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Sökning: L773:0175 7598 OR L773:1432 0614 > (2010-2014)

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1.
  • Almstrand, Robert, et al. (författare)
  • Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Verlag (Germany). - 0175-7598 .- 1432-0614. ; 97:5, s. 2183-2191
  • Tidskriftsartikel (refereegranskat)abstract
    • Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.
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2.
  • Begerow, D., et al. (författare)
  • Current state and perspectives of fungal DNA barcoding and rapid identification procedures
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 87:1, s. 99-108
  • Forskningsöversikt (refereegranskat)abstract
    • Fungal research is experiencing a new wave of methodological improvements that most probably will boost mycology as profoundly as molecular phylogeny has done during the last 15 years. Especially the next generation sequencing technologies can be expected to have a tremendous effect on fungal biodiversity and ecology research. In order to realise the full potential of these exciting techniques by accelerating biodiversity assessments, identification procedures of fungi need to be adapted to the emerging demands of modern large-scale ecological studies. But how should fungal species be identified in the near future? While the answer might seem trivial to most microbiologists, taxonomists working with fungi may have other views. In the present review, we will analyse the state of the art of the so-called barcoding initiatives in the light of fungi, and we will seek to evaluate emerging trends in the field. We will furthermore demonstrate that the usability of DNA barcoding as a major tool for identification of fungi largely depends on the development of high-quality sequence databases that are thoroughly curated by taxonomists and systematists.
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3.
  • Blomqvist, Johanna, et al. (författare)
  • Fermentation characteristics of Dekkera bruxellensis strains
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - New York, USA : Springer. - 0175-7598 .- 1432-0614. ; 87:4, s. 1487-1497
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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4.
  • Dimarogona, Maria, et al. (författare)
  • Recalcitrant polysaccharide degradation by novel oxidative biocatalysts
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 97:19, s. 8455-8465
  • Tidskriftsartikel (refereegranskat)abstract
    • 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
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5.
  • Doan Van, Thuoc, et al. (författare)
  • Ectoine-mediated protection of enzyme from the effect of pH and temperature stress: a study using Bacillus halodurans xylanase as a model.
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:14, s. 6271-6278
  • Tidskriftsartikel (refereegranskat)abstract
    • Compatible solutes are small, soluble organic compounds that have the ability to stabilise proteins against various stress conditions. In this study, the protective effect of ectoines against pH stress is examined using a recombinant xylanase from Bacillus halodurans as a model. Ectoines improved the enzyme stability at low (4.5 and 5.0) and high pH (11 and 12); stabilisation effect of hydroxyectoine was superior to that of ectoine and trehalose. In the presence of hydroxyectoine, residual activity (after 10 h heating at 50 °C) increased from about 45 to 86 % at pH 5 and from 33 to 89 % at pH 12. When the xylanase was incubated at 65 °C for 5 h with 50 mM hydroxyectoine at pH 10, about 40 % of the original activity was retained while no residual activity was detected in the absence of additives or in the presence of ectoine or trehalose. The xylanase activity was slightly stimulated in the presence of 25 mM ectoines and then gradually decreased with increase in ectoines concentration. The thermal unfolding of the enzyme in the presence of the compatible solutes showed a modest increase in denaturation temperature but a larger increase in calorimetric enthalpy.
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6.
  • Dopson, Mark, et al. (författare)
  • Metal resistance in acidophilic microorganisms and its significance for biotechnologies.
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 98:19, s. 8133-8144
  • Tidskriftsartikel (refereegranskat)abstract
    • Extremely acidophilic microorganisms have an optimal pH of <3 and are found in all three domains of life. As metals are more soluble at acid pH, acidophiles are often challenged by very high metal concentrations. Acidophiles are metal-tolerant by both intrinsic, passive mechanisms as well as active systems. Passive mechanisms include an internal positive membrane potential that creates a chemiosmotic gradient against which metal cations must move, as well as the formation of metal sulfate complexes reducing the concentration of the free metal ion. Active systems include efflux proteins that pump metals out of the cytoplasm and conversion of the metal to a less toxic form. Acidophiles are exploited in a number of biotechnologies including biomining for sulfide mineral dissolution, biosulfidogenesis to produce sulfide that can selectively precipitate metals from process streams, treatment of acid mine drainage, and bioremediation of acidic metal-contaminated milieux. This review describes how acidophilic microorganisms tolerate extremely high metal concentrations in biotechnological processes and identifies areas of future work that hold promise for improving the efficiency of these applications.
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7.
  • Ganachaud, C., et al. (författare)
  • An anomalous behavior of trypsin immobilized in alginate network
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:10, s. 4403-4414
  • Tidskriftsartikel (refereegranskat)abstract
    • Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N (alpha)-benzoyl-l-arginine-4-nitroanilide hydrochloride (l-BAPNA), and in the presence or absence of d-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 A degrees C. It was found that at 37 and 42 A degrees C a strong acceleration of the reaction was obtained, whereas at 10 A degrees C and at room temperature, the presence of d-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with d-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus l-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.
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8.
