccharomyces microorganism and coupled to a glucose-utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two-stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae-based fermentations.
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29.
  • Gustafsson, N O, et al. (författare)
  • Measurement of diffusion coefficients in gels using holographic laser interferometry
  • 1993
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 9:4, s. 436-441
  • Tidskriftsartikel (refereegranskat)abstract
    • The accuracy and precision of holographic interferometry as a method to measure diffusion coefficients in gels are investigated both experimentally and theoretically. The standard deviations in the experimentally determined diffusion coefficient for ethanol in 4% (w/v) agarose gel were 3.3% for diffusion into the gel and 6.1% for diffusion out of the gel. These are in good agreement with the standard deviations obtained using Monte Carlo simulations. Systematic errors derived from an assumption of constant diffusion coefficients were also investigated.
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30.
  • Hagström, Anna, et al. (författare)
  • Chemo-enzymatic epoxidation-process options for improving biocatalytic productivity.
  • 2011
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 27:1, s. 67-76
  • Tidskriftsartikel (refereegranskat)abstract
    • The reactor choice is crucial when designing a process where inactivation of the biocatalyst is a problem. The main bottleneck for the chemo-enzymatic epoxidation has been found to be enzyme inactivation by the hydrogen peroxide, H(2)O(2), substrate. In the work reported here, the effect of reaction parameters on the reaction performance have been investigated and used to establish suitable operating strategies to minimize the inactivation of the enzyme, using rapeseed methyl ester (RME) as a substrate in a solvent-free system. The use of a controlled fed-batch reactor for maintaining H(2)O(2) concentration at 1.5 M resulted in increased productivity, up to 76 grams of product per gram of biocatalyst with higher retention of enzyme activity. Further investigation included a multistage design that separated the enzymatic reaction and the saturation of the RME substrate with H(2)O(2) into different vessels. This setup showed that the reaction rate as well as enzyme inactivation is strongly dependent on the H(2)O(2) concentration. A 20-fold improvement in enzymatic efficiency is required for reaching an economically feasible process. This will require a combination of enzyme modification and careful process design. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010.
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31.
  • Ivanov, Alexander, et al. (författare)
  • Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: a step towards thermosensitive industrial biocatalyst.
  • 2003
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 19:4, s. 1167-1175
  • Tidskriftsartikel (refereegranskat)abstract
    • Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 106 and, presumably, cross-linked protein-polymer aggregates of MW > 107. Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 M) was found to be slightly higher than that of the free enzyme (12 M), and evaluation of Vmax testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from D-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of -lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).
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32.
  • Jahic, Mehmedalija, et al. (författare)
  • Process technology for production and recovery of heterologous proteins with Pichia pastoris
  • 2006
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 22:6, s. 1465-1473
  • Forskningsöversikt (refereegranskat)abstract
    • Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.
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33.
  • Johansson, Louise, et al. (författare)
  • A study of long-term effects on plasmid-containing Escherichia coli in carbon-limited chemostat using 2D-fluorescence spectrofluorimetry
  • 2006
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 22:4, s. 1132-1139
  • Tidskriftsartikel (refereegranskat)abstract
    • Strain stability of plasmid-containing recombinant organisms is clearly important for industrial applications. Stability is normally assessed by methods such as selective colony forming units or by simply measuring the recombinant product. These methods are typically performed off-line, are time-consuming, and do not give detailed information on the changes in the metabolism. In the current work, long-term stability of a plasmid-containing strain of Escherichia coli (W3110.shik1) capable of shikimic acid overproduction was studied by means of a 2D-fluorescence sensor (BioView) able to emit and detect light in ranges of 260-560 nm and 300600 nm, respectively. Long-term carbon-limited chemostat experiments were made under both selective (tetracycline-containing medium) and nonselective conditions. It is shown that the fluorescence spectra provide information about metabolic changes at an earlier stage, thereby giving a noninvasive method for monitoring of strain stability. Further, the fluorescence measurements showed that (i) the metabolic changes in the strain W3110.shik1 with time were qualitatively different in selective and nonselective environment, (ii) plasmid recombination resulted primarily in increased biomass yield, and (iii) a change in metabolism probably involving FAD/FMN and pyridoxal-5-P occurred in all experiments. It was concluded that the strain was not stable in any growth condition for more than about 25 growth generations and even less if plasmid recombination took place.
