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  • 1.
    Abrahamsson, Louise
    Linköping University, Department of Thematic Studies, Tema Environmental Change.
    Improving methane production using hydrodynamic cavitation as pre-treatment2016Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    To develop anaerobic digestion (AD), innovative solutions to increase methane yields in existing AD processes are needed. In particular, the adoption of low energy pre-treatments to enhance biomass biodegradability is needed to provide efficient digestion processes increasing profitability. To obtain these features, hydrodynamic cavitation has been evaluated as an innovative solutions for AD of waste activated sludge (WAS), food waste (FW), macro algae and grass, in comparison with steam explosion (high energy pre-treatment). The effect of these two pre-treatments on the substrates, e.g. particle size distribution, soluble chemical oxygen demand (sCOD), biochemical methane potential (BMP) and biodegradability rate, have been evaluated. After two minutes of hydrodynamic cavitation (8 bar), the mean fine particle size decreased from 489- 1344 nm to 277- 381 nm (≤77% reduction) depending of the biomasses. Similar impacts were observed after ten minutes of steam explosion (210 °C, 30 bar) with a reduction in particle size between 40% and 70% for all the substrates treated.  In terms of BMP value, hydrodynamic cavitation caused significant increment only within the A. nodosum showing a post treatment increment of 44% compared to the untreated value, while similar values were obtained before and after treatment within the other tested substrates. In contrast, steam explosion allowed an increment for all treated samples, A. nodosum (+86%), grass (14%) and S. latissima (4%). However, greater impacts where observed with hydrodynamic cavitation than steam explosion when comparing the kinetic constant K. Overall, hydrodynamic cavitation appeared an efficient pre-treatment for AD capable to compete with the traditional steam explosion in terms om kinetics and providing a more efficient energy balance (+14%) as well as methane yield for A. nodosum.

  • 2.
    Ahlén, Gustaf
    et al.
    Recopharma AB.
    Strindelius, Lena
    Recopharma AB.
    Johansson, Tomas
    Recopharma AB.
    Nilsson, Anki
    Rrecopharma AB.
    Chatzissavidou, Nathalie
    Recopharma AB.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Holsgersson, Jan
    Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy.
    Mannosylated mucin-type immunoglobulin fusion proteins enhance antigen-specific antibody and T lymphocyte responses2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10Article in journal (Refereed)
    Abstract [en]

    Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented.OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays.Immunizations with the OVA − mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect.Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses.

  • 3. Anasontzis, George
    et al.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Challenges in ethanol production with Fusarium oxysporum through consolidated bioprocessing2014In: Bioengineered, ISSN 2165-5979, E-ISSN 2165-5987, Vol. 5, no 6, 393-395 p.Article in journal (Other (popular science, discussion, etc.))
    Abstract [en]

    Fusarium oxysporum has been reported as being able to both produce the enzymes necessary to degrade lignocellulosic biomass to sugars and also ferment the monosaccharides to ethanol under anaerobic or microaerobic conditions. However, in order to become an economically feasible alternative to other ethanol-producing microorganisms, a better understanding of its physiology, metabolic pathways, and bottlenecks is required, together with an improvement in its efficiency and robustness. In this report, we describe the challenges for the future and give additional justification for our recent publication.

  • 4.
    Anasontzis, George E.
    et al.
    National and Kapodistrian University of Athens, Chalmers University of Technology, Department of Chemical and Biological Engineering, Microbial Biotechnology Unit, Sector of Botany, Department of Biology, National and Kapodistrian University of Athens, Zografou.
    Kourtoglou, Elisavet
    National Technical University of Athens, BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Villas-Boâs, Silas G
    Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, Technical University of Denmark.
    Hatzinikolaou, Dimitris G.
    Department of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Metabolic Engineering of Fusarium oxysporum to Improve Its Ethanol-Producing Capability2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, 632Article in journal (Refereed)
    Abstract [en]

    Fusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharomyces cerevisiae is used-namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast-its low growth rate and low ethanol productivity-hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the γ-aminobutyric acid shunt, and an excess in NADPH regeneration

  • 5.
    Anasontzis, George
    et al.
    Chalmers University of Technology, Department of Chemical and Biological Engineering.
    Kourtoglou, Elisavet
    BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Mamma, Diomi
    BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Villas-Boâs, Silas G
    Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland.
    Hatzinikolaou, Dimitris
    Microbial Biotechnology Unit, Sector of Botany, Department of Biology, National and Kapodistrian University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum2014In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 13, 43Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism.RESULTS: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher xylose to biomass yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source.CONCLUSIONS: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

  • 6.
    Anderson, Mattias
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Afewerki, Samson
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Cõrdova, Armando
    Total Synthesis of Capsaicin Analogues from Lignin-Derived Compounds by Combined Heterogeneous Metal, Organocatalytic and Enzymatic Cascades in One Pot2014In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 356, no 9, 2113-2118 p.Article in journal (Refereed)
    Abstract [en]

    The total synthesis of capsaicin analogues was performed in one pot, starting from compounds that can be derived from lignin. Heterogeneous palladium nanoparticles were used to oxidise alcohols to aldehydes, which were further converted to amines by an enzyme cascade system, including an amine transaminase. It was shown that the palladium catalyst and the enzyme cascade system could be successfully combined in the same pot for conversion of alcohols to amines without any purification of intermediates. The intermediate vanillyl-amine, prepared with the enzyme cascade system, could be further converted to capsaicin analogues without any purification using either fatty acids and a lipase, or Schotten-Baumann conditions, in the same pot. An aldol compound (a simple lignin model) could also be used as starting material for the synthesis of capsaicin analogues. Using l-alanine as organocatalyst, vanillin could be obtained by a retro-aldol reaction. This could be combined with the enzyme cascade system to convert the aldol compound to vanillylamine in a one-step one-pot reaction.