  • Geladi, Paul (författare)
  • Growth characteristics of three Fusarium species evaluated by near-infrared hyperspectral imaging and multivariate image analysis
  • 2012
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 96, s. 803-813
  • Tidskriftsartikel (refereegranskat)abstract
    • Colony growth of three Fusarium spp. on potato dextrose agar was followed by collecting near-infrared (NIR) hyperspectral images of the colonies at regular intervals after inoculation up to 55 h. After principal component analysis (PCA), two clusters were apparent in the score plot along principal component 1. Using the brushing technique, these clusters were divided into four groups of pixels with similar score values. These could be visualised as growth zones within the colonies in the corresponding score image. Three spectral bands, i.e. 1,166, 1,380 and 1,918 nm, were prominent in the multiplicative scatter corrected and Savitzky-Golay second derivative spectra. These indicated chemical changes, associated with carbohydrates (1,166 and 1,380 nm) and protein (1,918 nm), that occurred as the mycelium grew and matured. The protein band was more prominent in the mature fungal material while the carbohydrate band was less pronounced. The younger material and the agar were characterised by the carbohydrate spectral band. Integrating whole mycelium colonies as the sum of pixels over time made it possible to construct curves that resembled growth curves; this included the lag phase, active growth phase, deceleration phase and phase of constant growth. Growth profiles constructed from individual growth zones indicated more detailed growth characteristics. The use of NIR hyperspectral imaging and multivariate image analysis (MIA) allowed one to visualise radial growth rings in the PCA score images. This would not have been possible with bulk spectroscopy. Interpreting spectral data enabled better understanding of microbial growth characteristics on agar medium. NIR hyperspectral imaging combined with MIA is a powerful tool for the evaluation of growth characteristics of fungi.
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9.
  • Hjort, Karin, et al. (författare)
  • Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 98:6, s. 2819-2828
  • Tidskriftsartikel (refereegranskat)abstract
    • Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.
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10.
  • Hou, Jin, 1982, et al. (författare)
  • Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:8, s. 3559-3568
  • Tidskriftsartikel (refereegranskat)abstract
    • The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous alpha-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.
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11.
  • Hou, J., et al. (författare)
  • Using regulatory information to manipulate glycerol metabolism in Saccharomyces cerevisiae
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 85:4, s. 1123-1130
  • Tidskriftsartikel (refereegranskat)abstract
    • Metabolic engineering has emerged as an attractive alternative to random mutagenesis and screening to design cell factories for industrial fermentation processes. The design of metabolic networks has been realized by gene deletions or strong overexpression of heterologous genes. There is an increasing body of evidence that indicates complete inactivation of native genes and high-level activity of heterologous enzymes may be deleterious to the cell. To moderately implement their expression, genes of interest are expressed under the control of promoters with different strengths. Constructing a promoter library is labor-intensive and requires precise quantification of the promoter strength. However, when the mechanisms of pathway regulation are known, it is possible to exploit this information to effect genetic changes efficiently. We report the implementation of this concept to reducing glycerol production during aerobic growth of Saccharomyces cerevisiae. Glycerol is produced to dispose excess cytosolic reduced nicotinamide adenine dinucleotide (NADH), and the regulating step in the pathway is mediated by glycerol 3-phosphate dehydrogenase (encoded by GPD1 and GPD2 genes). We expressed NADH oxidase in S. cerevisiae under the control of the GPD2 promoter to modulate the decrease in cytosolic NADH to the right level where the heterologous enzyme does not compete with oxidative phosphorylation while at the same time, decreasing glycerol production. This metabolic design resulted in substantially decreasing glycerol production and indeed, the excess carbon was redirected to biomass, resulting in a 14% increase in the specific growth rate. We believe that such strategies are more efficient than conventional methods and will find applications in bioprocesses.
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12.
  • Jirjis, Raida (författare)
  • Microbial communities in large-scale wood piles and their effects on wood quality and the environment
  • 2012
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 95, s. 551-563
  • Forskningsöversikt (refereegranskat)abstract
    • The demand of renewable energy sources, i.e. biomass, is steadily increasing worldwide to reduce the need of fossil energy sources. Biomass such as energy crops, woody species, forestry and agricultural residues are the most common renewable energy sources. Due to uneven demand for wood fuel, the material is mostly stored outdoors in chip piles or as logs until utilisation. Storage of biomass is accompanied by chemical, physical and biological processes which can significantly reduce the fuel quality. However, heating plants require high-quality biomass to ensure efficient operation, thereby minimising maintenance costs. Therefore, optimised storage conditions and duration times for chipped wood and tree logs have to be found. This paper aims at reviewing available knowledge on the pathways of microbial effects on stored woody biomass and on investigations of the fungal and bacterial community structure and identity. Moreover, potential functions of microorganisms present in wood chip piles and logs are discussed in terms of (1) reduction of fuel quality, (2) catalysing self-ignition processes, and (3) constituting health risk and unfriendly work environment.
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13.
  • Kadow, Maria, et al. (författare)
  • Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E-coli
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer-Verlag Berlin Heidelberg. - 0175-7598 .- 1432-0614. ; 98:9, s. 3975-3986
  • Tidskriftsartikel (refereegranskat)abstract
    • The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.
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14.
  • Karnaouri, Anthi C., et al. (författare)
  • Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 98:1, s. 231-242
  • Tidskriftsartikel (refereegranskat)abstract
    • An endoglucanase gene from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 7, was functionally expressed in methylotrophic yeast Pichia pastoris. The putative endoglucanase from the genomic DNA was successfully cloned in P. pastoris X-33 and the recombinant enzyme was purified to its homogeneity (65 kDa) and subsequently characterized. Substrate specificity analysis revealed that the enzyme exhibits high activity on substrates containing β-1,4-glycosidic bonds such as carboxymethyl cellulose, barley β-glucan, and cello-oligosaccharides, as well as activity on xylan-containing substrates, including arabinoxylan and oat spelt xylan. MtEG7a was proved to liquefy rapidly and efficiently pretreated wheat straw, indicating its key role to the initial step of hydrolysis of high-solids lignocellulose substrates. High thermostability of the endoglucanase reflects potential commercial significance of the enzyme
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15.
