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  • 1. Andersson, M.
    et al.
    Andersson, T.
    Adlercreutz, P.
    Nielsen, Tim
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Sveriges tekniska forskningsinstitut, SIK – Institutet för livsmedel och bioteknik.
    Hornsten, E.G.
    Toward an enzyme-based oxygen scavenging laminate.: Influence of industrial lamination conditions on the performance of glucose oxidase2002In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 79, no 1, p. 37-42Article in journal (Refereed)
    Abstract [en]

    The laminate consisted of several polymer layers, aluminium, and one cellulose-based layer containing the active enzymatic system (e.g., glucose oxidase, catalase, glucose, and CaCO3). During the industrial lamination process, the enzyme layer was exposed to three temperature spikes up to 325°C without significant enzyme inactivation. Ninety-seven percent of the glucose oxidase activity still remained after the lamination process. The best laminate had an oxygen absorbing capacity of 7.6 ± 1.0 L/m2. A reference that was not laminated expressed a corresponding oxygen absorbing capacity of 7.1 ± 0.8 L/m2. © 2002 Wiley Periodicals, Inc.

  • 2. Bjornsson, L.
    et al.
    Hornsten, E.G.
    Mattiasson, B.
    Utilization of a palladium-metal oxide semiconductor (Pd-MOS) sensor for on-line monitoring of dissolved hydrogen in anaerobic digestion2001In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 73, no 1, p. 35-43Article in journal (Refereed)
    Abstract [en]

    The use of a hydrogen-sensitive palladium-metal oxide semiconductor (Pd-MOS) sensor in combination with a membrane for liquid-to-gas transfer for the detection of dissolved hydrogen was investigated. The system was evaluated with known concentrations of dissolved hydrogen in water. The lowest concentration detected with this set-up was 160 nM. The method was applied to monitoring of a laboratory-scale anaerobic digestion process employing mixed sludge containing mainly food/industrial waste. Pulse loads of glucose were added to the system at different levels of microbial activity, and the microbial status of the culture was reflected in the dissolved hydrogen response. Simultaneous headspace hydrogen measurements were performed, and at the lower levels of dissolved hydrogen no corresponding headspace hydrogen could be detected. When glucose was added to a resting culture the dissolved hydrogen response was rapid and the first response could be detected 9 min after addition of glucose, whereas headspace hydrogen concentrations increased only after 80 to 110 min. This indicates limitations in the liquid-to-gas hydrogen transfer and illustrates the importance of hydrogen monitoring in the liquid. The sensor system developed is flexible, the membrane is easily replaceable, and the probe for liquid-to-gas hydrogen transfer can be adjusted easily to large-scale applications. © 2001 John Wiley & Sons, Inc.

  • 3. Boer, H.
    et al.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Koivula, A.
    Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters2000In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 69, no 5, p. 486-494Article in journal (Refereed)
    Abstract [en]

    Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) pro meter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermenter. Production of Ce17A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K-m values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

  • 4. Bramble, J L
    et al.
    Graves, D J
    Brodelius, Peter
    Department of Plant Biotechnology, University of Lund.
    Calcium and Phosphate Effects on Growth and Alkaloid Production in Coffea arabica: Experimental Results and Mathematical Model.1991In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 37, no 9, p. 859-868Article in journal (Refereed)
    Abstract [en]

    Plant, mammalian, and microbial cells are commonly immobilized in calcium alginate gels for the production of valuable secondary metabolites. However, calcium ions are known to inhibit growth in various type of cells, and calcium is an integral part of such gels. Therefore, an investigation was conducted to evaluate the effect of calcium on the growth and alkaloid production of a model cell-line, Coffea arabica, in suspension culture before attempting to immobilize such cells in alginate. A kinetic model was then developed from the results to describe cell growth and alkaloid production and the mechanism by which calcium influences these variables. In addition, it was observed that there was a characteristic relationship between the concentration of calcium in the external medium and the concentration of extracellular and intracellular phosphate. The intracellular phosphate level was, in turn, related to the production of alkaloids. Using these results, a dynamic mathematical model of cell growth and alkaloid production was developed based on the proposed roles of calcium and phosphate. The model showed satisfactory agreement with three sets of experiments at different calcium concentrations. A possible linkage between the calcium and phosphate results is postulated based on the limited solubility of calcium phosphate. 

  • 5. Brandberg, T.
    et al.
    Karimi, K.
    University of Borås, School of Engineering.
    Taherzadeh, M.J.
    University of Borås, School of Engineering.
    Franzén, C.J.
    Gustafsson, L.
    Continuous fermentation of wheat-supplemented lignocellulose hydrolysate with different types of cell retention2007In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 98, no 1, p. 80-Article in journal (Refereed)
    Abstract [en]

    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.

  • 6. Bylund, F.
    et al.
    Castan, A.
    Mikkola, R.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Larsson, G.
    Influence of scale-up on the quality of recombinant human growth hormone2000In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 69, no 2, p. 119-128Article in journal (Refereed)
    Abstract [en]

    The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer.

  • 7.
    Cassimjee, Karim Engelmark
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Trummer, Martin
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Silica-immobilized His(6)-tagged enzyme: Alanine racemase in hydrophobic solvent2008In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 99, no 3, p. 712-716Article in journal (Refereed)
    Abstract [en]

    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.

  • 8. Castan, A.
    et al.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli2002In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 77, no 3, p. 324-328Article in journal (Refereed)
    Abstract [en]

    Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.

  • 9. Cavka, Adnan
    et al.
    Alriksson, Björn
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Processum.
    Ahnlund, Maria
    Jönsson, Leif J.
    Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors2011In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 108, no 11, p. 2592-2599Article in journal (Refereed)
    Abstract [en]

    Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10mM dithionite to medium containing 2.5mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5). © 2011 Wiley Periodicals, Inc.

  • 10.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Björn
    Processum Biorefinery Initiative AB, SE-891 22 Örnsköldsvik, Sweden.
    Ahnlund, Maria
    Umeå Plant Science Center, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden.
    Jönsson, Leif J
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors2011In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 108, no 11, p. 2592-2599Article in journal (Refereed)
    Abstract [en]

    Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).

  • 11. Christakopoulos, Paul
    et al.
    Koullas, Dimitrios P.
    Department of Chemical Engineering, National Technical University of Athens.
    Kekos, Dimitris
    Department of Chemical Engineering, National Technical University of Athens.
    Macris, Basil J
    Department of Chemical Engineering, National Technical University of Athens.
    Koukios, Emmanuel G.
    Department of Chemical Engineering, National Technical University of Athens.
    Correlating the effect of pretreatment on the enzymatic hydrolysis of straw1992In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 39, no 1, p. 113-116Article in journal (Refereed)
    Abstract [en]

    Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120°C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (x(max)), and the enzymic hydrolysis time corresponding to 50% of x(max) (t( 1/2 )). An empirical correlation between x(max) and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content. Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120°C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (xmax), and the enzymic hydrolysis time corresponding to 50% of Xmax (t1/2). An empirical correlation between Xmax and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content.

