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Träfflista för sökning "L773:0175 7598 OR L773:1432 0614 srt2:(2005-2009)"

Search: L773:0175 7598 OR L773:1432 0614 > (2005-2009)

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1.
  • Adomas, Aleksandra, et al. (author)
  • WS-5995 B, an antifungal agent inducing differential gene expression in the conifer pathogen Heterobasidion annosum but not in Heterobasidion abietinum
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 85, s. 347-358
  • Journal article (peer-reviewed)abstract
    • The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 A mu M WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5'-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.
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2.
  • Almeida, Joao, et al. (author)
  • Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction.
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 751-761
  • Journal article (peer-reviewed)abstract
    • Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD(+)-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD(+) deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.
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3.
  • Almeida, Joao, et al. (author)
  • Metabolic effects of furaldehydes and impacts on biotechnological processes.
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 82:4, s. 625-638
  • Research review (peer-reviewed)abstract
    • There is a growing awareness that lignocellulose will be a major raw material for production of both fuel and chemicals in the coming decades-most likely through various fermentation routes. Considerable attention has been given to the problem of finding efficient means of separating the major constituents in lignocellulose (i.e., lignin, hemicellulose, and cellulose) and to efficiently hydrolyze the carbohydrate parts into sugars. In these processes, by-products will inevitably form to some extent, and these will have to be dealt with in the ensuing microbial processes. One group of compounds in this category is the furaldehydes. 2-Furaldehyde (furfural) and substituted 2-furaldehydes-most importantly 5-hydroxymethyl-2-furaldehyde-are the dominant inhibitory compounds found in lignocellulosic hydrolyzates. The furaldehydes are known to have biological effects and act as inhibitors in fermentation processes. The effects of these compounds will therefore have to be considered in the design of biotechnological processes using lignocellulose. In this short review, we take a look at known metabolic effects, as well as strategies to overcome problems in biotechnological applications caused by furaldehydes.
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4.
  • Almeida, Joao, et al. (author)
  • NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae.
  • 2008
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 78:6, s. 939-945
  • Journal article (peer-reviewed)abstract
    • Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.
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5.
  • Andersson, Sofia, et al. (author)
  • Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 82:3, s. 535-543
  • Journal article (peer-reviewed)abstract
    • Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3-37%), nucleic acids (9-50%), and carbohydrates (3-21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.
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6.
  • Bolivar, Juan M, et al. (author)
  • Immobilization-stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus
  • 2008
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 80:1, s. 49-58
  • Journal article (peer-reviewed)abstract
    • The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.
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7.
  • Boström, Maria, et al. (author)
  • Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 68:1, s. 82-90
  • Journal article (peer-reviewed)abstract
    • The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.
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8.
  • Efremenko, E, et al. (author)
  • Purification of His(6)-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 70:5, s. 558-563
  • Journal article (peer-reviewed)abstract
    • Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His(6)-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His(6)-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.
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9.
  • Elegir, Graziano, et al. (author)
  • Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor
  • 2007
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 77:4, s. 809-817
  • Journal article (peer-reviewed)abstract
    • In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2'azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.
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10.
  • Ernest, Chi Fru, 1972, et al. (author)
  • In situ bacterial colonization of compacted bentonite under deep geological high-level radioactive waste repository conditions
  • 2008
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 79:3, s. 499-510
  • Journal article (peer-reviewed)abstract
    • Subsurface microorganisms are expected to invade, colonize, and influence the safety performance of deep geological spent nuclear fuel (SNF) repositories. An understanding of the interactions of subsurface dwelling microbial communities with the storage is thus essential. For this to be achieved, experiments must be conducted under in situ conditions. We investigated the presence of groundwater microorganisms in repository bentonite saturated with groundwater recovered from tests conducted at the Äspö underground Hard Rock Laboratory in Sweden. A 16S ribosomal RNA and dissimilatory bisulfite reductase gene distribution between the bentonite and groundwater samples suggested that the sulfate-reducing bacteria widespread in the aquifers were not common in the clay. Aerophilic bacteria could be cultured from samples run at ≤55°C but not at ≥67°C. Generally, the largely gram-negative groundwater microorganisms were poorly represented in the bentonite while the gram-positive bacteria commonly found in the clay predominated. Thus, bentonite compacted to a density of approximately 2 g cm−3 together with elevated temperatures might discourage the mass introduction of the predominantly mesophilic granitic aquifer bacteria into future SNF repositories in the long run.