  • Katsimpouras, Constantinos, et al. (författare)
  • Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 98:12, s. 5507-5516
  • Tidskriftsartikel (refereegranskat)abstract
    • Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl d-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three d-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-d-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.
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16.
  • Kazemi Seresht, Ali, 1980, et al. (författare)
  • Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:9, s. 3939-3948
  • Tidskriftsartikel (refereegranskat)abstract
    • The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.
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17.
  • Khoomrung, Sakda, 1978, et al. (författare)
  • Fast and accurate preparation fatty acid methyl esters by microwave-assisted derivatization in the yeast Saccharomyces cerevisiae
  • 2012
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 94:6, s. 1637-1646
  • Tidskriftsartikel (refereegranskat)abstract
    • We present a fast and accurate method for preparation of fatty acid methyl esters (FAMEs) using microwave-assisted derivatization of fatty acids present in yeast samples. The esterification of free/bound fatty acids to FAMEs was completed within 5 min, which is 24 times faster than with conventional heating methods. The developed method was validated in two ways: (1) through comparison with a conventional method (hot plate) and (2) through validation with the standard reference material (SRM) 3275-2 omega-3 and omega-6 fatty acids in fish oil (from the Nation Institute of Standards and Technology, USA). There were no significant differences (P > 0.05) in yields of FAMEs with both validations. By performing a simple modification of closed-vessel microwave heating, it was possible to carry out the esterification in Pyrex glass tubes kept inside the closed vessel. Hereby, we are able to increase the number of sample preparations to several hundred samples per day as the time for preparation of reused vessels was eliminated. Pretreated cell disruption steps are not required, since the direct FAME preparation provides equally quantitative results. The new microwave-assisted derivatization method facilitates the preparation of FAMEs directly from yeast cells, but the method is likely to also be applicable for other biological samples.
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18.
  • Knuf, Christoph, 1984, et al. (författare)
  • Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:8, s. 3517-3527
  • Tidskriftsartikel (refereegranskat)abstract
    • Malic acid is a C-4 dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C-4 dicarboxylic acids, the chemical industry has a growing interest in this chemical compound. As malic acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for malic acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a malic acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under malic acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete malic acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))(-1) h(-1) and with a yield of 1.49 mol mol(-1). Intracellular fluxes were obtained using C-13 flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce malic acid at high titers, rates, and yields.
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19.
  • Krogh, Kristian B. R. M., et al. (författare)
  • Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 86:1, s. 143-154
  • Tidskriftsartikel (refereegranskat)abstract
    • A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60A degrees C at pH 4-6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-beta-d-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-C-13(6) as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.
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20.
  • Krogh, K.B.R.M., et al. (författare)
  • Characterization and kinetic analysis of a thermostable GH3 β-glucosidase from Penicillum brasilianum
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 86:86, s. 143-154
  • Tidskriftsartikel (refereegranskat)abstract
    • A GH3 β-glucosidase (BGL) from Penicillum brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60°C at pH 4-6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower afinity for cellobiose compared with the artificial substrate para-nitrophenyl-β-D-glucopyranoside (pNP-Glc)and further, pronounced substrate inhibition using p-NP-Glc. Kinetic studies demonstrated the high importance of using cellbiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolosis. The assay uses labelled glucose-13C6 as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates
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21.
  • Kroll, Jens, et al. (författare)
  • A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium
  • 2011
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 89:3, s. 593-604
  • Tidskriftsartikel (refereegranskat)abstract
    • Introduction: Hitherto the production of the biopolymer cyanophycin (CGP) using recombinant Escherichia coli strains and cheap mineral salts medium yielded only trace amounts of CGP (<0.5%, w/w) of the cell dry matter (CDM). This was probably due to the instability of the plasmids encoding the cyanophycin synthetase. Material and methods: In this study, we developed an anabolism-based media-dependent plasmid addiction system (PAS) to enhance plasmid stability, and we established a process based on a modified mineral salts medium yielding a CGP content of 42% (w/w) at the maximum without the addition of amino acids to the medium for the first time. This PAS is based on different lysine biosynthesis pathways and consists of two components: (1) a knockout of the chromosomal dapE disrupts the native succinylase pathway in E. coli and (2) the complementation by the plasmid-encoded artificial aminotransferase pathway mediated by the dapL gene from Synechocystis sp. PCC 6308, which allows the synthesis of the essential lysine precursor L,L-2,6-diaminopimelate. In addition, this plasmid also harbors cphAC595S, an engineered cyanophycin synthetase gene responsible for CGP production. Results: Cultivation experiments in Erlenmeyer flask and also in bioreactors in mineral salts medium without antibiotics revealed an at least 4.5-fold enhanced production of CGP in comparison to control cultivations without PAS. Discussion: Fermentation experiments with culture volume of up to 400 l yielded a maximum of 18% CGP (w/w) and a final cell density of 15.2 g CDM/l. Lactose was used constantly as an effective inducer and carbon source. Thus, we present a convenient option to produce CGP with E. coli at a technical scale without the need to add antibiotics or amino acids using the mineral salts medium designed in this study.
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22.
  • Liu, Zihe, 1984, et al. (författare)
  • Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:20, s. 8955-8962
  • Tidskriftsartikel (refereegranskat)abstract
    • With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e.g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner, whereas alpha-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h(-1) that influences protein production. Thus, for lower specific growth rates, the alpha-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas for higher specific growth rates, the two strains respond differently to changes in the specific growth rate.
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23.