  • 12.
    Dopson, Mark
    et al.
    Umeå University.
    Halinen, A.-K.
    Tampere Univ Technol, Finland.
    Rahunen, N.
    Tampere Univ Technol, Finland.
    Boström, Dan
    Umeå University.
    Sundkvist, J.-E.
    Boliden Mineral AB, Boliden, Sweden.
    Riekkola-Vanhanen, M.
    Talvivaara Project Ltd, Sotkamo, Finland.
    Kaksonen, A.H.
    Tampere Univ Technol, Finland.
    Puhakka, J. A.
    Tampere Univ Technol, Finland.
    Silicate mineral dissolution during heap bioleaching2008In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 99, no 4, p. 811-820Article in journal (Refereed)
    Abstract [en]

    Silicate minerals are present in association with metal sulfides in ores and their dissolution occurs when the sulfide minerals are bioleached in heaps for metal recovery. It has previously been suggested that silicate mineral dissolution can affect mineral bioleaching by acid consumption, release of trace elements, and increasing the viscosity of the teach solution. In this study, the effect of silicates present in three separate samples in conjunction with chalcopyrite and a complex multi-metal sulfide ore on heap bioleaching was evaluated in column bioreactors. Fe2+ oxidation was inhibited in columns containing chalcopyrite samples A and C that leached 1.79 and 1.11 mM fluoride, respectively but not in sample B that contained 0.14 mM fluoride. Microbial Fe2+ oxidation inhibition experiments containing elevated fluoride concentrations and measurements of fluoride release from the chalcopyrite ores supported that inhibition of Fe2+ oxidation during column leaching of two of the chalcopyrite ores was due to fluoride toxicity. Column bioleaching of the complex sulfide ore was carried out at various temperatures (7-50 degrees C) and pH values (1.5-3.0). Column leaching at pH 1.5 and 2.0 resulted in increased acid consumption rates and silicate dissolutionsuch that it became difficult to filter the leach solutions and for the leach liquor to percolate through the column. However, column temperature (at pH 2.5) only had a minor effect on the acid consumption and silicate dissolution rates. This study demonstrates the potential negative impact of silicate mineral dissolution on heap bioleaching by microbial inhibition and liquid flow.

  • 13.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Halinen, Anna-Kaisa
    Rahrmen, Nelli
    Boström, Dan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics, Energy Technology and Thermal Process Chemistry.
    Sundkvist, Jan-Eric
    Riekkola-Vanhanen, Marja
    Kaksonen, Anna H.
    Puhakka, Jaakko A.
    Silicate mineral dissolution during heap bioleaching2008In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 99, no 4, p. 811-820Article in journal (Refereed)
    Abstract [en]

    Silicate minerals are present in association with metal sulfides in ores and their dissolution occurs when the sulfide minerals are bioleached in heaps for metal recovery. It has previously been suggested that silicate mineral dissolution can affect mineral bioleaching by acid consumption, release of trace elements, and increasing the viscosity of the teach solution. In this study, the effect of silicates present in three separate samples in conjunction with chalcopyrite and a complex multi-metal sulfide ore on heap bioleaching was evaluated in column bioreactors. Fe2+ oxidation was inhibited in columns containing chalcopyrite samples A and C that leached 1.79 and 1.11 mM fluoride, respectively but not in sample B that contained 0.14 mM fluoride. Microbial Fe2+ oxidation inhibition experiments containing elevated fluoride concentrations and measurements of fluoride release from the chalcopyrite ores supported that inhibition of Fe2+ oxidation during column leaching of two of the chalcopyrite ores was due to fluoride toxicity. Column bioleaching of the complex sulfide ore was carried out at various temperatures (7-50 degrees C) and pH values (1.5-3.0). Column leaching at pH 1.5 and 2.0 resulted in increased acid consumption rates and silicate dissolution such that it became difficult to filter the leach solutions and for the leach liquor to percolate through the column. However, column temperature (at pH 2.5) only had a minor effect on the acid consumption and silicate dissolution rates. This study demonstrates the potential negative impact of silicate mineral dissolution on heap bioleaching by microbial inhibition and liquid flow.

  • 14.
    Dopson, Mark
    et al.
    Umeå University.
    Halinen, Anna-Kaisa
    Tampere University of Technology, Tampere, Finland.
    Rahunen, Nelli
    Tampere University of Technology, Tampere, Finland.
    Özkaya, Bestamin
    Tampere University of Technology, Tampere, Finland.
    Sahinkaya, Erkan
    Tampere University of Technology, Tampere, Finland.
    Kaksonen, Anna H
    Tampere University of Technology, Tampere, Finland.
    Lindström, E Börje
    Umeå University.
    Puhakka, Jaakko A
    Tampere University of Technology, Tampere, Finland.
    Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms.2007In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 97, no 5, p. 1205-1215Article in journal (Refereed)
    Abstract [en]

    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.

  • 15. Du, Liping
    et al.
    Villarreal, Seth
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymerase2012In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 109, no 4, p. 1043-1050Article in journal (Refereed)
    Abstract [en]

    Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TF large (class I) terminator with adjacent copies of the vesicular stomatitis virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within Escherichia coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e., more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TF terminators for BioBrick synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.

  • 16.
    Gahan, Chandra Sekhar
    et al.
    Luleå University of Technology.
    Sundkvist, Jan-Eric
    Luleå University of Technology ; Boliden Mineral AB, Boliden, Sweden.
    Dopson, Mark
    Umeå University.
    Sandström, Åke
    Luleå University of Technology.
    Effect of chloride on ferrous iron oxidation by a Leptospirillum ferriphilum-dominated chemostat culture2010In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 106, no 3, p. 422-431Article in journal (Refereed)
    Abstract [en]

    Biomining is the use of microorganisms to catalyze metal extraction from sulfide ores. However, the available water in some biomining environments has high chloride concentrations and therefore, chloride toxicity to ferrous oxidizing microorganisms has been investigated. Batch biooxidation of Fe(2+) by a Leptospirillum ferriphilum dominated culture was completely inhibited by 12gL(-1) chloride. In addition, the effects of chloride on oxidation kinetics in a Fe(2+) limited chemostat were studied. Results from the chemostat modeling suggest that the chloride toxicity was attributed to affects on the Fe2+ oxidation system, pH homeostasis, and lowering of the proton motive force. Modeling showed a decrease in the maximum specific growth rate (mu(max)) and an increase in the substrate constant (K(s)) with increasing chloride concentrations, indicating an effect on the Fe(2+) oxidation system. The model proposes a lowered maintenance activity when the media was fed with 2-3 g L(-1) chloride with a concomitant drastic decrease in the true yield (Y(true)). This model helps to understand the influence of chloride on Fe(2+) biooxidation kinetics.