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34.
  • Johnsson, Ola, et al. (författare)
  • Feed rate control in fed-batch fermentations based on frequency content analysis.
  • 2013
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 29:3, s. 817-824
  • Tidskriftsartikel (refereegranskat)abstract
    • A new strategy for controlling substrate feed in the exponential growth phase of aerated fed-batch fermentations is presented. The challenge in this phase is typically to maximize specific growth rate while avoiding the accumulation of overflow metabolites which can occur at high substrate feed rates. In the new strategy, regular perturbations to the feed rate are applied and the proximity to overflow metabolism is continuously assessed from the frequency spectrum of the dissolved oxygen signal. The power spectral density for the frequency of the external perturbations is used as a control variable in a controller to regulate the substrate feed. The strategy was implemented in an industrial pilot scale fermentation set up and calibrated and verified using an amylase producing Bacillus licheniformis strain. It was shown that a higher biomass yield could be obtained without excessive accumulation of harmful overflow metabolites. The general applicability of the strategy was further demonstrated by implementing the controller in another process utilizing a Bacillus licheniformis strain currently used in industrial production processes. Also in this case a higher growth rate and decreased accumulation of overflow metabolites in the exponential growth phase was achieved in comparison to the reference controller. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
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35.
  • Karlsson, David, et al. (författare)
  • Electronic speckle pattern interferometry: A tool for determining diffusion and partition coefficients for proteins in gels
  • 2002
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 18:6, s. 1423-1430
  • Tidskriftsartikel (refereegranskat)abstract
    • The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the,reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 +/- 1.6, 4.8 +/- 0.6, and 3.0 +/- 0.3 m(2)/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 +/- 0.04, 0.44 +/- 0.06, and 0.51 +/- 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.
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36.
  • Keener, RN, et al. (författare)
  • Mechanical deformation of compressible chromatographic columns
  • 2002
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 18:3, s. 587-596
  • Tidskriftsartikel (refereegranskat)abstract
    • A one-dimensional model of mechanical deformation of compressible chromatography columns is presented. The model is based on linear elasticity and continuum mechanics and is compared to a more complete two-dimensional model and one-dimensional porosity profiles measured by NMR imaging methods. The model provides a quantitative description of compression and the effects of wall support during scale-up. A simple criterion for the significance of wall support as a function of both diameter and length is also developed. Although the model accounts only for mechanical deformation, flow compression can be included, and validation presented here suggests that a more complete model may be valuable for anticipating the effects of scale and aspect ratio on pressure-flow behavior of compressible columns.
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37.
  • Komaraiah, Palle, et al. (författare)
  • Growth behavior in plant cell cultures based on emissions detected by a multisensor array
  • 2004
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 20:4, s. 1245-1250
  • Tidskriftsartikel (refereegranskat)abstract
    • The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.
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38.
  • Lequeux, Gaspard, et al. (författare)
  • MFA for overdetermined systems reviewed and compared with RNA expression data to elucidate the difference in shikimate yield between carbon- and phosphate-limited continuous cultures of E. coli W3110.shik1
  • 2006
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 22:4, s. 1056-1070
  • Tidskriftsartikel (refereegranskat)abstract
    • The present contribution focuses on the mathematical techniques used to solve steady state metabolic models for the case of an overdetermined system. Even when parts of the system are underdetermined it is possible to solve the model partially and obtain statistically meaningful results. This is illustrated with data gathered from a set of E. coli W3110.shik1 phosphate- or carbon-limited continuous cultures. It is shown that the low yield in shikimate for C-limited cultures is not due to a lower flux going to the shikimate pathway but is caused by a high secretion of byproducts. Carbon-limited cultures could be better for shikimate production than carbon-abundant cultures provided the byproduct secretion is reduced. Finally, flux calculations are compared with RNA expression data.
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39.
  • Lindberg, Jenny, et al. (författare)
  • Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein
  • 2002
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 18:6, s. 1176-1182
  • Tidskriftsartikel (refereegranskat)abstract
    • In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.
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40.