  • 7.
    Andersson, Klara
    KTH, School of Biotechnology (BIO).
    Development of a shake flask method suitable for effective screening of Escherichia coli expression constructs2011Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Screening of expression constructs suitable for protein pharmaceuticals is often done in batch cultivations. But the production of the recombinant protein is made during fed-batch cultivations. The two types of cultivations are different and therefore may good expression constructs that grow poorly in batch cultivations but good in fed-batch cultivations be rejected. Therefore would it be desirable to develop a fed-batch method that can be used in shake flasks. Biosilta has developed a method where starch is broken down into glucose by an enzyme creating fed-batch conditions. This method has been tried out and analyzed during this project. It is shown that the cells grown under these conditions can be glucose limited. However, at a later stage of the cultivation the cells produce a large amount of acetate and pH is not stable. The system builds on a booster tablet which content is unknown. If the booster is not added to the cultivations the cells stop growing, this indicates that there is some other limitation than just glucose. It is also seen that the amount of protein that is produced during this fed-batch mimic cultivation is much lower than that is produced during normal batch cultivations. I would therefore not recommend EnBase as a screening method.

  • 8.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Leonov, Laura
    DuPont Industrial Biosciences.
    Jûtten, Peter
    Taros Chemicals GmbH & Co.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Papadopoulou, Adamantia
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Kletsas, Dimitris
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Ralli, Marianna
    Korres Natural Products.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions2017In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, no 8, 3213-3226 p.Article in journal (Refereed)
    Abstract [en]

    Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

  • 9.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Varriale, Simona
    Department of Chemical Sciences, University of Naples "Federico II".
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Faraco, Voncenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 15, 6519-6543 p.Article in journal (Refereed)
    Abstract [en]

    Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry

  • 10.
    Bansal, Namita
    et al.
    DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Bhalla, Aditya
    DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Pattathil, Sivakumar
    University of Georgia, Complex Carbohydrate Research Center, University of Georgia, Athens, GA.
    Adelman, Sara L.
    DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Hahn, Michael G
    University of Georgia, Complex Carbohydrate Research Center, University of Georgia, Athens, GA.
    Hodge, David
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hegg, Eric L.
    Michigan State University, DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison.
    Cell wall-associated transition metals improve alkaline-oxidative pretreatment in diverse hardwoods2016In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 18, no 5, 1405-1415 p.Article in journal (Refereed)
    Abstract [en]

    The responses of four diverse hardwoods (hybrid poplar, silver birch, hybrid aspen, and sugar maple) to alkaline hydrogen peroxide (AHP) pretreated at ambient temperature and pressure were analyzed to gain a deeper understanding of the cell wall properties that contribute to differences in enzymatic hydrolysis efficacy following alkaline-oxidative pretreatment. The enzymatic hydrolysis yields of these diverse hardwoods increased significantly with increasing the cell wall-associated, redox-active transition metal content. These increases in hydrolysis yields were directly correlated with improved delignification. Furthermore, we demonstrated that these improvements in hydrolysis yields could be achieved either through elevated levels of naturally-occurring metals, namely Cu, Fe, and Mn, or by the addition of a homogeneous transition metal catalyst (e.g. Cu 2,2′-bipyridine complexes) capable of penetrating into the cell wall matrix. Removal of naturally-occurring cell wall-associated transition metals by chelation resulted in substantial decreases in the hydrolysis yields following AHP pretreatment, while re-addition of metals in the form of Cu 2,2′-bipyridine complexes and to a limited extent Fe 2,2′-bipyridine complexes prior to pretreatment restored the improved hydrolysis yields. Glycome profiles showed improved extractability of xylan, xyloglucan, and pectin epitopes with increasing hydrolysis yields for the diverse hardwoods subjected to the alkaline-oxidative pretreatment, demonstrating that the strength of association between cell wall matrix polymers decreased as a consequence of improved delignification

  • 11.
    Bauer, Fredric
    et al.
    Lund University.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Hulteberg, Christian
    Lund University.
    Lundgren, Joakim
    Luleå University of Technology, Department of Engineering Sciences and Mathematics, Energy Science.
    Mesfun, Sennai
    Luleå University of Technology, Department of Engineering Sciences and Mathematics, Energy Science.
    Nilsson, Robert
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Wännström, Sune
    SP Technical Research Institute of Sweden.
    Comparative system analysis of carbon preserving fermentations for biofuels production2013Report (Refereed)
  • 12.
    Bhalla, Aditya
    et al.
    DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Bansal, Namita
    DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Stoklosa, Ryan J.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Fountain, Mackenzie
    Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing.
    Ralph, John P.
    DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison.
    Hodge, David
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hegg, Eric L.
    Michigan State University, DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing.
    Effective alkaline metal-catalyzed oxidative delignification of hybrid poplar2016In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 9, 34Article in journal (Refereed)
    Abstract [en]

    BackgroundStrategies to improve copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment of hybrid poplar were investigated. These improvements included a combination of increasing hydrolysis yields, while simultaneously decreasing process inputs through (i) more efficient utilization of H2O2 and (ii) the addition of an alkaline extraction step prior to the metal-catalyzed AHP pretreatment. We hypothesized that utilizing this improved process could substantially lower the chemical inputs needed during pretreatment.ResultsHybrid poplar was pretreated utilizing a modified process in which an alkaline extraction step was incorporated prior to the Cu-AHP treatment step and H2O2 was added batch-wise over the course of 10 h. Our results revealed that the alkaline pre-extraction step improved both lignin and xylan solubilization, which ultimately led to improved glucose (86 %) and xylose (95 %) yields following enzymatic hydrolysis. An increase in the lignin solubilization was also observed with fed-batch H2O2 addition relative to batch-only addition, which again resulted in increased glucose and xylose yields (77 and 93 % versus 63 and 74 %, respectively). Importantly, combining these strategies led to significantly improved sugar yields (96 % glucose and 94 % xylose) following enzymatic hydrolysis. In addition, we found that we could substantially lower the chemical inputs (enzyme, H2O2, and catalyst), while still maintaining high product yields utilizing the improved Cu-AHP process. This pretreatment also provided a relatively pure lignin stream consisting of ≥90 % Klason lignin and only 3 % xylan and 2 % ash following precipitation. Two-dimensional heteronuclear single-quantum coherence (2D HSQC) NMR and size-exclusion chromatography demonstrated that the solubilized lignin was high molecular weight (Mw ≈ 22,000 Da) and only slightly oxidized relative to lignin from untreated poplar.ConclusionsThis study demonstrated that the fed-batch, two-stage Cu-AHP pretreatment process was effective in pretreating hybrid poplar for its conversion into fermentable sugars. Results showed sugar yields near the theoretical maximum were achieved from enzymatically hydrolyzed hybrid poplar by incorporating an alkaline extraction step prior to pretreatment and by efficiently utilizing H2O2 during the Cu-AHP process. Significantly, this study reports high sugar yields from woody biomass treated with an AHP pretreatment under mild reaction conditions.