  • 17. Gahan, Chandra Sekhar
    et al.
    Sundkvist, Jan-Eric
    Boliden Mineral AB.
    Dopson, Mark
    Department of Molecular Biology, Umeå University.
    Sandström, Åke
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Effect of chloride on ferrous iron oxidation by a Leptospirillum ferriphilum-dominated chemostat culture2010In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 106, no 3, p. 422-431Article in journal (Refereed)
    Abstract [en]

    Biomining is the use of microorganisms to catalyze metal extraction from sulfide ores. However, the available water in some biomining environments has high chloride concentrations and therefore, chloride toxicity to ferrous oxidizing microorganisms has been investigated. Batch biooxidation of Fe2+ by a Leptospirillum ferriphilum-dominated culture was completely inhibited by 12 g L-1 chloride. In addition, the effects of chloride on oxidation kinetics in a Fe2+ limited chemostat were studied. Results from the chemostat modeling suggest that the chloride toxicity was attributed to affects on the Fe2+ oxidation system, pH homeostasis, and lowering of the proton motive force. Modeling showed a decrease in the maximum specific growth rate (µmax) and an increase in the substrate constant (Ks) with increasing chloride concentrations, indicating an effect on the Fe2+ oxidation system. The model proposes a lowered maintenance activity when the media was fed with 2 to 3 g L-1 chloride with a concomitant drastic decrease in the true yield (Ytrue). This model helps to understand the influence of chloride on Fe2+ biooxidation kinetics.

  • 18. Gahan, CS
    et al.
    Sundkvist, JE
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sandström, A
    Effect of chloride on ferrous iron oxidation by a leptospirillum ferriphilum-dominated chemostat culture2010In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 106, no 3, p. 422-431Article in journal (Refereed)
    Abstract [en]

    Biomining is the use of microorganisms to catalyze metal extraction from sulfide ores. However, the available water in some biomining environments has high chloride concentrations and therefore, chloride toxicity to ferrous oxidizing microorganisms has been investigated. Batch biooxidation of Fe2+ by a Leptospirillum ferriphilum dominated culture was completely inhibited by 12gL(-1) chloride. In addition, the effects of chloride on oxidation kinetics in a Fe2+ limited chemostat were studied. Results from the chemostat modeling suggest that the chloride toxicity was attributed to affects on the Fe2+ oxidation system, pH homeostasis, and lowering of the proton motive force. Modeling showed a decrease in the maximum specific growth rate (mu(max)) and an increase in the substrate constant (K-s) with increasing chloride concentrations, indicating an effect on the Fe2+ oxidation system. The model proposes a lowered maintenance activity when the media was fed with 2-3 g L-1 chloride with a concomitant drastic decrease in the true yield (Y-true). This model helps to understand the influence of chloride on Fe2+ biooxidation kinetics. Biotechnol. Bioeng. 2010;106: 422-431. (C) 2010 Wiley Periodicals, Inc.

  • 19.
    Grabherr, R.
    et al.
    Institute of Applied Microbiology, University of Agricultural Science, Vienna, Muthgasse 18, A-1190 Vienna, Austria.
    Nilsson, Erik
    Linköping University, The Institute of Technology.
    Striedner, G.
    Institute of Applied Microbiology, University of Agricultural Science, Vienna, Muthgasse 18, A-1190 Vienna, Austria.
    Bayer, K.
    Institute of Applied Microbiology, University of Agricultural Science, Vienna, Muthgasse 18, A-1190 Vienna, Austria.
    Stabilizing plasmid copy number to improve recombinant protein production2002In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 77, no 2, p. 142-147Article in journal (Refereed)
    Abstract [en]

    The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization. © 2002 John Wiley & Sons, Inc.

  • 20.
    Horváth, I. S.
    et al.
    Department of Chemical Reaction Engineering, Chalmers University of Technology.
    Taherzadeh, Mohammad J
    Department of Chemical Reaction Engineering, Chalmers University of Technology.
    Niklasson, C.
    Department of Chemical Reaction Engineering, Chalmers University of Technology.
    Lidén, G.
    Department of Chemical Engineering II, Lund Institute of Technology.
    Effects of furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae2001In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 75, no 5, p. 540-549Article in journal (Refereed)
    Abstract [en]

    Furfural is an important inhibitor of yeast metabolism in lignocellulose-derived substrates. The effect of furfural on the physiology of Saccharomyces cerevisiae CBS 8066 was investigated using anaerobic continuous cultivations. Experiments were performed with furfural in the feed medium (up to 8.3 g/L) using three dierent dilution rates (0.095, 0.190, and 0.315 h-1). The measured concentration of furfural was low (<0.1 g/L) at all steady states obtained. However, it was not possible to achieve a steady state at a specific conversion rate of furfural, qf, higher than approximately 0.15 g/g·h. An increased furfural concentration in the feed caused a decrease in the steady-state glycerol yield. This agreed well with the decreased need for glycerol production as a way to regenerate NAD+, i.e., to function as a redox sink because furfural was reduced to furfuryl alcohol. Transient experiments were also performed by pulse addition of furfural directly into the fermentor. In contrast to the situation at steady-state conditions, both glycerol and furfuryl alcohol yields increased after pulse addition of furfural to the culture. Furthermore, the maximum specific conversion rate of furfural (0.6 g/g·h) in dynamic experiments was significantly higher than what was attainable in the chemostat experiments. The dynamic furfural conversion could be described by the use of a simple Michaelis-Menten-type kinetic model. Also furfural conversion under steady-state conditions could be explained by a Michaelis-Menten-type kinetic model, but with a higher anity and a lower maximum conversion rate. This indicated the presence of an additional component with a higher anity, but lower maximum capacity, either in the transport system or in the conversion system of furfural.

  • 21. Jahic, M.
    et al.
    Knoblechner, J.
    Charoenrat, T.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Interfacing Pichia pastoris cultivation with expanded bed adsorption2006In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 93, no 6, p. 1040-1049Article in journal (Refereed)
    Abstract [en]

    For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L (.) h.

  • 22.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Simultaneous Uptake of Lignocellulose- Based Monosaccharides by Escherichia Coli2014In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 111, no 6, p. 1108-1115Article in journal (Refereed)
    Abstract [en]

    Lignocellulosic waste is a naturally abundant biomass and is therefore an attractive material to use in second generation biorefineries. Microbial growth on the monosaccharides present in hydrolyzed lignocellulose is however associated with several obstacles whereof one is the lack of simultaneous uptake of the sugars. We have studied the aerobic growth of Escherichia coli on D-glucose, D-xylose, and L-arabinose and for simultaneous uptake to occur, both the carbon catabolite repression mechanism (CCR) and the AraC repression of xylose uptake and metabolism had to be removed. The strain AF1000 is a MC4100 derivative that is only able to assimilate arabinose after a considerable lag phase, which is unsuitable for commercial production. This strain was successfully adapted to growth on L-arabinose and this led to simultaneous uptake of arabinose and xylose in a diauxic growth mode following glucose consumption. In this strain, a deletion in the phosphoenolpyruvate:phosphotransferase system (PTS) for glucose uptake, the ptsG mutation, was introduced. The resulting strain, PPA652ara simultaneously consumed all three monosaccharides at a maximum specific growth rate of 0.59h(-1), 55% higher than for the ptsG mutant alone. Also, no residual sugar was present in the cultivation medium. The potential of PPA652ara is further acknowledged by the performance of AF1000 during fed-batch processing on a mixture of D-glucose, D-xylose, and L-arabinose. The conclusion is that without the removal of both layers of carbon uptake control, this process results in accumulation of pentoses and leads to a reduction of the specific growth rate by 30%.