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11.
  • Faulds, C.B., et al. (author)
  • Synergy between xylanases from glycoside hydrolase family 10 and family 11 and feruloyl esterase in the release of phenolic acids from cereal arabinoxylan
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 71:5, s. 622-629
  • Journal article (peer-reviewed)abstract
    • The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1→4)-β-d-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5′ form of diferulic acid from arabinoxylan (AX) derived from brewers’ spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.
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12.
  • Fonseca, Cesar, et al. (author)
  • L-Arabinose metabolism in Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation
  • 2007
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 75:2, s. 303-310
  • Journal article (peer-reviewed)abstract
    • L-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to D-glucose and D-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from D-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated-arabitol from L-arabinose and xylitol from D-xylose. Different L-arabinose concentrations and oxygen conditions were tested to better understand L-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from L-arabinose (and also xylitol from D-xylose) than C arabinofermentans PYCC 5603(T). In contrast to the latter, P guilliermondii PYCC 3012 did not produce any traces of ethanol in complex L-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in L-arabinose-assimilating yeasts.
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13.
  • Ghebremichael, K. A., et al. (author)
  • Single-step ion exchange purification of the coagulant protein from Moringa oleifera seed
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 70:5, s. 526-532
  • Journal article (peer-reviewed)abstract
    • The coagulant protein from Moringa oleifera (MO) seed was purified using a single-step batch ion exchange (IEX) method. Adsorption and elution parameters were optimized. Impact of the purification on the reduction of organic and nutrient release to the water was studied. The matrix was equilibrated using ammonium acetate buffer, and the optimum ionic strength of NaCl for elution was 0.6 M. The time for adsorption equilibrium was between 90 and 120 min. Maximum adsorption capacity of the matrix, estimated with the Langmuir model, was 68 mg protein/g adsorbent. The purified protein does not release organic and nutrient loads to the water, which are the main concerns of the crude extract. This work suggests that a readily scalable single-step IEX purification method can be used to produce the coagulant protein and it can be carried out with locally available facilities. This will promote the use of MO in large water treatment plants and other industries.
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14.
  • Guzmán, Hector, et al. (author)
  • A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis.
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 1069-1077
  • Journal article (peer-reviewed)abstract
    • The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L(-1)) at 10-15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L(-1)) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L(-1), respectively, were obtained at 5-7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed-the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH (4) (+) , and PO (4) (3-) . In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L(-1) h(-1) and ectoine concentration, content, and volumetric productivity of 4.3 g L(-1), 7.2 wt.%, and 2.8 g L(-1) day(-1), respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L(-1) day(-1).
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15.
  • Görgens, Johann F, et al. (author)
  • Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 67:5, s. 684-691
  • Journal article (peer-reviewed)abstract
    • Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.
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16.
  • Hahn-Hägerdal, Bärbel, et al. (author)
  • Towards industrial pentose-fermenting yeast strains
  • 2007
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 74:5, s. 937-953
  • Research review (peer-reviewed)abstract
    • Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.
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17.
  • Hou, Jin, 1982, et al. (author)
  • Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 82:82, s. 909-919
  • Journal article (peer-reviewed)abstract
    • During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.
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18.
  • Johanson, Ted, et al. (author)
  • Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone
  • 2008
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 77:5, s. 1111-1118
  • Journal article (peer-reviewed)abstract
    • Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.
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19.
  • Karhumaa, Kaisa, et al. (author)
  • High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae
  • 2007
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 73:5, s. 1039-1046
  • Journal article (peer-reviewed)abstract
    • Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.
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20.
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21.