  • Ljunggren, Mattias, et al. (författare)
  • Techno-economic evaluation of a two-step biological process for hydrogen production.
  • 2010
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 26, s. 496-504
  • Tidskriftsartikel (refereegranskat)abstract
    • An integrated biological process for the production of hydrogen based on thermophilic and photo-heterotrophic fermentation was evaluated from a technical and economic standpoint. Besides the two fermentation steps the process also includes pretreatment of the raw material (potato steam peels) and purification of hydrogen using amine absorption. The study aimed neither at determining the absolute cost of biohydrogen nor at an economic optimization of the production process, but rather at studying the effects of different parameters on the production costs of biohydrogen as a guideline for future improvements. The effect of the key parameters, hydrogen productivity and yield and substrate concentration in the two fermentations on the cost of the hydrogen produced was studied. The selection of the process conditions was based mainly on laboratory data. The process was simulated by use of the software Aspen Plus and the capital costs were estimated using the program Aspen Icarus Process Evaluator. The study shows that the photo-fermentation is the main contributor to the hydrogen production cost mainly because of the cost of plastic tubing, for the photo-fermentors, which represents 40.5% of the hydrogen production cost. The costs of the capital investment and chemicals were also notable contributors to the hydrogen production cost. Major economic improvements could be achieved by increasing the productivity of the two fermentation steps on a medium-term to long-term scale. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010.
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41.
  • Löfgren, Anton, et al. (författare)
  • Optimization of integrated chromatography sequences for purification of biopharmaceuticals
  • 2019
  • Ingår i: Biotechnology Progress. - : Wiley. - 8756-7938 .- 1520-6033. ; 35:6
  • Tidskriftsartikel (refereegranskat)abstract
    • With continued development of integrated and continuous downstream purification processes, tuning and optimization become increasingly complicated with additional parameters and codependent variables over the sequence. This article offers a novel perspective of nonlinear optimization of integrated sequences with regard to individual column sizes, flow rates, and scheduling. The problem setup itself is a versatile tool to be used in downstream design which is demonstrated in two case studies: a four-column integrated sequence and a continuously loaded twin-capture setup with five columns.
  •  
42.
  • Malisauskas, Mantas, et al. (författare)
  • Ultrathin silver nanowires produced by amyloid biotemplating
  • 2008
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 24:5, s. 1166-1170
  • Tidskriftsartikel (refereegranskat)abstract
    • By using a self-assembled amyloid from lysozyme as biotemplate we produced an ultrathin silver wire of 1 nm diameter and up to 2 μm in length, which is at the limit attainable in nanobiotechnological manufacturing. We showed that 2,2,2-trifluoroethanol produces a dual effect: it reduces ionic silver to colloidal nanoparticles with a regular size, depending on the length of incubation, and induces fibrillar assembly into the amyloid scaffold, forming the hollow channel filled with silver.
  •  
43.
  • Mandenius, Carl-Fredrik, et al. (författare)
  • Bioprocess Optimization Using Design-of-Experiments Methodology
  • 2008
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 24:6, s. 1191-1203
  • Forskningsöversikt (övrigt vetenskapligt/konstnärligt)abstract
    • This review surveys recent applications of design-of-experiments (DoE) methodology in the development of biotechnological processes. Methods such as factorial design, response surface methodology, and (DoE) provide powerful and efficient ways to optimize cultivations and other unit operations and procedures using a reduced number of experiments. The multitude of interdependent parameters involved within a unit operation or between units in a bioprocess sequence may be substantially refined and improved by the use of such methods. Other bioprocess-related applications include strain screening evaluation and cultivation media balancing. In view, of the emerging regulatory demands on pharmaceutical manufacturing processes, exemplified by the process analytical technology (PAT) initiative of the United States Food and Drug Administration, the use of experimental design approaches to improve process development for safer and more reproducible production is becoming increasingly important. Here, these options are highlighted and discussed with a few selected examples from antibiotic fermentation, expanded bed optimization, virus vector transfection of insect cell cultivation, feed profile adaptation, embryonic stem cell expansion protocols, and mammalian cell harvesting.
  •  
44.