  • 23. Jeihanipour, A.
    et al.
    Karimi, K.
    University of Borås, School of Engineering.
    Taherzadeh, Mohammad
    University of Borås, School of Engineering.
    Enhancement of ethanol and biogas production from high-crystalline cellulose by different modes of NMO pretreatment2010In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 105, no 3, p. 469-476Article in journal (Refereed)
    Abstract [en]

    Pretreatment of high-crystalline cellulose with N-methyl-morpholine-N-oxide (NMO or NMMO) to improve bioethanol and biogas production was investigated. The pretreatments were performed at 90 and 120°C for 0.5–15 h in three different modes, including dissolution (85% NMO), ballooning (79% NMO), and swelling (73% NMO). The pretreated materials were then enzymatically hydrolyzed and fermented to ethanol or anaerobically digested to biogas (methane). The pretreatment at 85% NMO, 120°C and 2.5 h resulted in 100% yield in the subsequent enzymatic hydrolysis and around 150% improvement in the yield of ethanol compared to the untreated and water-treated material. However, the best results of biogas production were obtained when the cellulose was treated with swelling and ballooning mode, which gave almost complete digestion in 15 days. Thus, the pretreatment resulted in 460 g ethanol or 415 L methane from each kg of cellulose. Analysis of the structure of treated and untreated celluloses showed that the dissolution mode can efficiently convert the crystalline cellulose I to cellulose II. However, it decreases the water swelling capacity of the cellulose. On the other hand, swelling and ballooning modes in NMO treatment were less efficient in both water swelling capacity and cellulose crystallinity. No cellulose loss, ambient pressure, relatively moderate conditions, and high efficiency make the NMO a good alternative for pretreatment of high-crystalline cellulosic materials.

  • 24.
    Kupka, Daniel
    et al.
    Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, SK-043 53 Koice, Slovakia.
    Rzhepishevska, Olena
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Lindström, Börje
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Karnachuk, Olia V
    3Department of Agriculture and Environmental Science, Tomsk State University, Prospekt Lenina 36, 634050 Tomsk, Russia.
    Tuovinen, Olli H
    Department of Microbiology, Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210.
    Bacterial oxidation of ferrous iron at low temperatures.2007In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 97, no 6, p. 1470-1478Article in journal (Refereed)
    Abstract [en]

    This study comprises the first report of ferrous iron oxidation by psychrotolerant, acidophilic iron-oxidizing bacteria capable of growing at 5 degrees C. Samples of mine drainage-impacted surface soils and sediments from the Norilsk mining region (Taimyr, Siberia) and Kristineberg (Skellefte district, Sweden) were inoculated into acidic ferrous sulfate media and incubated at 5 degrees C. Iron oxidation was preceded by an approximately 3-month lag period that was reduced in subsequent cultures. Three enrichment cultures were chosen for further work and one culture designated as isolate SS3 was purified by colony isolation from a Norilsk enrichment culture for determining the kinetics of iron oxidation. The 16S rRNA based phylogeny of SS3 and two other psychrotolerant cultures, SS5 from Norilsk and SK5 from Northern Sweden, was determined. Comparative analysis of amplified 16S rRNA gene sequences showed that the psychrotolerant cultures aligned within Acidithiobacillus ferrooxidans. The rate constant of iron oxidation by growing cultures of SS3 was in the range of 0.0162-0.0104 h(-1) depending on the initial pH. The oxidation kinetics followed an exponential pattern, consistent with a first order rate expression. Parallel iron oxidation by a mesophilic reference culture of Acidithiobacillus ferrooxidans was extremely slow and linear. Precipitates harvested from the 5 degrees C culture were identified by X-ray diffraction as mixtures of schwertmannite (ideal formula Fe(8)O(8)(OH)(6)SO(4)) and jarosite (KFe(3)(SO(4))(2)(OH)(6)). Jarosite was much more dominant in precipitates produced at 30 degrees C. (c) 2007 Wiley Periodicals, Inc.

  • 25.
    Kupka, Daniel
    et al.
    Slovak Academy of Sciences, Košice, Slovakia.
    Rzhepishevska, Olena I.
    Umeå University.
    Dopson, Mark
    Umeå University.
    Lindström, E. Börje
    Umeå University.
    Karnachuk, Olia V.
    Tomsk State University, Tomsk, Russia.
    Tuovinen, Olli H.
    Ohio State University, USA.
    Bacterial oxidation of ferrous iron at low temperatures2007In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 97, no 6, p. 1470-1478Article in journal (Refereed)
    Abstract [en]

    This study comprises the first report of ferrous iron oxidation by psychrotolerant, acidophilic iron-oxidizing bacteria capable of growing at 5 degrees C. Samples of mine drainage-impacted surface soils and sediments from the Norilsk mining region (Taimyr, Siberia) and Kristineberg (Skellefte district, Sweden) were inoculated into acidic ferrous sulfate media and incubated at 5 degrees C. Iron oxidation was preceded by an approximately 3-month lag period that was reduced in subsequent cultures. Three enrichment cultures were chosen for further work and one culture designated as isolate SS3 was purified by colony isolation from a Norilsk enrichment culture for determining the kinetics of iron oxidation. The 16S rRNA based phylogeny of SS3 and two other psychrotolerant cultures, SS5 from Norilsk and SK5 from Northern Sweden, was determined. Comparative analysis of amplified 16S rRNA gene sequences showed that the psychrotolerant cultures aligned within Acidithiobacillus ferrooxidans. The rate constant of iron oxidation by growing cultures of SS3 was in the range of 0.0162-0.0104 h(-1) depending on the initial pH. The oxidation kinetics followed an exponential pattern, consistent with a first order rate expression. Parallel iron oxidation by a mesophilic reference culture of Acidithiobacillus ferrooxidans was extremely slow and linear. Precipitates harvested from the 5 degrees C culture were identified by X-ray diffraction as mixtures of schwertmannite (ideal formula Fe(8)O(8)(OH)(6)SO(4)) and jarosite (KFe(3)(SO(4))(2)(OH)(6)). Jarosite was much more dominant in precipitates produced at 30 degrees C. Biotechnol. Bioeng. 2007;97:1470-1478. (c) 2007 Wiley Periodicals, Inc.