  • Lacayo, Martha, et al. (author)
  • Degradation of toxaphene by Bjerkandera sp. strain BOL13 using waste biomass as a cosubstrate
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 71:4, s. 549-554
  • Journal article (peer-reviewed)abstract
    • The white-rot fungus Bjerkandera sp. strain BOL13 was capable of degrading toxaphene when supplied with wood chips, wheat husk or cane molasses as cosubstrates in batch culture experiments. Approximately 85% of toxaphene was removed when wheat husk was the main substrate. The production of lignin peroxidase was only stimulated when wheat husk was present in the liquid medium. Although xylanase was always detected, wheat husk supported the highest xylanase production. A negligible amount of beta-glucosidase and cellulase were found in the batch culture medium. To the best of our knowledge, this is the first reported case of toxaphene degradation by white-rot fungi.
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22.
  • Lindskog, Eva, et al. (author)
  • A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 71:4, s. 444-449
  • Journal article (peer-reviewed)abstract
    • Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.
  •  
23.
  • Manolov, Taras, et al. (author)
  • Continuous acetonitrile degradation in a packed-bed bioreactor
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 66:5, s. 567-574
  • Journal article (peer-reviewed)abstract
    • A 20-l packed-bed reactor filled with foamed glass beads was tested for the treatment of acetonitrile HPLC wastes. Aeration was provided by recirculating a portion of the reactor liquid phase through an aeration tank, where the dissolved oxygen concentration was kept at 6 mg/l. At a feeding rate of 0.77 g acetonitrile l–1 reactor day–1, 99% of the acetonitrile was removed; and 86% of the nitrogen present in acetonitrile was released as NH3, confirming that acetonitrile volatilization was not significant. Increasing the acetonitrile loading resulted in lower removal efficiencies, but a maximum removal capacity of 1.0 g acetonitrile l–1 reactor day–1 was achieved at a feeding rate of 1.6 g acetonitrile l–1 reactor day–1. The removal capacity of the system was well correlated with the oxygenation capacity, showing that acetonitrile removal was likely to be limited by oxygen supply. Microbial characterization of the biofilm resulted in the isolation of a Comamonas sp. able to mineralize acetonitrile as sole carbon, nitrogen and energy source. This organism was closely related to C. testosteroni (91.2%) and might represent a new species in the Comamonas genus. This study confirms the potential of packed-bed reactors for the treatment of a concentrated mixture of volatile pollutants.
  •  
24.
  • Meijer, S. L., et al. (author)
  • Physiological characterisation of acuB deletion in Aspergillus niger
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84:1, s. 157-167
  • Journal article (peer-reviewed)abstract
    • The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the a dagger acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.
  •  
25.
  • Melin, Petter, et al. (author)
  • Optimisation and comparison of liquid and dry formulations of the biocontrol yeast Pichia anomala J121
  • 2007
  • In: Applied Microbiology and Biotechnology. - New York, USA : Springer. - 0175-7598 .- 1432-0614. ; 73:5, s. 1008-1016
  • Research review (peer-reviewed)abstract
    • The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 degrees C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 degrees C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.
  •  
26.
  • Modig, Tobias, et al. (author)
  • Anaerobic glycerol production by Saccharomyces cerevisiae strains under hyperosmotic stress.
  • 2007
  • In: Applied microbiology and biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 75:2, s. 289-96
  • Journal article (peer-reviewed)abstract
    • Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80-90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.
  •  
27.
  • Moukouli, Maria, et al. (author)
  • Cloning, characterization and functional expression of an alkalitolerant type C feruloyl esterase from Fusarium oxysporum
  • 2008
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 79:2, s. 245-254
  • Journal article (peer-reviewed)abstract
    • A hypothetical protein FoFaeC-12213 of Fusarium oxysporum was found to have high amino acid sequence identity with known type C feruloyl esterases (FAEs) containing a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase. The putative FAE from the genomic DNA was successfully cloned in frame with the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in Pichia pastoris X-33 to confirm that the enzyme exhibits FAE activity. The molecular weight (62 kDa) and pI (6.8) were in agreement with the theoretical calculated values indicating the correct processing of the secretion signal in P. pastoris. The recombinant FAE was purified to its homogeneity 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 is a type C FAE showing broad hydrolytic activity against the four methyl esters of hydroxycinnamic acids and strong preference for the hydrolysis of n-propyl ferulate. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from Trichoderma longibrachiatum (a maximum of 67% total FA released after 1-h incubation). The esterase showed broad pH stability making it an important candidate for alkaline applications such as pulp treatment in the paper industry.
  •  
28.