  • Mandenius, C. F., et al. (författare)
  • Predicting fermentability of wood hydrolyzates with responses from electronic noses
  • 1999
  • Ingår i: Biotechnology progress (Print). - New York, NY, United States : AIChE. - 8756-7938 .- 1520-6033. ; 15:4, s. 617-621
  • Tidskriftsartikel (refereegranskat)abstract
    • The fermentability of lignocellulose hydrolyzates have been predicted from the responses of a combination of chemical gas sensors. The hydrolyzates were prepared by dilute-acid hydrolysis of wood from pine, aspen, birch, and spruce. The volatile emission from the hydrolyzates before fermentation was measured, and the sensor array response pattern was compared with the observed fermentability of the hydrolyzates, i.e. with the final ethanol concentration after fermentation and the maximum specific ethanol production rate. Two concentration parameters in the hydrolyzates, furfural and the sum of furfural and 5-(hydroxymethyl)furfural (HMF), were also predicted from the responses. The sensors used were metal oxide semiconductor field effect transistors (MOSFET), tin oxide semiconductor devices, and conductive polymer sensors configured in two sensor arrays. The sensor array response pattern was analyzed by principal component analysis and artificial neural networks. Predictions from artificial neural networks deviated from measured values with less than 15%.The fermentability of lignocellulose hydrolyzates have been predicted from the responses of a combination of chemical gas sensors. The hydrolyzates were prepared by dilute-acid hydrolysis of wood from pine, aspen, birch, and spruce. The volatile emission from the hydrolyzates before fermentation was measured, and the sensor array response pattern was compared with the observed fermentability of the hydrolyzates, i.e. with the final ethanol concentration after fermentation and the maximum specific ethanol production rate. Two concentration parameters in the hydrolyzates, furfural and the sum of furfural and 5-(hydroxymethyl)furfural (HMF), were also predicted from the responses. The sensors used were metal oxide semiconductor field effect transistors (MOSFET), tin oxide semiconductor devices, and conductive polymer sensors configured in two sensor arrays. The sensor array response pattern was analyzed by principal component analysis and artificial neural networks. Predictions from artificial neural networks deviated from measured values with less than 15%.
  •  
45.
  • Miranda, A., et al. (författare)
  • Recovery of Clostridium thermosulfurogenes produced β-amylase by (hydroxypropyl)methylcellulose partition
  • 1990
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 6:3, s. 214-219
  • Tidskriftsartikel (refereegranskat)abstract
    • A procedure for recovering Clostridium thermosulfurogenes produced β-amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropyl)methylcellulose. The β-amylase-containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. The process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous-phase partitions, such as poly(ethylene glycol)/dextran, the current approach appeared to be biospecific.
  •  
46.
  • Miranda, Everson A., et al. (författare)
  • Evaluation of column flotation in the downstream processing of fermentation products : recovery of a genetically engineered α-amylase
  • 1993
  • Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 9:4, s. 411-420
  • Tidskriftsartikel (refereegranskat)abstract
    • Flotation is a simple, inexpensive, and versatile unit operation with a largely unexplored potential in biotechnology. There is a general lack of research concerning biotechnological applications in this area, especially in the recovery of fermentation products. Moreover, the few reports in the literature do not consider the modern concept of column flotation as practiced in the mineral industry. We report herein the application of column flotation for the recovery of a Bacillus stearothermophilus α-amylase expressed in Escherichia coli by the use of a food-grade polymer, (hydroxypropyl)methylcellulose (HPMC), and ammonium sulfate. First, the enzyme was removed from the liquid phase by partition to a salted-out HPMC phase. The enzyme-containing polymer flocs were then floated from the liquid. Recovery of active enzyme was as high as 90%, with throughput as high as 94 m3/(h·m2). The floatability of the enzyme from a periplasmic extract was higher than extracellular enzyme in the broth due to the presence of depressors of molecular weight lower than 10 000 in the broth.
  •  
47.
  • Monavari, Sanam, et al. (författare)
  • Improved one-step steam pretreatment of SO2-impregnated softwood with time-dependent temperature profile for ethanol production.
