| 1. |
- Andersson, Jonatan, et al.
(author)
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Isolation of potato proteins using simulated moving bed technology.
- 2008
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In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 101, s. 1256-1263
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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.
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| 2. |
- Bengtsson, Simon
(author)
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The utilization of glycogen accumulating organisms for mixed culture production of polyhydroxyalkanoates.
- 2009
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In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 104, s. 698-708
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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.
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| 3. |
- Bodin, Aase Katarina, 1977, et al.
(author)
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Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes
- 2007
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 97:2, s. 425-434
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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.
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| 4. |
- Brandberg, T., et al.
(author)
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Continuous fermentation of wheat-supplemented lignocellulose hydrolysate with different types of cell retention
- 2007
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In: Biotechnology and Bioengineering. - : John Wiley & Sons, Inc.. - 0006-3592 .- 1097-0290. ; 98:1, s. 80-
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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.
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| 5. |
- Caspeta-Guadarrama, Luis, 1974
(author)
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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
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In: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 102:2, s. 468-
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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.
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| 6. |
- Cassimjee, Karim Engelmark, et al.
(author)
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Silica-immobilized His(6)-tagged enzyme : Alanine racemase in hydrophobic solvent
- 2008
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 99:3, s. 712-716
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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.
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| 7. |
- Collén, Anna, et al.
(author)
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Protein production and induction of the unfolded protein response in Trichoderma reesei strain rut-c30 and its transformant expressing endoglucanase I with a hydrophobic tag
- 2005
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In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 89:3, s. 335-344
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Journal article (peer-reviewed)abstract
- The effect of induction of protein production was studied in bioreactor cultures of T. reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag. The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production. Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations. Induction of the cellulase genes followed a similar temporal pattern in both strains, The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed. Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter. The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions. However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain.
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| 8. |
- de Jongh, Willem A., et al.
(author)
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The Roles of Galactitol, Galactose-1-Phosphate, and Phosphoglucomutase in Galactose-Induced Toxicity in Saccharomyces cerevisiae
- 2008
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In: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 101:2, s. 317-326
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Journal article (peer-reviewed)abstract
- The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combiantions of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furtermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.
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| 9. |
- Diano, A., et al.
(author)
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Physiology of Aspergillus niger in Oxygen-Limited Continuous Cultures: Influence of Aeration, Carbon Source Concentration and Dilution Rate
- 2009
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In: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 103:5, s. 956-965
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Journal article (peer-reviewed)abstract
- In industrial production of enzymes using the filamentous fungus Aspergilhis niger supply of sufficient oxygen is often a limitation, resulting in the formation of by-products such as polyols. In order to identify the mechanisms behind formation of the different by-products we studied the effect of low oxygen availability, at different carbon source concentrations and at different specific growth rates, on the metabolism of A. niger, using continuous cultures. The results show that there is an increase in the production of tricarboxylic acid (TCA) cycle intermediates at low oxygen concentrations. Indeed, at these conditions, a decrease in the mitochondrial respiratory chain activity leads to an accumulation of NADH and to a decreased ATP production which uncouples catabolism and anabolism, influences the intracellular pH and leads to production and excretion of organic acids. Moreover, mannitol is being produced in order to ensure reoxidation of NADH, and this is the main cellular response to balance the ratio NADH/NAD at low oxygen availability. Mannitol production is also coupled to low specific growth rate, which suggests a control of carbon catabolite repression on the mannitol pathway. The roles of two other polyols, erythritol and glycerol, were also investigated. Both compounds are known to accumulate intracellularly, at high osmotic pressure, in order to restore the osmotic balance, but we show that the efficiency of this system is affected by a leakage of polyols through the membrane. Biotechnol. Bioeng. 2009;103: 956-965. (C) 2009 Wiley Periodicals, Inc.
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| 10. |
- Dopson, Mark, et al.
(author)
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Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms.
- 2007
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In: Biotechnol Bioeng. - : Wiley. - 0006-3592 .- 1097-0290. ; 97:5, s. 1205-15
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Journal article (peer-reviewed)abstract
- An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C. (c) 2006 Wiley Periodicals, Inc.
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