  • Majdejabbari, Sara, et al. (författare)
  • Synthesis and Properties of a Novel Biosuperabsorbent from Alkali Soluble Rhizomucor pusillus Proteins
  • 2011
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 92:6, s. 1171-1177
  • Tidskriftsartikel (refereegranskat)abstract
    • A novel biosuperabsorbent protein hydrogel was prepared from protein-rich alcoholic–alkali soluble parts of zygomycete Rhizomucor pusillus biomass. The fungal protein content was 46.8%, and the lipid content was 13.1%. Extraction of protein from this microorganism through the method applied prevents protein decomposition, resulting in maximum yield. After alcoholic–alkaline extraction, the proteins from the biomass were acylated using ethylenediaminetetraacetic dianhydride and subsequently treated with glutaraldehyde as a crosslinker for further experiments. Thermal consistency was investigated by means of two different methods: thermal denaturation via differential scanning calorimetry and thermal decomposition study via thermogravimetric analysis. The swelling behaviour of the crosslinked hydrogel was measured in deionised water, 0.9% NaCl solution and synthetic urine, which were 87.6, 43 and 38.6 g/g water after 24 h, respectively. Moreover, the isoelectric point (pI) of the hydrogel was determined as pH = 8 by studying swelling behaviour at different pHs. In addition, the dependencies of the swelling behaviour with regard to the chemical modification, the ionic strength, the degree of crosslinking, as well as water absorbency with or without load were studied.
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24.
  • Martinez Avila, Hector, 1985, et al. (författare)
  • Biocompatibility evaluation of densified bacterial nanocellulose hydrogel as an implant material for auricular cartilage regeneration
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:17, s. 7423-7435
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacterial nanocellulose (BNC), synthesized by the bacterium Gluconacetobacter xylinus, is composed of highly hydrated fibrils (99 % water) with high mechanical strength. These exceptional material properties make BNC a novel biomaterial for many potential medical and tissue engineering applications. Recently, BNC with cellulose content of 15 % has been proposed as an implant material for auricular cartilage replacement, since it matches the mechanical requirements of human auricular cartilage. This study investigates the biocompatibility of BNC with increased cellulose content (17 %) to evaluate its response in vitro and in vivo. Cylindrical BNC structures (48 Au 20 mm) were produced, purified in a built-in house perfusion system, and compressed to increase the cellulose content in BNC hydrogels. The reduction of endotoxicity of the material was quantified by bacterial endotoxin analysis throughout the purification process. Afterward, the biocompatibility of the purified BNC hydrogels with cellulose content of 17 % was assessed in vitro and in vivo, according to standards set forth in ISO 10993. The endotoxin content in non-purified BNC (2,390 endotoxin units (EU)/ml) was reduced to 0.10 EU/ml after the purification process, level well below the endotoxin threshold set for medical devices. Furthermore, the biocompatibility tests demonstrated that densified BNC hydrogels are non-cytotoxic and cause a minimal foreign body response. In support with our previous findings, this study concludes that BNC with increased cellulose content of 17 % is a promising non-resorbable biomaterial for auricular cartilage tissue engineering, due to its similarity with auricular cartilage in terms of mechanical strength and host tissue response.
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25.
  • Moirangthem, Lakshmipyari Devi, et al. (författare)
  • A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - Berlin : Springer Berlin/Heidelberg. - 0175-7598 .- 1432-0614. ; 98:8, s. 3809-3818
  • Tidskriftsartikel (refereegranskat)abstract
    • A spontaneous methyl viologen (MV)-resistant mutant of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 was isolated and the major enzymatic antioxidants involved in combating MV-induced oxidative stress were evaluated. The mutant displayed a high constitutive catalase activity as a consequence of which, the intracellular level of reactive oxygen species in the mutant was lower than the wild type (N. punctiforme) in the presence of MV. The superoxide dismutase (SOD) activity that consisted of a SodA (manganese-SOD) and a SodB (iron-SOD) was not suppressed in the mutant following MV treatment. The mutant was, however, characterised by a lower peroxidase activity compared with its wild type, and its improved tolerance to externally added H2O2 could only be attributed to enhanced catalase activity. Furthermore, MV-induced toxic effects on the wild type such as (1) loss of photosynthetic performance assessed as maximal quantum yield of photosystem II, (2) nitrogenase inactivation, and (3) filament fragmentation and cell lysis were not observed in the mutant. These findings highlight the importance of catalase in preventing MV-promoted oxidative damage and cell death in the cyanobacterium N. punctiforme. Such oxidative stress resistant mutants of cyanobacteria are likely to be a better source of biofertilisers, as they can grow and fix nitrogen in an unhindered manner in agricultural fields that are often contaminated with the herbicide MV, also commonly known as paraquat.
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26.
  • Mukherjee, Vaskar, 1986, et al. (författare)
  • Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:9483
  • Tidskriftsartikel (refereegranskat)abstract
    • Saccharomyces cerevisiae is the organism of choice for many food and beverage fermentations because it thrives in high-sugar and high-ethanol conditions. However, the conditions encountered in bioethanol fermentation pose specific challenges, including extremely high sugar and ethanol concentrations, high temperature, and the presence of specific toxic compounds. It is generally considered that exploring the natural biodiversity of Saccharomyces strains may be an interesting route to find superior bioethanol strains and may also improve our understanding of the challenges faced by yeast cells during bioethanol fermentation. In this study, we phenotypically evaluated a large collection of diverse Saccharomyces strains on six selective traits relevant for bioethanol production with increasing stress intensity. Our results demonstrate a remarkably large phenotypic diversity among different Saccharomyces species and among S. cerevisiae strains from different origins. Currently applied bioethanol strains showed a high tolerance to many of these relevant traits, but several other natural and industrial S. cerevisiae strains outcompeted the bioethanol strains for specific traits. These multitolerant strains performed well in fermentation experiments mimicking industrial bioethanol production. Together, our results illustrate the potential of phenotyping the natural biodiversity of yeasts to find superior industrial strains that may be used in bioethanol production or can be used as a basis for further strain improvement through genetic engineering, experimental evolution, or breeding. Additionally, our study provides a basis for new insights into the relationships between tolerance to different stressors.