  • 2010
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 26:4, s. 1054-1060
  • Tidskriftsartikel (refereegranskat)abstract
    • In the production of ethanol from lignocellulosic material, pretreatment of the raw material before enzymatic hydrolysis and fermentation is essential to obtain high overall yields of sugar and ethanol. Two-step steam pretreatment results in higher ethanol yields from softwood than the standard one-step pretreatment process. However, the difficulty with separation and washing of the material at high pressure between the two pretreatment steps is a major drawback. In this study, a new one-step pretreatment procedure was investigated, in which the time-temperature profile was varied during pretreatment. The efficiency of pretreatment was assessed by performing simultaneous saccharification and fermentation on the pretreated slurries. Pretreatment of SO(2)-impregnated softwood performed by varying the temperature (190-226 degrees C), the residence time (5-10 min), and the mode of temperature increase (linear or stepwise), resulted in recovery of about 90% of the mannose and glucose present in the raw material. The highest ethanol yield, 75% of theoretical based on the glucan and mannan content of the raw material, was obtained at pretreatment conditions of 190 degrees C for 12 min. Similar ethanol yields were achieved when running the pretreatment as one-step (190-200 degrees C), two levels of temperature, at shorter residence time (7 min), which results in lower capital costs for the process.
  •  
48.
  • Nilsang, Suthasinee, et al. (författare)
  • Monoclonal antibody production using a new supermacroporous cryogel bioreactor
  • 2007
  • Ingår i: Biotechnology Progress. - Malden, MA : Wiley-Blackwell. - 1520-6033 .- 8756-7938. ; 23:4, s. 932-939
  • Tidskriftsartikel (refereegranskat)abstract
    • A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (similar to 20 days), the hybridoma cell line consumed 0.75 mM day(-1) glucose, produced 2.48 mM day(-1) lactic acid, and produced 6.5 mu g mL(-1) day(-1) mAb during the exponential phase. The mAb concentration reached 130 mu g mL(-1) after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.
  •  
49.
  • Nilsang, Suthasinee, et al. (författare)
  • Three-dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles
  • 2008
  • Ingår i: Biotechnology progress (Print). - Malden, MA : Wiley-Blackwell Publishing Inc.. - 8756-7938 .- 1520-6033. ; 24:5, s. 1122-1131
  • Tidskriftsartikel (refereegranskat)abstract
    • Cell proliferation and long-term production of monoclonal antibody IgG(2b) by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 x 9 mm(2)). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two-dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine or L-glutamine + 2 mM alpha-ketoglutarate or L-alanyl-glutamine (GlutaMAXtrade mark) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L-glutamine + alpha-ketoglutarate was similar to cells grown on GlutaMAX containing medium, whereas D-MEM containing L-glutamine showed lower productivity. With the maximal viable cell density (6.85 x 10(6) cells mL(-1)) and highest specific mAb production rate (3.9 mug mL(-1) 10(-4) viable cell day(-1)), D-MEM-GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed-bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoclonal antibody production. © 2008 American Institute of Chemical Engineers.
  •  
50.
  • Palmarola Adrados, Beatriz, et al. (författare)
  • Hydrolysis of nonstarch carbohydrates of wheat-starch effluent for ethanol production
  • 2004
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 20:2, s. 474-479
  • Tidskriftsartikel (refereegranskat)abstract
    • A (polysaccharide-rich) waste stream derived from a combined starch and ethanol factory was investigated regarding hydrolysis of the nonstarch carbohydrates for ethanol production. The material was characterized and processed to yield the maximum amount of sugars. The starch fraction was hydrolyzed with amylolytic enzymes, and the resulting fibrous material was separated by filtration. This material, denoted starch-free fibers (SFF), was subjected to heat treatment followed by enzymatic hydrolysis to recover the other major carbohydrate components, namely, cellulose and hemicellulose, in monomeric form. Heat treatment in a microwave oven efficiently solubilized a fraction of these polysaccharides and made the material more accessible to the cellulolytic and hemicellulolytic enzymes used in the subsequent enzymatic hydrolysis. The maximum sugar yield after enzymatic hydrolysis, achieved with pretreatment at 170 degreesC for 40 min, was 34.1 g per 100 g SFF, comprising 12.8 g glucose, 13.9 g xylose and 7.4 g arabinose, corresponding to 66%, 71% and 51% of the theoretical, respectively.
  •  
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