  • 13.
    Blomqvist, Johanna
    et al.
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Eberhard, Thomas
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Schnürer, Johan
    Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Passoth, Volkmar
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Fermentation characteristics of Dekkera bruxellensis strains2010In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 87, no 4, 1487-1497 p.Article in journal (Refereed)
    Abstract [en]

    The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)(-1) and growth rates varied from 0.037 to 0.050 h(-1). The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0-13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72-84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.

  • 14.
    Bonturi, Nemailla
    et al.
    Department of Materials and Bioprocess Engineering, School of Chemical Engineering, State University of Campinas.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Nilsson, Robert
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Miranda, Everson Alves
    Department of Materials and Bioprocess Engineering, School of Chemical Engineering, State University of Campinas.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Single Cell Oil Producing Yeasts Lipomyces starkeyi and Rhodosporidium toruloides: Selection of Extraction Strategies and Biodiesel Property Prediction2015In: Energies, ISSN 1996-1073, E-ISSN 1996-1073, Vol. 8, no 6, 5040-5052 p.Article in journal (Refereed)
    Abstract [en]

    Single cell oils (SCOs) are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi. Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi). Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi). Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME) composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species.

  • 15.
    Boström, Karin
    KTH, School of Chemical Science and Engineering (CHE).
    Tillförsel av jäst till SSF i industriell skala2011Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [sv]

    Användning av etanol som drivmedel och en efterfråga på gröna kemikalier driver utvecklingen av bioetanol framåt. Etanolpiloten, SEKAB, i Örnsköldsvik är en av få anläggningar i världen med kompetens och kunskap att producera bioetanol baserat på lignocellulosa. På senare tid har det dock uppstått problem vid etanolframställningen på grund av att en del jästodlingar blivit kontaminerade av bakterier vilket lett till ett sämre utbyte av biomassa och etanol. Det huvudsakliga syftet med detta examensarbete var att ta reda på orsaken till dessa misslyckade jästodlingar.

     

    Examensarbetet delades upp i två huvudsakliga problemområden. Förutom orsaken till de kontaminerade odlingarna studerades även funktionen hos en ny jäststam, Saccaromyces cerevisiae torrjäst, i syfte att undersöka om det finns bättre alternativ till den jäststam som används i etanopiloten i nuläget.

     

    En specialstudie av rengöringen av odlingstankar och ledningar i etanolpiloten utfördes i syfte att kartlägga var i utrustningen som infektionsrisken är som störst. Försöken påvisade att det huvudsakliga problemet kan lokaliseras till den största jästodlingstanken. Där befinner sig jästen under en längre tid i en miljö som är gynnsam för tillväxt av både jäst och bakterier. En annan orsak till de infekterade odlingarna är att rengöringen av utrustningen inte har skett på rätt sätt, samt att temperaturen hos tvättkemikalierna har varit för låg. En viktig slutsats är därför att bättre rutiner vid hanteringen av jästodlingsutrustningen samt att större noggrannhet i samband med rengöringen bör eftersträvas.

     

    En bidragande orsak till de infekterade odlingarna kan också härröra från uppodlingsprocessen av ympjäst som i dagens läge sker på laboratorium. Genom att använda en stam av S. cerevisiae som köps in i frystorkad form kan flera steg i jästodlingsprocessen elimineras. Det både förkortar odlingsprocessen och minskar infektionsrisken. S. cerevisiae torrjäst undersöktes både i laboratorium och i etanolpiloten. Tre olika odlingsskalor användes, skakflaskor (250 ml), labfermentorer (3 l) och pilotskala (10m3). Försöken påvisar höga utbyten av både biomassa och etanol. För att kunna hålla nere produktionskostnaderna för etanolframställningen är det viktigt att jästen som används går att odla på det hydrolysat som produceras vid förbehandlingen av råvaran. Försök i pilotskala visar på lovande resultat vid uppodling av S. cerevisiae torrjäst när hela 70 % av sockerkällan kommer från hydrolysat. Ytterligare utvärdering och optimering av odlingsprocessen samt en ekonomisk jämförelse mellan de tillgängliga jäststammarna krävs dock innan S. cerevisiae torrjäst eventuellt kan användas kontinuerligt i pilotskala. 

  • 16.
    Charavgi, Maria-Despoina
    et al.
    National Technical University of Athens.
    Dimarogona, Maria
    National Technical University of Athens.
    Topakas, Evangelos
    National Technical University of Athens.
    Christakopoulos, Paul
    Chrysina, Evangelia D.
    National Hellenic Research Foundation, Athens.
    The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst2013In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 69, no 1, 63-73 p.Article in journal (Refereed)
    Abstract [en]

    The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-[beta]-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an [alpha]/[beta]-hydrolase fold with a three-layer [alpha][beta][alpha]-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.