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27.
  • Narciso-da-Rocha, Carlos, et al. (författare)
  • Diversity and antibiotic resistance of Acinetobacter spp. in water from the source to the tap.
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 97:1, s. 329-340
  • Tidskriftsartikel (refereegranskat)abstract
    • Acinetobacter spp. are ubiquitous bacteria in the environment. Acinetobacter spp. isolated from a municipal drinking water treatment plant and from connected tap water were identified to the species level on the basis of rpoB gene partial sequence analysis. Intraspecies variation was assessed based on the analysis of partial sequences of housekeeping genes (rpoB, gyrB, and recA). Antibiotic resistance was characterized using the disk diffusion method and isolates were classified as wild or non-wild type (non-WT), according to the observed phenotype. The strains of Acinetobacter spp. were related to 11 different validly published species, although three groups of isolates, presenting low rpoB sequence similarities with previously described species, may represent new species. Most of the isolates were related to the species A. johnsonii and A. lwoffii. These two groups, as well as others related to the species A. parvus and A. tjernbergiae, were detected in the water treatment plant and in tap water. Other strains, related to the species A. pittii and A. beijerinckii, were isolated only from tap water. Most of the isolates (80%) demonstrated wild type (WT) to all of the 12 antibiotics tested. Non-WT for tetracycline, meropenem, and ceftazidime, among others, were observed in water treatment plant or in tap water samples. Although, in general, this study suggests a low prevalence of acquired antibiotic resistance in water Acinetobacter spp., the potential of some species to acquire and disseminate resistance via drinking water is suggested.
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28.
  • Papini, Marta, 1981, et al. (författare)
  • Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through (13)C-based metabolic flux analysis.
  • 2012
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 95:4, s. 1001-1010
  • Tidskriftsartikel (refereegranskat)abstract
    • Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden-Meyerhof-Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on (13)C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden-Meyerhof-Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.
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29.
  • Pawar, Sudhanshu, et al. (författare)
  • Thermophilic biohydrogen production: how far are we?
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:18, s. 7999-8009
  • Forskningsöversikt (refereegranskat)abstract
    • Apart from being applied as an energy carrier, hydrogen is in increasing demand as a commodity. Currently, the majority of hydrogen (H2) is produced from fossil fuels, but from an environmental perspective, sustainable H2 production should be considered. One of the possible ways of hydrogen production is through fermentation, in particular, at elevated temperature, i.e. thermophilic biohydrogen production. This short review recapitulates the current status in thermophilic biohydrogen production through fermentation of commercially viable substrates produced from readily available renewable resources, such as agricultural residues. The route to commercially viable biohydrogen production is a multidisciplinary enterprise. Microbiological studies have pointed out certain desirable physiological characteristics in H2-producing microorganisms. More process-oriented research has identified best applicable reactor types and cultivation conditions. Techno-economic and life cycle analyses have identified key process bottlenecks with respect to economic feasibility and its environmental impact. The review has further identified current limitations and gaps in the knowledge, and also deliberates directions for future research and development of thermophilic biohydrogen production.
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30.
  • Petersen, N., et al. (författare)
  • Bacterial cellulose-based materials and medical devices: current state and perspectives
  • 2011
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 91:5, s. 1277-1286
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacterial cellulose (BC) is a unique and promising material for use as implants and scaffolds in tissue engineering. It is composed of a pure cellulose nanofiber mesh spun by bacteria. It is remarkable for its strength and its ability to be engineered structurally and chemically at nano-, micro-, and macroscales. Its high water content and purity make the material biocompatible for multiple medical applications. Its biocompatibility, mechanical strength, chemical and morphologic controllability make it a natural choice for use in the body in biomedical devices with broader application than has yet been utilized. This paper reviews the current state of understanding of bacterial cellulose, known methods for controlling its physical and chemical structure (e.g., porosity, fiber alignment, etc.), biomedical applications for which it is currently being used, or investigated for use, challenges yet to be overcome, and future possibilities for BC.
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31.
  • Pisanelli, I., et al. (författare)
  • Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 86:2, s. 599-606
  • Tidskriftsartikel (refereegranskat)abstract
    • Pyranose dehydrogenase (PDH) is a flavin-dependant sugar oxidoreductase found in the family Agaricaceae, basidiomycetes that degrade lignocellulose-rich forest litter, and is catalytically related to the fungal enzymes pyranose 2-oxidase and cellobiose dehydrogenase. It has broad substrate specificity and displays similar activities with most sugar constituents of lignocellulose including disaccharides and oligosaccharides, a number of (substituted) quinones, and metal ions are suitable electron acceptors rather than molecular oxygen. In contrast to pyranose 2-oxidase and cellobiose dehydrogenase, which oxidize regioselectively at C-2 and C-1, respectively, PDH is capable of oxidation on C-1 to C-4 as well as double oxidations, depending on the nature of the substrate. This makes it a very interesting enzyme for biocatalytic applications, as many of the reaction products are otherwise unaccessible by chemical or enzymatic means. PDH was characterized in detail in a limited number of fungi, and the first encoding genes were isolated only recently. We report here, for the first time, the heterologous expression of one of these genes, encoding the major PDH protein in Agaricus meleagris, in the filamentous fungi Aspergillus nidulans, and Aspergillus niger.