  • 26.
    Li, Zhenglun
    et al.
    Michigan State University.
    Chen, Charles H.
    Michigan State University.
    Liu, Tongjun
    Michigan State University.
    Mathrubootham, Vaidyanathan
    Michigan State University.
    Hegg, Eric L.
    Michigan State University.
    Hodge, David
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Catalysis with Cuii(bpy) improves alkaline hydrogen peroxide pretreatment2013In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 110, no 4, p. 1078-1086Article in journal (Refereed)
    Abstract [en]

    Copper(II) 2,2′-bipyridine (CuII(bpy))-catalyzed alkaline hydrogen peroxide (AHP) pretreatment was performed on three biomass feedstocks including alkali pre-extracted switchgrass, silver birch, and a hybrid poplar cultivar. This catalytic approach was found to improve the subsequent enzymatic hydrolysis of plant cell wall polysaccharides to monosaccharides for all biomass types at alkaline pH relative to uncatalyzed pretreatment. The hybrid poplar exhibited the most significant improvement in enzymatic hydrolysis with monomeric sugar release and conversions more than doubling from 30% to 61% glucan conversion, while lignin solubilization was increased from 36.6% to 50.2% and hemicellulose solubilization was increased from 14.9% to 32.7%. It was found that CuII(bpy)-catalyzed AHP pretreatment of cellulose resulted in significantly more depolymerization than uncatalyzed AHP pretreatment (78.4% vs. 49.4% decrease in estimated degree of polymerization) and that carboxyl content the cellulose was significantly increased as well (fivefold increase vs. twofold increase). Together, these results indicate that CuII(bpy)-catalyzed AHP pretreatment represents a promising route to biomass deconstruction for bioenergy applications

  • 27.
    Liljeqvist, Maria
    et al.
    Umeå University.
    Sundkvist, Jan-Eric
    Boliden Mineral AB, Boliden, Sweden.
    Saleh, Amang
    Boliden Mineral AB, Boliden, Sweden.
    Dopson, Mark
    Umeå University.
    Low temperature removal of inorganic sulfur compounds from mining process waters2011In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 108, no 6, p. 1251-1259Article in journal (Refereed)
    Abstract [en]

    Process water and effluents from mining operations treating sulfide rich ores often contain considerable concentrations of metastable inorganic sulfur compounds such as thiosulfate and tetrathionate. These species may cause environmental problems if released to downstream recipients due to oxidation to sulfuric acid catalyzed by acidophilic microorganisms. Molecular phylogenic analysis of the tailings pond and recipient streams identified psychrotolerant and mesophilic inorganic sulfur compound oxidizing microorganisms. This suggested year round thiosalt oxidation occurs. Mining process waters may also contain inhibiting substances such as thiocyanate from cyanidation plants. However, toxicity experiments suggested their expected concentrations would not inhibit thiosalt oxidation by Acidithiobacillus ferrivorans SS3. A mixed culture from a permanently cold (4-6 degrees C) low pH environment was tested for thiosalt removal in a reactor design including a biogenerator and a main reactor containing a biofilm carrier. The biogenerator and main reactors were successively reduced in temperature to 5-6 degrees C when 43.8% of the chemical oxidation demand was removed. However, it was found that the oxidation of thiosulfate was not fully completed to sulfate since low residual concentrations of tetrathionate and trithionate were found in the discharge. This study has demonstrated the potential of using biotechnological solutions to remove inorganic sulfur compounds at 6 degrees C and thus, reduce the impact of mining on the environment.

  • 28.
    Liljeqvist, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sundkvist, Jan-Eric
    Saleh, Amang
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Low temperature removal of inorganic sulfur compounds from mining process waters2011In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 108, no 6, p. 1251-1259Article in journal (Refereed)
    Abstract [en]

    Process water and effluents from mining operations treating sulfide rich ores often contain considerable concentrations of metastable inorganic sulfur compounds such as thiosulfate and tetrathionate. These species may cause environmental problems if released to downstream recipients due to oxidation to sulfuric acid catalyzed by acidophilic microorganisms. Molecular phylogenic analysis of the tailings pond and recipient streams identified psychrotolerant and mesophilic inorganic sulfur compound oxidizing microorganisms. This suggested year round thiosalt oxidation occurs. Mining process waters may also contain inhibiting substances such as thiocyanate from cyanidation plants. However, toxicity experiments suggested their expected concentrations would not inhibit thiosalt oxidation by Acidithiobacillus ferrivorans SS3. A mixed culture from a permanently cold (4-6°C) low pH environment was tested for thiosalt removal in a reactor design including a biogenerator and a main reactor containing a biofilm carrier. The biogenerator and main reactors were successively reduced in temperature to 5-6°C when 43.8% of the chemical oxidation demand was removed. However, it was found that the oxidation of thiosulfate was not fully completed to sulfate since low residual concentrations of tetrathionate and trithionate were found in the discharge. This study has demonstrated the potential of using biotechnological solutions to remove inorganic sulfur compounds at 6°C and thus, reduce the impact of mining on the environment. Biotechnol. Bioeng. 2011; 108:1251-1259. © 2011 Wiley Periodicals, Inc.

  • 29. Lin, H. Y.
    et al.
    Hoffmann, F.
    Rozkov, Aleksei
    KTH, Superseded Departments, Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Rinas, U.
    Neubauer, P.
    Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli2004In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 87, no 5, p. 602-613Article in journal (Refereed)
    Abstract [en]

    Guanosine-3'5'-tetraphosphate (ppGpp) and as, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.

  • 30. Lin, H. Y.
    et al.
    Mathiszik, B.
    Xu, B.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Neubauer, P.
    Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli2001In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 73, no 5, p. 347-357Article in journal (Refereed)
    Abstract [en]

    A simple pulse-based method for the determination of the maximum uptake capacities for glucose a nd oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where mu was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when mu declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.

  • 31. Lindberg, Anna
    et al.
    Rasmuson, Åke C.
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Transport Phenomena.
    Selective desorption of carbon dioxide from sewage sludge for in situ methane enrichment - Part 1: Pilot-plant experiments2006In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 95, no 5, p. 794-803Article in journal (Refereed)
    Abstract [en]

    In situ methane enrichment in anaerobic digestion of sewage sludge has been investigated by experiments and by modeling. In this first part, the experimental work on the desorption of carbon dioxide and methane from sewage sludge is reported. The bubble column, had a diameter of 0.3 m and a variable height up to 1.8 m. At operation the dispersion height in the column was between 1 and 1.3 m. Outdoor air was used. The column was placed close to a full-scale sewage sludge digester, at a municipal wastewater treatment plant. The digester was operated at mesophilic conditions with a hydraulic retention time of about 20 days. The bubble column was operated to steady-state, at which carbon dioxide concentration and alkalinity were determined on the liquid side, and the concentration of carbon dioxide and methane on the gas side. Thirty-eight experiments were performed at various liquid and gas flow rates. The experimental results show that the desorption rates achieved for carbon dioxide ranges from 0.07 to 0.25 m(3) CO2/m(3) sludge per day, which is comparable to the rate of generation by the anaerobic digestion. With increasing liquid flow rate and decreasing gas flow rate the amount of methane desorbed per amount of carbon dioxide desorbed increases. The lowest methane loss achieved is approximately 2% of the estimated methane production in the digestion process.