  • 17.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Perfusion Processes2015In: Animal Cell Culture / [ed] Mohamed Al-Rubeai, Switzerland Springer: Springer , 2015, 407-443 p.Chapter in book (Refereed)
    Abstract [en]

    The interest for perfusion is increasing nowadays. This new focus has emerged from a synergy of a demand for disposable equipment and the availability of robust cell separation device, as well as the need for higher flexibility and lower investment cost. The cell separation devices mostly used today are based on filtration, i.e. alternating flow filtration, tangential flow filtration, spin-filter, or acceleration/gravity, i.e. inclined settler, centrifuge, acoustic settler. This paper gives an introduction to the basic concepts of perfusion and its practical implementation. It reviews the actual cell separation devices and describes the approaches used in the field to develop and optimize the perfusion processes.

  • 18.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Diana, Rafael
    Ge Healthcare Bio-sciences Ab.
    Kaisermayer, Christian
    Ge Healthcare Bio-sciences Ab.
    Lindskog, Eva
    Ge Healthcare Bio-sciences Ab.
    Robinson, Craig
    Ge Healthcare Bio-sciences Ab.
    Rucker, Jimmie L.
    Ge Healthcare Bio-sciences Ab.
    Walsh, Kieron D.
    Ge Healthcare Bio-sciences Ab.
    Flexible bag for cultivation of cells2011Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    An inflatable bioreactor bag for cell cultivation, which comprising a top and a bottom sheet of flexible material, joined together to form two end edges and two side edges, wherein one baffle or a plurality of baffles extend from the bottom sheet in a region where the shortest distance to any one of the two end edges is higher than about one fourth of the shortest distance between the two end edges.

  • 19.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Clincke, Marie-Francoise
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Very High Cell Density in Perfusion of CHO Cells by ATF, TFF, Wave Bioreactor, and/or CellTank Technologies: Impact of Cell Density and Applications2014In: Continuous Processing in Pharmaceutical Manufacturing / [ed] Ganapathy Subramanian, Germany Weinheim: Wiley-VCH Verlagsgesellschaft, 2014, 339-356 p.Chapter in book (Other academic)
  • 20.
    Chotteau, Véronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hjalmarsson, Håkan
    KTH, School of Electrical Engineering (EES), Automatic Control. KTH, School of Electrical Engineering (EES), Centres, ACCESS Linnaeus Centre.
    Tuning of dissolved oxygen and pH PID control parameters in large scale bioreactor by lag control2012In: Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT), 2012, 327-330 p.Conference paper (Refereed)
    Abstract [en]

    A method has been developed to tune the DO and pH controller PID parameters for pilot / large scale mammalian cultivation. Our approach is to identify a model of the variable to be controlled (e.g. DO, pH) and to design several possible PID controllers based on this model. The controllers were first tested in computer simulations, followed by wet simulation and finally the best controller was tested on the real process. The approach is developed for the tuning of the DO controller of a 50 L bioreactor using microbubble continuous oxygen flow. The method, called lag control here, is based on a lead lag control design using Bode analysis where the prediction part is omitted. Experiments show that the approach results in a highly satisfactory DO control. The oxygen microbubbles were almost completely consumed before reaching the liquid surface so the oxygen flow used to maintain the DO gave an excellent indication of the cellular oxygen consumption. The control system was robust against all the perturbations, i.e. cell growth, cell bleed, addition of air-saturated fresh medium, DO set point change and a second gas sparger used to strip out the carbon dioxide. This approach was also successfully used for the tuning of a 400 L bioreactor DO controller and pH controller.

  • 21.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: SOUnd-DRIven BIOtechnology (VR)2014Other (Other (popular science, discussion, etc.))
  • 22.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Bioconversion of Recalcitrant Polysaccharides by Novel Oxidative Biocatalysts for the production of ethanol and other novel products (SWEDISH ENERGY AGENCY)2014Other (Other (popular science, discussion, etc.))
  • 23.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Efficient bioconversion of forest biomass insoluble polyesters with a potential use in lignocellulosic feedstock biorefineries (FORMAS)2014Other (Other (popular science, discussion, etc.))
  • 24.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Optimized esterase biocatalysts for cost-effective industrial production (EU-FP7)2014Other (Other (popular science, discussion, etc.))
  • 25.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Synthesis of biological active compounds using carbohydrate esterases as biocatalysts2014In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, no Supplement, S90-S91 p.Article in journal (Refereed)
    Abstract [en]

    Various fungal and bacterial carbohydrate esterases represent appealing biocatalysts that have the ability not only to deconstruct plant biomass but also to modify compounds with a potential use in food, cosmetic and pharmaceutical industries. Feruloyl esterases (FAEs, E.C. 3.1.1.73) have been proved promising candidates for the enzymatic synthesis of antioxidants allowing more flexible process configurations. Among the advantages they provide are use of lower temperatures (50-60 °C) comparing to the counterpart chemical process (150οC), one step production of one product instead of mixtures and no need of by-product and catalyst residues removal in order to produce clean and high quality substances. Glucuronoyl esterase (GE) synthetic ability needs to be explored towards the production of alkyl branched glucuronic acid derivatives which are non-ionic surfactants and have good surface properties, including biodegradability. In addition, due to their tastelessness, non skin-irritation and non toxicity, these bioactive compounds find diverse uses in the cosmetic and pharmaceutical industries.Aim of this work is the development of competitive and eco-friendly bioconversions based on transesterification reactions catalyzed by FAEs and GEs, for the production of molecules with antioxidant activity, such as phenolic fatty and sugar esters. The synthesis of four biological active compounds (prenyl ferulate, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, and glyceryl ferulate) was evaluated using recombinant FAEs from Myceliopthora thermophila and Fusarium oxysporum, while the synthesis of benzyl D-glucuronate and prenyl-D-glucuronate was evaluated using recombinant GEs from M. thermophila. All reactions were carried out in ternary systems of n-hexane/alcohol/water forming surfactantless microemulsions.