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32.
  • Quillaguaman, Jorge, et al. (författare)
  • Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 85:6, s. 1687-1696
  • Forskningsöversikt (refereegranskat)abstract
    • Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.
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33.
  • Rosengren, Anna, et al. (författare)
  • An Aspergillus nidulans β-mannanase with high transglycosylation capacity revealed through comparative studies within glycosidase family 5.
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:24, s. 10091-10104
  • Tidskriftsartikel (refereegranskat)abstract
    • β-Mannanases are involved in the conversion and modification of mannan-based saccharides. Using a retaining mechanism, they can, in addition to hydrolysis, also potentially perform transglycosylation reactions, synthesizing new glyco-conjugates. Transglycosylation has been reported for β-mannanases in GH5 and GH113. However, although they share the same fold and catalytic mechanism, there may be differences in the enzymes' ability to perform transglycosylation. Three GH5 β-mannanases from Aspergillus nidulans, AnMan5A, AnMan5B and AnMan5C, which belong to subfamily GH5_7 were studied. Comparative studies, including the GH5_7 TrMan5A from Trichoderma reesei, showed some differences between the enzymes. All the enzymes could perform transglycosylation but AnMan5B stood out in generating comparably higher amounts of transglycosylation products when incubated with manno-oligosaccharides. In addition, AnMan5B did not use alcohols as acceptor, which was also different compared to the other three β-mannanases. In order to map the preferred binding of manno-oligosaccharides, incubations were performed in H2 (18)O. AnMan5B in contrary to the other enzymes did not generate any (18)O-labelled products. This further supported the idea that AnMan5B potentially prefers to use saccharides as acceptor instead of water. A homology model of AnMan5B showed a non-conserved Trp located in subsite +2, not present in the other studied enzymes. Strong aglycone binding seems to be important for transglycosylation with saccharides. Depending on the application, it is important to select the right enzyme.
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34.
  • Sanchez Nogue, Violeta, et al. (författare)
  • Short-term adaptation improves the fermentation performance of Saccharomyces cerevisiae in the presence of acetic acid at low pH.
  • 2013
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:16, s. 7517-7525
  • Tidskriftsartikel (refereegranskat)abstract
    • The release of acetic acid due to deacetylation of the hemicellulose fraction during the treatment of lignocellulosic biomass contributes to the inhibitory character of the generated hydrolysates. In the present study, we identified a strain-independent adaptation protocol consisting of pre-cultivating the strain at pH 5.0 in the presence of at least 4 g L(-1) acetic acid that enabled aerobic growth and improved fermentation performance of Saccharomyces cerevisiae cells at low pH (3.7) and in the presence of inhibitory levels of acetic acid (6 g L(-1)). During anaerobic cultivation with adapted cells of strain TMB3500, the specific ethanol production rate was increased, reducing the fermentation time to 48 %.
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35.
  • Sandström, Anders, et al. (författare)
  • Saccharomyces cerevisiae: a potential host for carboxylic acid production from lignocellulosic feedstock?
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:17, s. 7299-7318
  • Forskningsöversikt (refereegranskat)abstract
    • Carboxylic acids are important bulk chemicals that can be used as building blocks for the production of polymers, as acidulants, preservatives and flavour compound or as precursors for the synthesis of pharmaceuticals. Today, their production mainly takes place through catalytic processing of petroleum-based precursors. An appealing alternative would be to produce these compounds from renewable resources, using tailor-made microorganisms. Saccharomyces cerevisiae has already demonstrated its value for bioethanol production from renewable resources. In this review, we discuss Saccharomyces cerevisiae engineering potential, current strategies for carboxylic acid production as well as the specific challenges linked to the use of lignocellulosic biomass as carbon source.
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36.
  • Skorupa Parachin, Nadia, et al. (författare)
  • Flotation as a tool for indirect DNA extraction from soil
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 87:5, s. 1927-1933
  • Tidskriftsartikel (refereegranskat)abstract
    • Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 mug DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.
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37.
  • Tobias, Joshua, 1969, et al. (författare)
  • Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines.
  • 2010
  • Ingår i: Applied microbiology and biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 87:4, s. 1355-65
  • Tidskriftsartikel (refereegranskat)abstract
    • Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal-mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.
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38.
  • Tobias, Joshua, 1969, et al. (författare)
  • Strategies to overexpress enterotoxigenic Escherichia coli (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates.
  • 2012
  • Ingår i: Applied microbiology and biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 93:6, s. 2291-300
  • Forskningsöversikt (refereegranskat)abstract
    • Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveler's diarrhea (TD). Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. The most extensively studied ETEC candidate vaccine is the rCTB-CF ETEC vaccine, containing recombinantly produced cholera B subunit and the most commonly encountered ETEC CFs on the surface of whole inactivated bacteria. Initial clinical trials with this vaccine showed significant immune responses against the key antigens in different age groups in Bangladesh and Egypt and protection against more severe TD in Western travelers. However, when tested in a phase-III trial in Egyptian infants, the protective efficacy of the vaccine was found to be low, indicating the need to improve the immunogenicity of the vaccine, e.g., by increasing the levels of the protective antigens. This review describes different strategies for the construction of recombinant nontoxigenic E. coli and Vibrio cholerae candidate vaccine strains over-expressing higher amounts of ETEC CFs than clinical ETEC isolates selected to produce high levels of the respective CF, e.g., those ETEC strains which have been used in the rCTB-CF ETEC vaccine. Several different expression vectors containing the genes responsible for the expression and assembly of the examined CFs, all downstream of the powerful tac promoter, which could be maintained either with or without antibiotic selection, were constructed. Expression from the tac promoter was under the control of the lacI(q) repressor present on the plasmids. Following induction with isopropyl-β-D-thiogalactopyranoside, candidate vaccine strains over-expressing single CFs, unnatural combinations of two CFs, and also hybrid forms of ETEC CFs were produced. Specific monoclonal antibodies against the major subunits of the examined CF were used to quantify the amount of the surface-expressed CF by a dot-blot assay and inhibition ELISA. Oral immunization with formalin- or phenol-inactivated recombinant bacteria over-expressing the CFs was found to induce significantly higher antibody responses compared to immunization with the previously used vaccine strains. We therefore conclude that our constructs may be useful as candidate strains in an oral whole-cell inactivated CF ETEC vaccine.