  • 32. Lindberg, Anna
    et al.
    Rasmuson, Åke C.
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Transport Phenomena.
    Selective desorption of carbon dioxide from sewage sludge for in situ methane enrichment - Part II: Modelling and evaluation of experiments2007In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 97, no 5, p. 1039-1052Article in journal (Refereed)
    Abstract [en]

    In situ methane enrichment in anaerobic digestion of sewage sludge has been investigated by experiments and by modelling. Sludge from a full scale digester was fed to a pilot scale bubble column having 0.3 m diameter for preferential desorption of carbon dioxide. In this second part, a model describing the steady-state performance of the bubble column for desorption of carbon dioxide and methane has been developed. The column is assumed to operate in the homogeneous flow regime, and with respect to carbon dioxide and methane both phases are described by the axial dispersion model.. The model treats the chemical reaction equilibrium between carbon dioxide and bicarbonate as being slow and the bicarbonate concentration as being constant. The model is correlated to previously reported experimental, results to determine the liquid side mass transfer coefficient in each experiment. A simple power law model is used to correlate, the mass transfer coefficient to the gas flow rate. In general, the model allows for a reasonable qualitative description of the behaviour of the bubble column performance but the quantitative agreement with experimental results is not satisfactory. It is believed, though that the main. problem is actually not in the model but is related to experimental uncertainties concerning inlet concerttrations of liquid phase methane and bicarbonate..

  • 33.
    Morales, Teresita A
    et al.
    Stockholm University.
    Dopson, Mark
    Umeå University.
    Athar, Rana
    Umeå University.
    Herbert, Roger B
    Uppsala University.
    Analysis of bacterial diversity in acidic pond water and compost after treatment of artificial acid mine drainage for metal removal.2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 5, p. 543-551Article in journal (Refereed)
    Abstract [en]

    The microbial population of a sludge amended leaf compost material utilized for treatment of artificial acid mine drainage was studied by culture-independent molecular methods. Iron-rich and sulfurous wastewater (artificial acid mine drainage) was circulated through a column bioreactor for 16 months. After 12 months the column was inoculated with a mixed culture from an acidic pond receiving acid mine drainage from a tailings impoundment at a decommissioned site in Kristineberg, North Sweden. Hydrogen sulfide odor and the formation of black precipitates indicated that sulfate-reduction occurred in the column. 16S rDNA gene analysis by denaturing gradient gel electrophoresis, cloning, and sequencing as well as fluorescent in situ hybridization confirmed the presence of microorganisms closely related to sulfate-reducing bacteria and microorganisms from the genera Pseudoxanthmonas, Dechlorosoma, Desulfovibrio, Agrobacterium, Methylocapsa, Rhodococcus, Sulfobacillus, and some unidentified bacteria. Sulfate-reducing bacteria were found in the column bioreactor 2 weeks after inoculation, but not thereafter. This suggests they were in low abundance, even though sulfate remediation rates were significant. Instead, the population contained species similar to those previously found to utilize humic substances released from the compost material.

  • 34. Morales, Teresita
    et al.
    Dopson, Mark
    Athar, R.
    Herbert, Roger
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Air and Water Science.
    Analysis of bacterial diversity in acidic pond water and compost after treatment of artificial acid mine drainage for metal removal2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 5, p. 543-551Article in journal (Refereed)
    Abstract [en]

    The microbial population of a sludge amended leaf compost material utilized for treatment of artificial acid mine drainage was studied by culture-independent molecular methods. Iron-rich and sulfurous wastewater (artificial acid mine drainage) was circulated through a column bioreactor for 16 months. After 12 months the column was inoculated with a mixed culture from an acidic pond receiving acid mine drainage from a tailings impoundment at a decommissioned site in Kristineberg, North Sweden. Hydrogen sulfide odor and the formation of black precipitates indicated that sulfate-reduction occurred in the column. 16S rDNA gene analysis by denaturing gradient gel electrophoresis, cloning, and sequencing as well as fluorescent in situ hybridization confirmed the presence of microorganisms closely related to sulfate-reducing bacteria and microorganisms from the genera Pseudoxanthmonas, Dechlorosoma, Desulfovibrio, Agrobacterium, Methylocapsa, Rhodococcus, Sulfobacillus, and some unidentified bacteria. Sulfate-reducing bacteria were found in the column bioreactor 2 weeks after inoculation, but not thereafter. This suggests they were in low abundance, even though sulfate remediation rates were significant. Instead, the population contained species similar to those previously found to utilize humic substances released from the compost material.

  • 35.
    Piletsky, Sergey
    et al.
    Institute of BioScience and Technology, Cranfield University at Silsoe, Bedfordshire, UK.
    Piletska, Elena
    Institute of BioScience and Technology, Cranfield University at Silsoe, Bedfordshire, UK.
    Bossi, Alessandra
    Deparment of Science and Technology, University of Verona, Verona, Italy.
    Turner, Nicholas
    Institute of BioScience and Technology, Cranfield University at Silsoe, Bedfordshire, UK.
    Turner, Anthony
    Cranfield University, UK.
    Surface functionalization of porous polypropylene membranes with polyaniline for protein immobilization2003In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 82, no 1, p. 86-92Article in journal (Refereed)
    Abstract [en]

    Commercial porous polypropylene membranes were chemically modified with polyaniline (PANI) using ammonium persulfate as the oxidizer. The influence of polymerization conditions on the membrane properties was studied by adsorption analysis and membrane permeability. The PANI-coated polypropylene (PANI/PP) membranes possessed high affinity toward the proteins, which can be immobilized onto the membrane surface through physical adsorption or covalent immobilization. The quantity of immobilized horseradish peroxidase (HRP) and its activity depended on the quantity and quality (oxidation level) of PANI. The storage conditions for PANI/PP membranes containing immobilized HRP were studied. HRP immobilized on the PANI/PP membrane was shown to retain 70% of its activity after 3-month storage at +5degreesC, suggesting that this material can be used for practical application, such as in bioreactors as enzyme membranes.

  • 36.
    Prytz, Ingela
    et al.
    KTH, Superseded Departments, Biotechnology.
    Sandén, Anna Maria
    KTH, Superseded Departments, Biotechnology.
    Nyström, Thomas
    Farewell, Anne
    Wahlström, Åsa
    Förberg, Cecilia
    Pragai, Zoltan
    Barer, Mike
    Harwood, Colin
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations2003In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 83, no 5, p. 595-603Article in journal (Refereed)
    Abstract [en]

    A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, beta-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the sigma(70) regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fedbatch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell.