  • 26.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Organosolv Biomass Pretreatment for Flexible Fuel Production (Swedish Energy Agency)2016Other (Other (popular science, discussion, etc.))
  • 27.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: BIOcatalytic Carbon Capture and Conversion of steel flue gas to liquid hydrocarbons (FORMAS)2016Other (Other (popular science, discussion, etc.))
  • 28.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Editorial note: Advances in enzymology and enzyme engineering2012In: Computational and Structural Biotechnology Journal, ISSN 2001-0370, Vol. 2, no 3, e201209001Article in journal (Refereed)
  • 29.
    Danielsson, Per-Erik
    Linköping University, Department of Electrical Engineering. Linköping University, The Institute of Technology.
    Implementations of the Convolution Operation1982Report (Other academic)
    Abstract [en]

    The first part of this article surveys a large number of implementations of the convolution operation (which is also known as the sum-of-products, the inner product) based on a systematic exploration of index permutations. First we assume a limited amount of parallelism in the form of an adder. Next, multipliers and RAM:s are utilized. The so called distributed arithmetic follows naturally from this approach.

    The second part brings in the concept of pipelining on the bitlevel to obtain high throughput convolvers adapted for VLSI-design (systolic arrays). The serial/parallel multiplier is analyzed in a way that unravels a vast amount new variations. Even more interesting, all these new variations can be carried over to serial/parallel convolvers. These novel devices can be implemented as linear structures of identical cells where the multipliers are embedded at equidistant intervals.

  • 30.
    de Maré, L
    et al.
    Lund Institute of Technology, Sweden.
    Velut, S
    Lund Institute of Technology, Sweden.
    Ledung, Erika
    Mälardalen University, Department of Biology and Chemical Engineering.
    Cimander, C
    Novozymes Biopharma AB, Lund, Sweden .
    Norrman, Bo
    Mälardalen University, Department of Biology and Chemical Engineering.
    Karlsson, E
    Lund Institute of Technology, Lund, Sweden.
    Holst, O
    Lund Institute of Technology, Lund, Sweden.
    Hagander, P
    Lund Institute of Technology, Sweden.
    A cultivation technique for E. coli fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor2005In: Biotechnology Letters, ISSN 0141-5492, Vol. 27, no 14, 983-990 p.Article in journal (Refereed)
    Abstract [en]

    A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.

  • 31.
    Dimarogona, M
    et al.
    Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation.
    Topakas, E
    School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Chrysina, E.D
    Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation.
    The structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the catalytic cleft2012In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 68, no 7, 735-742 p.Article in journal (Refereed)
    Abstract [en]

    Xylanase enzymes have been the focus of considerable research in recent decades owing to their extensive use in a variety of biotechnological applications. Previous structural studies of a number of GH10 xylanases revealed that all GH10 family members have the (β/α)8-barrel fold and their catalytic site is conserved. The structure of a new GH10 xylanase from Fusarium oxysporum (FoXyn10a) was determined at 1.94 Å resolution from crystals belonging to the tetragonal space group P41212 with five molecules per asymmetric unit. Comparison of the structure of FoXyn10a with previously determined structures of GH10 family members indicated that most of the differences were located in the loop regions between the ordered secondary-structure elements of the barrel, as expected. However, alignment of FoXyn10a with sequence and structural homologues denoted an atypically long loop connecting strand β6b and helix 6 that was only present in one other GH10 xylanase, the structure of which is not known. This structural feature may be of functional importance, with potential implications in the catalytic efficiency of the enzyme.

  • 32.
    Dimarogona, Maria
    et al.
    National Technical University of Athens, Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Nikolaivits, Efstratios
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Kanelli, Maria
    National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sandgren, Mats
    Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences (SLU).
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential2015In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, no 11, 2308–2317- p.Article in journal (Refereed)
    Abstract [en]

    BackgroundCutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers.MethodsA cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9 Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues.ResultsThe highest production of recombinant FoCut5a was achieved using periplasmic expression at 16οC. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40οC and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C2, C4, C12), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers.ConclusionsThe first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers.General significanceFoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.

  • 33.
    Dimarogona, Maria
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Topakas, Evangelos
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Recalcitrant polysaccharide degradation by novel oxidative biocatalysts2013In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 97, no 19, 8455–8465- p.Article in journal (Refereed)
    Abstract [en]

    The classical hydrolytic mechanism for the degradation of plant polysaccharides by saprophytic microorganisms has been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases (LPMOs). LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals. They act on recalcitrant polysaccharides by a combination of hydrolytic and oxidative function, generating oxidized and non-oxidized chain ends. They are copper-dependent and require molecular oxygen and an external electron donor for their proper function. In this review, we present the recent findings concerning the mechanism of action of these oxidative enzymes and identify issues and questions to be addressed in the future

  • 34. Dimarogona, Maria
    et al.
    Topakas, Evangelos
    Christakopoulos, Paul
    Cellulose degradation by oxidative enzymes2012In: Computational and Structural Biotechnology Journal, ISSN 2001-0370, Vol. 2, no 3Article in journal (Refereed)
    Abstract [en]

    Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

  • 35.
    Dimarogona, Maria
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Topakas, Evangelos
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Olsson, Lisbeth
    Chalmers University of Technology.
    Christakopoulos, Paul
    Lignin boosts the cellulase performance of a GH-61 enzyme from Sporotrichum thermophile2012In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 110, 480-487 p.Article in journal (Refereed)
    Abstract [en]

    An enzyme belonging to the glycoside hydrolase family 61 from the thermophilic fungus Sporotrichum thermophile, was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter. The enzyme hydrolyzed barley β-glucan, carboxymethyl cellulose, lichenan, wheat arabinoxylan and birchwood xylan showing optimal activity at pH 8 and 65 °C. A 2:1 mixture of Celluclast 1.5 L and StCel61a was capable of increasing the degree of spruce conversion by 42%. The use of substrates with varying lignin content permitted the detection of a dependence of the enhancing capacity of StCel61a on the radical scavenging capacity of the different lignocellulosics. In the presence of a reductant, StCel61a boosted the efficiency of a mixture of purified cellulases (EGII, CBHI, β-GLUC) by 20%. The synergistic activity exhibited by StCel61a and its dependence on reducing substances provide guidelines for process design towards the production of economically viable bioethanol.