  • Munoz, Raul, et al. (author)
  • Combined carbon and nitrogen removal from acetonitrile using algal-bacterial bioreactors
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 67:5, s. 699-707
  • Journal article (peer-reviewed)abstract
    • When compared with Chlorella vulgaris, Scenedesmus obliquus and Selenastrum capricornutum, C. sorokiniana presented the highest tolerance to acetonitrile and the highest O-2 production capacity. It also supported the fastest acetonitrile biodegradation when mixed with a suitable acetonitrile-degrading bacterial consortium. Consequently, this microalga was tested in symbiosis with the bacterial culture for the continuous biodegradation of acetonitrile at 2 g l(-1) in a stirred tank photobioreactor and in a column photobioreactor under continuous illumination (250 mu E m(-2) s(-1)). Acetonitrile removal rates of up to 2.3 g l(-1) day(-1) and 1.9 g l(-1) day(-1) were achieved in the column photobioreactor and the stirred-tank photobioreactor, respectively, when operated at the shortest retention times tested (0.4 days, 0.6 days, respectively). In addition, when the stirred-tank photobioreactor was operated with a retention time of 3.5 days, the microbial culture was capable of assimilating up to 71% and nitrifying up to 12% of the NH4+ theoretically released through the biodegradation of acetonitrile, thus reducing the need for subsequent nitrogen removal. This study suggests that complete removal of N-organics can be combined with a significant removal of nitrogen by using algal - bacterial systems and that further residual biomass digestion could pay-back part of the operation costs of the treatment plant.
  •  
29.
  • Pérez-Torrado, Roberto, et al. (author)
  • Fermentative capacity of dry active wine yeast requires an efficient oxidative stress response during industrial biomass growth
  • 2008
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 81:5, s. 951-960
  • Journal article (peer-reviewed)abstract
    • Induction of the oxidative stress response has been described under many physiological conditions in Saccharomyces cerevisiae, including industrial fermentation for wine yeast biomass production where cells are grown through several batch and fed-batch cultures on molasses. Here, we investigate the influence of aeration on the expression changes of different gene markers for oxidative stress and compare the induction profiles to the accumulation of several intracellular metabolites in order to correlate the molecular response to physiological and metabolic changes. We also demonstrate that this specific oxidative response is relevant for wine yeast performance by construction of a genetically engineered wine yeast strain overexpressing the TRX2 gene that codifies a thioredoxin, one of the most important cellular defenses against oxidative damage. This modified strain displays an improved fermentative capacity and lower levels of oxidative cellular damages than its parental strain after dry biomass production. © 2008 Springer-Verlag.
  •  
30.
  • Piddocke, Maya P, et al. (author)
  • Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84:3, s. 453-464
  • Journal article (peer-reviewed)abstract
    • High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.
  •  
31.
  • Quillaguaman, Jorge, et al. (author)
  • Optimizing conditions for poly(beta-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source
  • 2007
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 74:5, s. 981-986
  • Journal article (peer-reviewed)abstract
    • Halomonas boliviensis LC1 is able to accumulate poly(beta-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l(-1) CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l(-1) and 14 g l(-1), respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l(-1) in 17 h but reduced the PHB content in the cells to 52 wt.%.
  •  
32.
  • Quillaguaman, Jorge, et al. (author)
  • Poly(3-hydroxybutyrate) production by Halomonas boliviensis in fed-batch culture.
  • 2008
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 78:2, s. 227-232
  • Journal article (peer-reviewed)abstract
    • High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l(-1)). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH(4)Cl and K(2)HPO(4) on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH(4)Cl and 0.22% (w/v) K(2)HPO(4) and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l(-1), respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH(4)Cl, and K(2)HPO(4) were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l(-1), PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l(-1) h(-1).
  •  
33.
  • Ramchuran, Santosh, et al. (author)
  • Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 71:4, s. 463-472
  • Journal article (peer-reviewed)abstract
    • A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZ alpha B vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (alpha-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a "batch-induced" cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.
  •  
34.
  • Runquist, David, et al. (author)
  • Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae.