  • 37.
    Rzhepishevska, Olena I
    et al.
    Umeå University.
    Lindström, E Börje
    Umeå University.
    Tuovinen, Olli H
    Ohio State University, USA.
    Dopson, Mark
    Umeå University.
    Bioleaching of sulfidic tailing samples with a novel, vacuum-positive pressure driven bioreactor.2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 92, no 5, p. 559-567Article in journal (Refereed)
    Abstract [en]

    This study presents a design for a novel bioreactor that uses alternating vacuum and positive pressure cycles to transfer acidic leach solution in and out of contact with finely ground sulfidic mine tailings. These tailings constitute an environmental problem that needs experimental data to support the development of management and control strategies. A conventional stirred tank bioreactor was used as a reference system. Both bioreactors were inoculated with mixed cultures of acidophilic iron and sulfur oxidizers. The rate of the bioleaching of tailings was 0.50 +/- 0.14 g Fe/L . day in the stirred tank bioreactor and 0.17 +/- 0.05 g Fe/L . day in the novel bioreactor. Microbial populations were identified in the two-bioreactor systems by analysis of 16S rRNA genes involving amplification, denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. The inoculum contained sulfur-oxidizing Acidithiobacillus caldus and Acidithiobacillus thiooxidans, iron oxidizers from the genera Leptospirillum and Ferroplasma, and a chemoorganotrophic Alicyclobacillus sp. During bioleaching of the tailings, the microbial populations in both bioreactors were similar to the inoculum culture, except that At. thiooxidans outgrew At. caldus. Sequences consistent with a Sulfobacillus sp. were amplified from both bioreactor samples although this bacterium was initially below the level of detection in the inoculum. After prolonged operation, Ferroplasma acidiphilum and an uncultured bacterium related to the CFB group were also detected in the novel bioreactor, whereas Sulfobacillus sp. was no longer detected. The novel bioreactor has potential uses in other areas of environmental biotechnology that involves periodic contact of liquids with solid substrates.

  • 38.
    Rzhepishevska, Olena
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lindström, Börje
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tuovinen, Olli H
    Department of Microbiology, Ohio State University, 484 W 12th Avenue, Columbus, Ohio 43210.
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bioleaching of sulfidic tailing samples with a novel, vacuum-positive pressure driven bioreactor.2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 92, no 5, p. 559-67Article in journal (Refereed)
    Abstract [en]

    This study presents a design for a novel bioreactor that uses alternating vacuum and positive pressure cycles to transfer acidic leach solution in and out of contact with finely ground sulfidic mine tailings. These tailings constitute an environmental problem that needs experimental data to support the development of management and control strategies. A conventional stirred tank bioreactor was used as a reference system. Both bioreactors were inoculated with mixed cultures of acidophilic iron and sulfur oxidizers. The rate of the bioleaching of tailings was 0.50 +/- 0.14 g Fe/L . day in the stirred tank bioreactor and 0.17 +/- 0.05 g Fe/L . day in the novel bioreactor. Microbial populations were identified in the two-bioreactor systems by analysis of 16S rRNA genes involving amplification, denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. The inoculum contained sulfur-oxidizing Acidithiobacillus caldus and Acidithiobacillus thiooxidans, iron oxidizers from the genera Leptospirillum and Ferroplasma, and a chemoorganotrophic Alicyclobacillus sp. During bioleaching of the tailings, the microbial populations in both bioreactors were similar to the inoculum culture, except that At. thiooxidans outgrew At. caldus. Sequences consistent with a Sulfobacillus sp. were amplified from both bioreactor samples although this bacterium was initially below the level of detection in the inoculum. After prolonged operation, Ferroplasma acidiphilum and an uncultured bacterium related to the CFB group were also detected in the novel bioreactor, whereas Sulfobacillus sp. was no longer detected. The novel bioreactor has potential uses in other areas of environmental biotechnology that involves periodic contact of liquids with solid substrates. (c) 2005 Wiley Periodicals, Inc.

  • 39.
    Sandén, Anna Maria
    et al.
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Boström, Maria
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Markland, Katrin
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 2, p. 239-247Article in journal (Refereed)
    Abstract [en]

    From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full-length protein, accumulation of the model fusion proteins Zb-MalE and Zb-MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed-batch cultivation.

    Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb-MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure.

    It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a "high" feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (<= 5 mu M), this formation is almost abolished while at a comparatively high concentration (>= 300 mu M) 50% of the total product accumulated was in the form of inclusion bodies.

  • 40.
    Sandén, Anna Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Prytz, Ingela
    KTH, Superseded Departments, Biotechnology.
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    The influence of time and method of induction on recombinant protein productivity and acetic acid formation in Escherichia coli2004In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290Article in journal (Other academic)
  • 41.
    Sandén, Anna Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Prytz, Ingela
    KTH, Superseded Departments, Biotechnology.
    Tubulekas, I.
    Förberg, C.
    Le, H.
    Hektor, A.
    Neubauer, P.
    Pragai, Z.
    Harwood, C.
    Ward, A.
    Picon, A.
    de Mattos, J. T.
    Postma, P.
    Farewell, A.
    Nyström, T.
    Reeh, S.
    Pedersen, S.
    Larsson, Gen
    KTH, Superseded Departments, Biotechnology.
    Limiting factors in Escherichia coli fed-batch production of recombinant proteins2003In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 81, no 2, p. 158-166Article in journal (Refereed)
    Abstract [en]

    Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high (mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

  • 42.
    Spens, Erika
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Defined protein and animal component-free NS0 fed-batch culture2007In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 98, no 6, p. 1183-1194Article in journal (Refereed)
    Abstract [en]

    A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a stepwise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for nonmeasurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L-1 was obtained as compared to 2.3 X 10(6) cells mL(-1) and 70 mg antibody L-1 in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6-fold increase in total cell yield, a 10.5-fold increase 6.2 in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not i significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.

  • 43.
    Sundkvist, Jan-Eric
    et al.
    Boliden Mineral AB.
    Gahan, Chandra Sekhar
    Sandström, Åke
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Modeling of ferrous iron oxidation by a Leptospirillum ferrooxidans-dominated chemostat culture2008In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 99, no 2, p. 378-389Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to evaluate a direct classical bioengineering approach to model data generated from continuous bio-oxidation of Fe2+ by a Leptospirillum ferrooxidans-dominated culture fed with either 9 g or 18 g Fe2+ L-1 under chemostat conditions (dilution rates were between 0.051 and 0.094 h-1). The basic Monod and Pirt equations have successfully been integrated in an overall mass balance procedure, which has not been previously presented in this detail for Fe2+ oxidation. To ensure chemostat conditions, it was found that the range of the dilution rates had to be limited. A too long retention time might cause starvation or non-negligible death rate whereas, a too short retention time may cause a significant alteration in solution chemistry and culture composition. Modeling of the experimental data suggested that the kinetic- and yield parameters changed with the overall solution composition. However, for respective feed solutions only minor changes of ionic strength and chemical speciation can be expected within the studied range of dilution rates, which was confirmed by thermodynamic calculations and conductivity measurements. The presented model also suggests that the apparent Fe3+ inhibition on specific Fe2+ utilization rate was a direct consequence of the declining biomass yield on Fe2+ due to growth uncoupled Fe2+ oxidation when the dilution rate was decreased. The model suggested that the maintenance activities contributed up to 90% of the maximum specific Fe2+ utilization rate, which appears close to the critical dilution rate.