  • 36.
    El-Gamal, Rehab
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Nikolaivits, Efstratios
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Zervakis, Georgios I.
    Agricultural University of Athens, Laboratory of General and Agricultural Microbiology.
    Abdel-Maksoud, Gomaa
    Conservation Department, Faculty of Archaeology, Cairo University, Giza.
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    The use of chitosan in protecting wooden artifacts from damage by mold fungi2016In: Electronic Journal of Biotechnology, ISSN 0717-3458, E-ISSN 0717-3458, Vol. 24, 70-78 p.Article in journal (Refereed)
    Abstract [en]

    Background

    Many buildings in Egypt e.g. museums, mosques and churches, do not possess controlled environments for minimizing the risks of damage of wooden artifacts due to the growth of fungi. Fungal damage usually appears as change in wood color, appearance of stains, and sometimes deformation of wooden surfaces. In this study we focused on the effect that some fungi exert on the properties of wooden artifacts and evaluated the effectiveness of different concentrations of chitosan on their protection against damage by mold fungi.

    Results

    Samples were collected from different monuments and environments, and fungi growing on them were isolated and identified. The isolated Penicillium chrysogenum, Aspergillus flavus and A. niger strains were used for the infestation of new pitch pine samples. The results revealed that the lightness of samples infected with any of the tested fungi decreased with increasing incubation times. XRD analysis showed that the crystallinity of incubated samples treated individually with the different concentrations of chitosan was lower than the crystallinity of infected samples. The crystallinity index measured by the first and the second method decreased after the first and second months but increased after the third and fourth months. This may due to the reducing of amorphous part by enzymes or acids produced by fungi in wooden samples.

    Conclusions

    The growth of fungi on the treated wood samples decreased with increasing the concentration of chitosan. Hence, it was demonstrated that chitosan prevented fungal growth, and its use could be recommended for the protection of archaeological wooden artifacts.

    Keywords

    • archaeological wood;
    • Chitosan protection;
    • crystallinity index;
    • damage of wooden artifacts;
    • filamentous fungi;
    • FTIR;
    • Fungal damage;
    • prevention of fungal growth;
    • UV spectrophotometry;
    • wood deterioration;
    • XRD
  • 37.
    Enman, Josefine
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hodge, David
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Growth promotive conditions for enhanced eritadenine production during submerged cultivation of Lentinus edodes2012In: Journal of chemical technology and biotechnology (1986), ISSN 0268-2575, E-ISSN 1097-4660, Vol. 87, no 7, 903-907 p.Article in journal (Refereed)
    Abstract [en]

    Background: Mycelium of the medicinal mushroom shiitake, Lentinus edodes, is a potential source for production of the blood cholesterol reducing compound eritadenine. To increase the mycelial biomass and in turn the production of eritadenine, a potential growth promoting substance in the form of a water extract of distillers dried grains with solubles (DDGS) was added to the culture media. Results: The hot water extract of DDGS was shown to considerably increase the growth of shiitake mycelia in bioreactor cultivations; the mycelial yield was 2-3 times higher than in the control, and the highest final biomass concentration obtained was 3.4 g L -1. Further, by using shake flask cultures as inoculums the bioreactor cultivation time could be reduced by 1 week for some of the experiments. The highest final titer of eritadenine in the present study was 25.1 mg L -1, which was about 2 times higher than in the control, and was also obtained when a water extract of DDGS was added to the culture medium. Conclusion: It was demonstrated that a water extract of DDGS promoted the growth of shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production

  • 38.
    Faisal, Abrar
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Zarebska, Agata
    Saremi, Pardis
    Korelskiy, Danil
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Ohlin, Lindsay
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Hedlund, Jonas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Grahn, Mattias
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    MFI zeolite as adsorbent for selective recovery of hydrocarbons from ABE fermentation broths2014In: Adsorption, ISSN 0929-5607, E-ISSN 1572-8757, Vol. 20, no 2-3, 465-470 p.Article in journal (Refereed)
    Abstract [en]

    1-Butanol and butyric acid are two interesting compounds that may be produced by acetone, butanol, and ethanol fermentation using e.g. Clostridium acetobutylicum. The main drawback, restricting the commercialization potential of this process, is the toxicity of butanol for the cell culture resulting in low concentrations of this compound in the broth. To make this process economically viable, an efficient recovery process has to be developed. In this work, a hydrophobic MFI type zeolite with high silica to alumina ratio was evaluated as adsorbent for the recovery of butanol and butyric acid from model solutions. Dual component adsorption experiments revealed that both butanol and butyric acid showed a high affinity for the hydrophobic MFI zeolite when adsorbed from aqueous model solutions. Multicomponent adsorption experiments using model solutions, mimicking real fermentation broths, revealed that the adsorbent was very selective to the target compounds. Further, the adsorption of butyric and acetic acid was found to be pH dependent with high adsorption below, and low adsorption above, the respective pKa values of the acids. Thermal desorption of butanol from MFI type zeolite was also studied and a suitable desorption temperature was identified.

  • 39.
    Gerlach, Inga
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Operator Training Simulators towards Industrial Biotechnology2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Biotechnological processes are carried out by applying advanced and demanding process strategies. Thus, fundamental understanding and knowledge about the applied microorganism is required to achieve high productivity and profitability. Therefore, computer-based simulation has been integrated into training methodologies for supplementing and expanding academic education and industrial operator training, in order to provide more effective as well as cost- and time-efficient training.