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39.
  • Topakas, Evangelos, et al. (författare)
  • Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila
  • 2012
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 94:2, s. 399-411
  • Tidskriftsartikel (refereegranskat)abstract
    • A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39 kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1 h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.
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40.
  • Topakas, Evangelos, et al. (författare)
  • Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile : identification of the nucleophilic serine
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 87:5, s. 1765-1772
  • Tidskriftsartikel (refereegranskat)abstract
    • A glucuronoyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55°C. The esterase displayed broad pH range stability between 4–10 and temperature stability up to 50°C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.
  •  
41.
  • Tyo, Keith, 1979, et al. (författare)
  • Impact of protein uptake and degradation on recombinant protein secretion in yeast
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:16, s. 7149-7159
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize the protein uptake and degradation pathways of S. cerevisiae to better understand its impact on protein secretion titers. We do find that S. cerevisiae can consume significant (in the range of 1 g/L/day) quantities of whole proteins. Characterizing the physiological state and combining metabolomics and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion systems, and the current data should help formulate strategies to mitigate product loss.
  •  
42.
  • Vassileva, Maria, et al. (författare)
  • Multifunctional properties of phosphate-solubilizing microorganisms grown on agro-industrial wastes in fermentation and soil conditions
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 85:5, s. 1287-1299
  • Forskningsöversikt (refereegranskat)abstract
    • One of the most studied approaches in solubilization of insoluble phosphates is the biological treatment of rock phosphates. In recent years, various techniques for rock phosphate solubilization have been proposed, with increasing emphasis on application of P-solubilizing microorganisms. The P-solubilizing activity is determined by the microbial biochemical ability to produce and release metabolites with metal-chelating functions. In a number of studies, we have shown that agro-industrial wastes can be efficiently used as substrates in solubilization of phosphate rocks. These processes were carried out employing various technologies including solid-state and submerged fermentations including immobilized cells. The review paper deals critically with several novel trends in exploring various properties of the above microbial/agro-wastes/rock phosphate systems. The major idea is to describe how a single P-solubilizing microorganism manifests wide range of metabolic abilities in different environments. In fermentation conditions, P-solubilizing microorganisms were found to produce various enzymes, siderophores, and plant hormones. Further introduction of the resulting biotechnological products into soil-plant systems resulted in significantly higher plant growth, enhanced soil properties, and biological (including biocontrol) activity. Application of these bio-products in bioremediation of disturbed (heavy metal contaminated and desertified) soils is based on another important part of their multifunctional properties.
  •  
43.
  • Weber, Nora, et al. (författare)
  • Exploiting cell metabolism for biocatalytic whole-cell transamination by recombinant Saccharomyces cerevisiae.
  • 2014
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:10, s. 4615-4624
  • Tidskriftsartikel (refereegranskat)abstract
    • The potential of Saccharomyces cerevisiae for biocatalytic whole-cell transamination was investigated using the kinetic resolution of racemic 1-phenylethylamine (1-PEA) to (R)-1-PEA as a model reaction. As native yeast do not possess any ω-transaminase activity for the reaction, a recombinant yeast biocatalyst was constructed by overexpressing the gene coding for vanillin aminotransferase from Capsicum chinense. The yeast-based biocatalyst could use glucose as the sole co-substrate for the supply of amine acceptor via cell metabolism. In addition, the biocatalyst was functional without addition of the co-factor pyridoxal-5'-phosphate (PLP), which can be explained by a high inherent cellular capacity to sustain PLP-dependent reactions in living cells. In contrast, external PLP supplementation was required when cell viability was low, as it was the case when using pyruvate as a co-substrate. Overall, the results indicate a potential for engineered S. cerevisiae as a biocatalyst for whole-cell transamination and with glucose as the only co-substrate for the supply of amine acceptor and PLP.
  •  
44.
  • Westman, Johan, 1983, et al. (författare)
  • Effects of encapsulation of microorganisms on product formation during microbial fermentations
  • 2012
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 96:6, s. 1441-1454
  • Forskningsöversikt (refereegranskat)abstract
    • This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.
  •  
45.