  • 44.
    Svennebring, Jessica
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Skafte-Pedersen, Peder
    Bruus, Henrik
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Selective bioparticle retention and characterization in a chip-integrated confocal ultrasonic cavity2009In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 103, p. 323-328Article in journal (Refereed)
    Abstract [en]

    We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field at the chamber center, where the cells are trapped. We investigate the resonant modes in the expansion chamber and its connecting inlet channel by theoretical modeling and experimental verification during no-flow conditions. Furthermore, by triple-frequency ultrasonic actuation during continuous microfluidic sample feeding, a set of several manipulation functions performed in series is demonstrated: sample bypass-injection-aggregation and retention-positioning. Finally, we demonstrate transillumination microscopy imaging Of Ultrasonically trapped COS-7 cell aggregates.

  • 45.
    Taherzadeh, Mohammad J
    et al.
    Dept. of Chem. Reaction Engineering, Chalmers University of Technology.
    Niklasson, C.
    Dept. of Chem. Reaction Engineering, Chalmers University of Technology.
    Lidén, G.
    Dept. of Chemical Engineering II, Lund Institute of Technology.
    On-line control of fed-batch fermentation of dilute-acid hydrolyzates2000In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 69, no 3, p. 330-338Article in journal (Refereed)
    Abstract [en]

    Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%). (C) 2000 John Wiley and Sons, Inc.Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).

  • 46.
    Talebnia, F.
    et al.
    Department of Chemical Engineering and Environmental Science, Chalmers University of Technology.
    Niklasson, C.
    Department of Chemical Engineering and Environmental Science, Chalmers University of Technology.
    Taherzadeh, Mohammad J
    University of Borås, Faculty of Textiles, Engineering and Business.
    Ethanol production from glucose and dilute-acid hydrolyzates by encapsulated S. cerevisiae2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 3, p. 345-353Article in journal (Refereed)
    Abstract [en]

    The performance of encapsulated Saccharomyces cerevisiae CBS 8066 in anaerobic cultivation of glucose, in the presence and absence of furfural as well as in dilute-acid hydrolyzates, was investigated. The cultivation of encapsulated cells in 10 sequential batches in synthetic media resulted in linear increase of biomass up to 106 g/L of capsule volume, while the ethanol productivity remained constant at 5.15 (±0.17) g/L·h (for batches 6-10). The cells had average ethanol and glycerol yields of 0.464 and 0.056 g/g in these 10 batches. Addition of 5 g/L furfural decreased the ethanol productivity to a value of 1.31 (±0.10) g/L·h with the encapsulated cells, but it was stable in this range for five consecutive batches. On the other hand, the furfural decreased the ethanol yield to 0.41-0.42 g/g and increased the yield of acetic acid drastically up to 0.068 g/g. No significant lag phase was observed in any of these experiments. The encapsulated cells were also used to cultivate two different types of dilute-acid hydrolyzates. While the free cells were not able to ferment the hydrolyzates within at least 24 h, the encapsulated yeast successfully converted glucose and mannose in both of the hydrolyzates in less than 10 h with no significant lag phase. However, since the hydrolyzates were too toxic, the encapsulated cells lost their activity gradually in sequential batches. 

  • 47.
    Tomek, Kyle J.
    et al.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Saldarriaga, Carlos Rafael Castillo
    Department of Chemical and Environmental Engineering, Universidad Nacional de Colombia, Bogotá.
    Velasquez, Fernando Peregrino Cordoba
    Department of Chemical and Environmental Engineering, Universidad Nacional de Colombia, Bogotá.
    Liu, Tongjun
    DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Hodge, David
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Whitehead, Timothy A.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Removal and upgrading of lignocellulosic fermentation inhibitors by in situ biocatalysis and liquid-liquid extraction2015In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 112, no 3, p. 627-632Article in journal (Refereed)
    Abstract [en]

    Hydroxycinnamic acids are known to inhibit microbial growth during fermentation of lignocellulosic biomass hydrolysates, and the ability to diminish hydroxycinnamic acid toxicity would allow for more effective biological conversion of biomass to fuels and other value-added products. In this work, we provide a proof-of-concept of an in situ approach to remove these fermentation inhibitors through constituent expression of a phenolic acid decarboxylase combined with liquid-liquid extraction of the vinyl phenol products. As a first step, we confirmed using simulated fermentation conditions in two model organisms, Escherichia coli and Saccharomyces cerevisiae, that the product 4-vinyl guaiacol is more inhibitory to growth than ferulic acid. Partition coefficients of ferulic acid, p-coumaric acid, 4-vinyl guaiacol, and 4-ethyl phenol were measured for long-chain primary alcohols and alkanes, and tetradecane was identified as a co-solvent that can preferentially extract vinyl phenols relative to the acid parent and additionally had no effect on microbial growth rates or ethanol yields. Finally, E. coli expressing an active phenolic acid decarboxylase retained near maximum anaerobic growth rates in the presence of ferulic acid if and only if tetradecane was added to the fermentation broth. This work confirms the feasibility of donating catabolic pathways into fermentative microorganisms in order to ameliorate the effects of hydroxycinnamic acids on growth rates, and suggests a general strategy of detoxification by simultaneous biological conversion and extraction.

  • 48.
    TURNER, APF
    et al.
    Cranfield University, UK.
    ASTON, WJ
    UNIV OXFORD,INORGAN CHEM LAB,OXFORD OX1 3QR,ENGLAND; .
    HIGGINS, IJ
    UNIV OXFORD,INORGAN CHEM LAB,OXFORD OX1 3QR,ENGLAND; .
    DAVIS, G
    UNIV OXFORD,INORGAN CHEM LAB,OXFORD OX1 3QR,ENGLAND; .
    HILL, HAO
    UNIV OXFORD,INORGAN CHEM LAB,OXFORD OX1 3QR,ENGLAND; .
    APPLIED ASPECTS OF BIOELECTROCHEMISTRY - FUEL-CELLS, SENSORS, AND BIOORGANIC SYNTHESIS1982In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, p. 401-412Article in journal (Refereed)
    Abstract [en]

    n/a

  • 49.
    Wang, Jinfan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kinetics of tRNAPyl-mediated amber suppression in E. coli translation reveals unexpected limiting steps and competing reactions: Kinetics of tRNAPyl-mediated amber suppression2016In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 113, no 7, p. 1552-1559Article in journal (Refereed)
    Abstract [en]

    The utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNA(Pyl). Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified Escherichia coli translation system. Dipeptide formation from initiator fMet-tRNA(fMet) and tRNA(Pyl) charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, approximate to 30-fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF-Tu concentration, allowing measurement of K-d values of EF-Tu binding, both of which were approximate to 25-fold weaker than normal. However, binding could be increased approximate to 30-fold by lowering temperature. The fast phase rates were limited by the surprisingly approximate to 10-fold less efficient binding of EF-Tu:GTP:AA-tRNA(Pyl) ternary complex to the ribosomes, not GTP hydrolysis or peptide bond formation. Furthermore, processivity was unexpectedly impaired as approximate to 40% of the dipeptidyl-tRNA(Pyl) could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non-cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. Biotechnol. Bioeng. 2016;113: 1552-1559.

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