    Operator training simulators (OTS) are computer-based training tools that represent the real laboratory or plant within an interactive, virtual environment. Trainees can learn about cause-and-effect relationships during the virtual process to improve process understanding. Also, trainees and operators get the opportunity to develop, maintain and improve their operational skills. The virtual environment allows delayed operational actions and operational faults, as they are not interfering with the real process. Acceleration of the simulation speed makes training more effective. For instance, different procedures such as start-up and shut-down can be trained in a limited period of training time. Incident and trouble-shooting training can be realized in a safe, virtual environment without exposing the operator to potential risks. So far, OTS are mainly used in military, aviation, medicine and chemical industries. However, there are only a few examples where OTS are applied in biotechnological process applications.

    The aim of this thesis is to expose the benefits of using OTS for biotechnological process applications in academic education and industrial operator training. For this purpose, the feasibility of developing and applying OTS was investigated and discussed. Three OTS for the production of bio-ethanol and recombinant protein production were developed in order to investigate the benefits and their requirements as well as to assess the training effectiveness of these. To visualize the technical system including equipment and distributed control system (DCS), graphical user interfaces (GUIs) were designed, allowing the user to interact with the simulator. Mathematical models were developed and implemented in the OTS to ensure dynamic simulation of the process, where cause-and-effect relationships are realistically described. The essential part of an OTS for biotechnological process applications is the embedded sub-model which describes the biological production system. It is important that the cell growth behaviour within a bioreactor can be  simulated with sufficient accuracy in order to ensure high training effectiveness.

    In the assessment of the training effectiveness in academic education, it was observed that the students’ capability of understanding and controlling complex biotechnological processes improved after OTS training. Students showed better performance in laboratory experiments after participating in OTS pre-training. Moreover, the development of an OTS for an industrial, large-scale bio-ethanol plant illuminates the needs of OTS in biotechnological process industries. It was shown how the conceptual design methodology can be applied in designing an OTS, based on training needs from the industrial user perspective. Also, construction of the OTS including the formulation of mathematical models, the model structure which is embedded in the OTS and the design of the GUIs is covered in this thesis. Whether the use of the OTS will become cost-effective and assure maximum transfer of training depends on long term evaluation. However, application and evaluation of the developed OTS reveal the benefits and training effectiveness of these systems. The findings of the thesis might considerably facilitate the introduction of OTS into academic education and industrial operator training for biotechnological process applications.

  • 40.
    Gerlach, Inga
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, The Institute of Technology. University of Applied Sciences, Bremen, Germany.
    Bruening, Simone
    University of Applied Sciences, Bremen, Germany .
    Gustavsson, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, The Institute of Technology.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, The Institute of Technology.
    Hass, Volker C.
    University of Applied Sciences Furtwangen, Villingen-Schwenningen, Germany .
    Operator training in recombinant protein production using a structured simulator model2014In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 177, 53-59 p.Article in journal (Refereed)
    Abstract [en]

    Model-based operator training simulators ( OTS) could be powerful tools for virtual training of operational procedures and skills of production personnel in recombinant protein processes. The applied model should describe critical events in the bioprocess so accurately that the operators ability to observe and alertly act upon these events is trained with a high degree of efficiency. In this work is shown how this is accomplished in a structured multi-compartment model for the production of a recombinant protein in an Escherichia coli fed-batch process where in particular the induction procedure, the stress effects and overflow metabolism were highlighted. The structured model was applied on the OTS platform that virtually simulated the operational bioreactor procedures in real or accelerated time. Evaluation of training using the model-based OTS showed that trained groups of operators exhibited improved capability compared with the untrained groups when subsequently performing real laboratory scale cultivations. The results suggest that this model-based OTS may provide a valuable resource for enhancing operator skills in large scale recombinant protein manufacturing.

  • 41.
    Gerlach, Inga
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Hass, Volker C.
    Hochschule Furtwangen, University of Applied Sciences Furtwangen, Germany.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Conceptual Design of an Operator Training Simulator for a Bio-Ethanol Plant2015In: Processes, ISSN 2227-9717, Vol. 3, no 3, 664-683 p.Article in journal (Refereed)
    Abstract [en]

    Conceptual design methodology for the configuration and procedural training with an operating training simulator (OTS) in a large-scale plant for commercial bio-ethanol production is described. The aim of the study is to show how the methodology provides a powerful way for finding the best configuration and training structure of the OTS before constructing and implementing the software of the OTS. The OTS principle, i.e., to use a computer-based virtual representation of the real process plant intended for efficient training of process operators, has long since been applied in aviation and process industries for more efficient and flawless operations. By using the conceptual design methodology (sometimes referred to as bio-mechatronics) a variety of OTS configurations with this capacity was generated. The systematic approach of for targeting the users’ (i.e., the plant management and process operators) needs resulted in better understanding and efficiency in training of hands-on skills in operating the plant. The training included general standard operating procedures for running the plant under normal operation conditions with different starch materials, handling of typical frequent disturbances as well as acting in situations not described in the standard operation procedures and applying trouble-shooting

  • 42.
    Gerlach, Inga
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering. Department of Environmental- and Bio-Technology, Hochschule Bremen University of Applied Sciences Bremen, Bremen, Germany.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Hass, Volker C.
    Faculty of Medical and Life Sciences, Hochschule Furtwangen University of Applied Sciences Furtwangen, Germany.
    Operator training simulation for integrating cultivation and homogenisation in protein production2015In: Biotechnology Reports, ISSN 2215-017X, Vol. 6, 91-99 p.Article in journal (Refereed)
    Abstract [en]

    Operating training simulators (OTS) are virtual simulation tools used for training of process operators in industry in performing procedures and running processes. Based on structured mathematical models of the unit operations of a bioprocess an OTS can train a process operator by visualising changing conditions during the process, allow testing operator actions, testing controller settings, experience unexpected technical problems and getting practice in using prescribed standard procedures for a plant. This work shows the design of an OTS where two sequential steps of a recombinant protein production process, a fed-batch cultivation and a high-pressure homogenisation, are integrated. The OTS was evaluated on a user test group and showed that the OTS promoted and developed their understanding of the process, their capability to identify parameters influencing process efficiency and the skills of operating it.