  • Zammit, Carla M., et al. (författare)
  • Bioleaching in brackish waters-effect of chloride ions on the acidophile population and proteomes of model species
  • 2012
  • Ingår i: Applied Microbiology and Biotechnology. - New York : Springer. - 0175-7598 .- 1432-0614. ; 93:1, s. 319-329
  • Tidskriftsartikel (refereegranskat)abstract
    • High concentrations of chloride ions inhibit the growth of acidophilic microorganisms used in biomining, a problem particularly relevant to Western Australian and Chilean biomining operations. Despite this, little is known about the mechanisms acidophiles adopt in order to tolerate high chloride ion concentrations. This study aimed to investigate the impact of increasing concentrations of chloride ions on the population dynamics of a mixed culture during pyrite bioleaching and apply proteomics to elucidate how two species from this mixed culture alter their proteomes under chloride stress. A mixture consisting of well-known biomining microorganisms and an enrichment culture obtained from an acidic saline drain were tested for their ability to bioleach pyrite in the presence of 0, 3.5, 7, and 20 g L(-1) NaCl. Microorganisms from the enrichment culture were found to out-compete the known biomining microorganisms, independent of the chloride ion concentration. The proteomes of the Gram-positive acidophile Acidimicrobium ferrooxidans and the Gram-negative acidophile Acidithiobacillus caldus grown in the presence or absence of chloride ions were investigated. Analysis of differential expression showed that acidophilic microorganisms adopted several changes in their proteomes in the presence of chloride ions, suggesting the following strategies to combat the NaCl stress: adaptation of the cell membrane, the accumulation of amino acids possibly as a form of osmoprotectant, and the expression of a YceI family protein involved in acid and osmotic-related stress.
  •  
46.
  • Ödman, Peter, et al. (författare)
  • Sensor combination and chemometric variable selection for online monitoring of Streptomyces coelicolor fed-batch cultivations
  • 2010
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 86:6, s. 1745-1759
  • Tidskriftsartikel (refereegranskat)abstract
    • Fed-batch cultivations of Streptomyces coelicolor, producing the antibiotic actinorhodin, were monitored online by multiwavelength fluorescence spectroscopy and off-gas analysis. Partial least squares (PLS), locally weighted regression, and multilinear PLS (N-PLS) models were built for prediction of biomass and substrate (casamino acids) concentrations, respectively. The effect of combination of fluorescence and gas analyzer data as well as of different variable selection methods was investigated. Improved prediction models were obtained by combination of data from the two sensors and by variable selection using a genetic algorithm, interval PLS, and the principal variables method, respectively. A stepwise variable elimination method was applied to the three-way fluorescence data, resulting in simpler and more accurate N-PLS models. The prediction models were validated using leave-one-batch-out cross-validation, and the best models had root mean square error of cross-validation values of 1.02 g l(-1) biomass and 0.8 g l(-1) total amino acids, respectively. The fluorescence data were also explored by parallel factor analysis. The analysis revealed four spectral profiles present in the fluorescence data, three of which were identified as pyridoxine, NAD(P)H, and flavin nucleotides, respectively.
  •  
47.
  • Jänicke, Ralf, 1980, et al. (författare)
  • Minimal loading conditions for higher order numerical homogenisation schemes
  • 2012
  • Ingår i: Archive of Applied Mechanics. - : Springer Science and Business Media LLC. - 0939-1533 .- 1432-0681. ; 82:8, s. 1075-1088
  • Tidskriftsartikel (refereegranskat)abstract
    • The present study deals with the formulation of minimal loading conditions for microscale applications in numerical two-scale modelling (FE2) approaches. From the homogenisation concept, a set of volume average rules constrains the microscale PDE to be solved. They are considered to be the minimal set of loading conditions and can be specified by additional polynomial or periodic assumptions, for example, on the microscale displacement field. Whereas the resulting volume integrals can be transformed into surface integrals for so-called first-order homogenisation schemes, this is not possible for a second-order homogenisation of second gradient or micromorphic effective media substituting a heterogeneous microcontinuum represented by a volume element on the microscale. Several numerical examples compare the minimal loading condition concept with standard techniques discussed in literature.
  •  
48.
  • Yildiz, U. A., et al. (författare)
  • Deep observations of O-2 toward a low-mass protostar with Herschel-HIFI
  • 2013
  • Ingår i: Astronomy and Astrophysics. - : EDP Sciences. - 0004-6361 .- 1432-0746. ; 558
  • Tidskriftsartikel (refereegranskat)abstract
    • Context. According to traditional gas-phase chemical models, O-2 should be abundant in molecular clouds, but until recently, attempts to detect interstellar O-2 line emission with ground-and space-based observatories have failed. Aims. Following the multi-line detections of O-2 with low abundances in the Orion and. Oph A molecular clouds with Herschel, it is important to investigate other environments, and we here quantify the O-2 abundance near a solar-mass protostar. Methods. Observations of molecular oxygen, O-2, at 487 GHz toward a deeply embedded low-mass Class 0 protostar, NGC 1333IRAS 4A, are presented, using the Heterodyne Instrument for the Far Infrared (HIFI) on the Herschel Space Observatory. Complementary data of the chemically related NO and CO molecules are obtained as well. The high spectral resolution data are analysed using radiative transfer models to infer column densities and abundances, and are tested directly against full gas-grain chemical models.Results. The deep HIFI spectrum fails to show O-2 at the velocity of the dense protostellar envelope, implying one of the lowest abundance upper limits of O-2/H-2 at = 6x 10-9 (3s). The O-2/CO abundance ratio is less than 0.005. However, a tentative (4.5s) detection of O-2 is seen at the velocity of the surrounding NGC 1333 molecular cloud, shifted by 1 km s-1 relative to the protostar. For the protostellar envelope, pure gas-phase models and gas-grain chemical models require a long pre-collapse phase (similar to 0.7-1 x 106 years), during which atomic and molecular oxygen are frozen out onto dust grains and fully converted to H2O, to avoid overproduction of O2 in the dense envelope. The same model also reproduces the limits on the chemically related NO molecule if hydrogenation of NO on the grains to more complex molecules such as NH2OH, found in recent laboratory experiments, is included. The tentative detection of O-2 in the surrounding cloud is consistent with a low-density PDR model with small changes in reaction rates.
  •  
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