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 82:1, s. 123-130
  • Journal article (peer-reviewed)abstract
    • The glucose/xylose facilitator Gxf1 from Candida intermedia was expressed in the recombinant xylose-fermenting Saccharomyces cerevisiae strain TMB 3057. The new strain, TMB 3411, displayed approximately two times lower K (m) for xylose transport compared to a control strain not expressing Gxf1. In aerobic batch cultivation, the specific growth rate was significantly higher at low xylose concentration, 4 g/L, when Gxf1 was expressed, whereas it remained unchanged at high xylose concentration, 40 g/L. Similarly, in aerobic-xylose-limited chemostat culture, the Gxf1-expressing strain consumed more xylose than the control strain at low dilution rates (low xylose concentration), whereas the situation was reversed at higher dilution rates (high xylose concentration). Also, under anaerobic conditions, the Gxf1-expressing strain showed faster xylose uptake and ethanol formation at low substrate concentrations. The results are discussed in relation to previous observations, which suggested that transport controlled xylose utilization in recombinant xylose-utilizing S. cerevisiae only at low xylose concentrations.
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35.
  • Shokri, Atefeh, et al. (author)
  • RelA1 gene control of Escherichia coli lipid structure and cell performace during glucose limited fed-batch conditions
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 73:2, s. 464-473
  • Journal article (peer-reviewed)abstract
    • At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids. Opposite to the wild type, the cardiolipin-depleted membrane is more fragile with respect to sonication and osmotic chock, at severe limitation, and results in extensive foaming during the process. Protein leakage and cell lysis is, however, lower in the mutant most likely due to the increased amounts of saturated fatty acids, which might be a possible strategy to overcome the reduced amounts of membrane-strengthening cardiolipin. The membrane potential of the outer surface is negative, however less negative for the mutant. This was supported by aqueous two-phase extraction experiments which, furthermore indicated a difference in outer surface hydrofobicity. These findings suggest that the relA1 gene has a defined, but ppGpp-independent, role in cells with a slowly decreasing metabolism of glucose to control the membrane morphology.
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36.
  • Skorupa Parachin, Nadia, et al. (author)
  • Comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli for the production of an optically pure keto alcohol.
  • 2009
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 487-497
  • Journal article (peer-reviewed)abstract
    • In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((-)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (-)-2 (>99% ee, 97-98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli.
  •  
37.
  • Soares, Ana, et al. (author)
  • Biodegradation of nonylphenol in a continuous bioreactor at low temperatures and effects on the microbial population
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 69:5, s. 597-606
  • Journal article (peer-reviewed)abstract
    • A packed-bed bioreactor inoculated with a mixed culture obtained from a contaminated site was used to continuously treat a saturated solution of nonylphenol. The reactor was operated at feeding rates of 13–112 ml h−1 and temperatures of 5.5, 10, and 15°C. Optimal bioreactor performance was achieved at 10°C and at a feeding rate of 84 ml h−1 (with a removal rate of 43 mg l−1 day−1 of nonylphenol). No endocrine activity was observed in the effluent of the bioreactor at any of the temperatures tested, and the only metabolic products found were branched carboxylic acids and alkanes (lacking an aromatic ring). The study of the microbial populations in the biofilm at the three temperatures tested using fluorescence in situ hybridization showed that all the bacterial species that could be identified belonged to the phylum Proteobacteria. The most abundant class identified at all three temperatures was β-Proteobacteria. The proportions of bacteria that bound to the specific probes among the total population, identified with the bacterial probe EUB338MIX, were 60, 43, and 24% at 15, 10, and 5.5°C, respectively.
  •  
38.
  • Soares, Ana, et al. (author)
  • The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 66:6, s. 719-725
  • Journal article (peer-reviewed)abstract
    • Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l–1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l–1 and 99 mg l–1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l–1 and 79 mg l–1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg–1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.
  •  
39.
  • Svensson, Ingrid, et al. (author)
  • Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 69:1, s. 92-98
  • Journal article (peer-reviewed)abstract
    • Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.
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40.