  • 43.
    Gerlach, Inga
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, The Institute of Technology. University of Applied Sciences, Bremen, Germany.
    Tholin, Sören
    Lantmännen Reppe AB, Lidköping, Sweden.
    Hass, Volker C.
    Hochschule Furtwangen University of Applied Sciences Furtwangen, Villingen- Schwenningen, Germany.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, The Institute of Technology.
    Operator training simulator for an industrial bio-ethanol plant2015Manuscript (preprint) (Other academic)
    Abstract [en]

    The development of a software-based operator training simulators (OTS) for an industrial bioethanol plant is described. OTS are used in the process industry for training of process operators since several years but few examples are reported for their use in biochemical and biotechnological industry. This study describes the implementation of an OTS at a large-scale bio-plant producing ethanol. The study includes the implementation of models and graphical user interfaces of the OTS as well as the experience of the operator training with it. The OTS encompasses the whole process, i.e. the sections for hydrolysis, fermentation, separation and distillation. The implementation was carried out on the commercial process control software WinErs. The graphical user interfaces, including all essential distributed control systems of the plant, show high fidelity with the real system. Dynamic process models were able to efficiently train operators in running and controlling the process under standard, start-up, shut-down and critical conditions. The models show a sufficient accuracy and robustness at different simulation speeds. Experiences of applying the OTS in the industrial operator environment of the large-scale plant implies that the OTS can be a useful tool for making operator training more time- and cost-efficient in the biochemical and biotechnological industry.

  • 44.
    Ginesy, Mireille
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Production of L-arginine by genetically modified Escherichia coli2015Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the recent years, the demand for environmental friendly produced L-arginine has risen with the increasing number of applications for this amino acid in pharmaceuticals, nutraceuticals, cosmetics, animal feed and fertilizers. Members of the Corynebacteriaceae family are usually used for microbial L-arginine production. However, Escherichia coli present the advantage of being able to utilize a wider range of substrates, including pentose sugars found in lignocellulosic feedstocks. The present thesis illustrates the first steps in the development of a sustainable process to produce L-arginine using E. coli. It starts with the constructions of a L-arginine overproducing strain, followed by optimization of the nitrogen supply for the fermentations.The first part of this thesis aimed at engineering an E. coli train able to produce high level of L-arginine. Mutations on key genes of the L-arginine biosynthesis pathway were step-wisely done. The mutants obtained at each step were tested in bioreactor fermentations to assess the effect of each genetic modification. The final strain was able to produce almost 12 g/l during fermentation, at a productivity of 0.24 g/l/h. In comparison the starting strain, E. coli K12 C600, was not able to excrete any L-arginine. To minimize nitrogen wastes and optimize the L-arginine production the impact of different nitrogen sources and concentration were investigated. It was shown that while ammonium phosphate dibasic was the most potent nitrogen source during cultivation on complex medium, all the sources were equivalent with minimal media; this probably reflected the phosphate deficiency of the complex medium used. In fermentation on minimal medium, a carbon to nitrogen ratio of 5 was demonstrated to be the most suitable, yielding up to 4.5 g/l L-arginine. At this ratio, both glucose and the nitrogen source were completely utilized during fermentation.

  • 45.
    Ginesy, Mireille
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Belotserkovsky, Jaroslav
    Department of Molecular Biosciences Wenner-Gren institute, Stockholm University.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Isaksson, Leif
    Department of Molecular Biosciences Wenner-Gren institute, Stockholm University.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, 29Article in journal (Refereed)
    Abstract [en]

    BackgroundArginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. ResultsFirstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. ConclusionsIn this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.

  • 46.
    Gowtham, Yogender Kumar
    et al.
    Clemson University.
    Miller, Kristen P.
    Clemson University.
    Hodge, David
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Henson, J. Michael
    Clemson University.
    Harcum, Sarah W.
    Clemson University.
    Novel two-stage fermentation process for bioethanol production using Saccharomyces pastorianus2014In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 30, no 2, 300-310 p.Article in journal (Refereed)
    Abstract [en]

    Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose-metabolizing pathways. In this study, a novel two-stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non-Saccharomyces microorganism and coupled to a glucose-utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two-stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae-based fermentations.

  • 47.
    Graiver, D.
    et al.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Dacomba, R.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Khawali, M.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Jaros, Adam
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Narayan, R.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Steel-corrosion inhibitors derived from soybean oil2012In: Journal of the American Oil Chemists Society, ISSN 0003-021X, E-ISSN 1558-9331, Vol. 89, no 10, 1895-1903 p.Article in journal (Refereed)
    Abstract [en]

    Soybean oil derivatives containing a Schiff-base (SOS-B) were prepared and evaluated as microbial corrosion inhibitors against sulfate-reducing bacteria using the gram-positive Desulfosporosinus orientis bacteria as a representative bacterium. These SOS-B compounds were also found to be excellent inhibitors against acidic corrosion of carbon steel. These soybean oil derivatives were prepared by ozonation of soybean oil to yield aldehyde functional intermediates which were then reacted with benzylamine to produce a mixture of imine functional triglycerides and linear compounds. The structure of these soy-based derivatives was confirmed by FTIR and NMR. It was found that the addition of these SOS-B compounds to D. orientis culture provided a complete inhibition of this bacterium. Furthermore, almost no corrosion of carbon steel panels was observed when the panels were aged in 2N HCl solution containing 10 ppm of these SOS-B compounds

  • 48.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements.

     

    A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed.

     

    Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.

  • 49.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Surface expression using the AIDA autotransporter:  Towards live vaccines and whole-cell biocatalysis2011Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation.

      

    However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.

  • 50.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Bäcklund, Emma
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Optimisation of surface expression using the AIDA autotransporter2011In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10Article in journal (Refereed)
    Abstract [en]

    Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

    Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

    Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

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