  • Svensson, Marie, et al. (author)
  • Osmotic stability of the cell membrane of Escherichia coli from a temperature-limited fed-batch process
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 67:3, s. 345-350
  • Journal article (peer-reviewed)abstract
    • The temperature-limited fed-batch (TLFB) process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. In Escherichia coli cultures, it has been proven to prevent an extensive release of endotoxins, i.e. lipopolysaccharides, that occurs in the glucose-limited fed-batch (GLFB) processes at specific growth rates below 0.1 h(-1). The TLFB and the GLFB process were compared to each other when applied to produce the periplasmic, constitutively expressed, enzyme beta-lactamase. The extraction of the enzyme was performed by osmotic shock. A higher production of beta-lactamase was achieved with the TLFB technique while no difference in the endotoxin release was found during the extraction procedure. Furthermore, it was found that growth at declining temperature, generated by the TLFB technique, gradually stabilizes the cytoplasmic membrane, resulting in a significantly increased product quality in the extract from the TLFB cultures in the osmotic shock treatment.
  •  
41.
  • Vassilev, N., et al. (author)
  • Simultaneous P-solubilizing and biocontrol activity of microorganisms : Potentials and future trends
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 71:2, s. 137-144
  • Journal article (peer-reviewed)abstract
    • Phosphate (P)-solubilizing microorganisms as a group form an important part of the microorganisms, which benefit plant growth and development. Growth promotion and increased uptake of phosphate are not the only mechanisms by which these microorganisms exert a positive effect on plants. Microbially mediated solubilization of insoluble phosphates through release of organic acids is often combined with production of other metabolites, which take part in biological control against soilborne phytopathogens. In vitro studies show the potential of P-solubilizing microorganisms for the simultaneous synthesis and release of pathogen-suppressing metabolites, mainly siderophores, phytohormones, and lytic enzymes. Further trends in this field are discussed, suggesting a number of biotechnological approaches through physiological and biochemical studies using various microorganisms.
  •  
42.
  • Vavilin, Vasily A., et al. (author)
  • Kinetic analysis of the transformation of phthalate esters in a series of stoichiometric reactions in anaerobic wastes
  • 2005
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 69:4, s. 474-484
  • Journal article (peer-reviewed)abstract
    • Phthalates such as dimethyl phthalate, dimethyl terephthalate (DMT), diethyl phthalate (DEP), di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate (MEHP) are degraded to varying degrees under anaerobic conditions in waste treatment systems. Here we kinetically analyse the enzymatic hydrolyses involved and the subsequent stoichiometric reactions. The resulting model indicates that the degradation of the alcohols released and the transformation of the phthalic acid (PA) result in biphasic kinetics for the methane formation during transformation of DMT, DEP and MEHP. The ester hydrolysis and the PA transformation to methane appear to be the two rate-limiting steps. The PA-fermenting bacteria, which have biomass-specific growth rates between 0.04 and 0.085 day−1, grow more slowly than the other bacteria involved. Anaerobic microorganisms that remove intermediate products during phthalic acid ester conversion appear to be important for the efficiency of the ultimate phthalate degradation and to be inhibited by elevated hydrogen partial pressures. The model was based on (and the simulations corresponded well with) data obtained from experimental waste treatment systems.
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43.
  • Vigliotta, Giovanni, et al. (author)
  • Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation
  • 2007
  • In: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 75:3, s. 633-645
  • Journal article (peer-reviewed)abstract
    • The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.
  •  
44.
  • Zilouei, Hamid, et al. (author)
  • Influence of temperature on process efficiency and microbial community response during the biological removal of chlorophenols in a packed-bed bioreactor
  • 2006
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 72:3, s. 591-599
  • Journal article (peer-reviewed)abstract
    • Two reactors, initially operated at 14 and 23 +/- 1 degrees C (RA and RB, respectively), were inoculated with a bacterial consortium enriched and acclimatized to the respective temperatures over 4 months. The biofilms, formed in the reactors, were studied using scanning electron microscopy, cultivation of the biofilm microflora, and physiological analysis of the isolates. Two bacteria able to mineralize chlorophenols under a large range of temperature (10-30 degrees C) were isolated from the biofilm communities of reactors RA and RB and characterized as Alcaligenaceae bacterium R14C4 and Cupriavidus basilensis R25C6, respectively. When temperature was decreased by 10 degrees C, the chlorophenols removal capacity was reduced from 51.6 to 22.8 mg l(-1) h(-1) in bioreactor RA (from 14 to 4 degrees C) and from 59.3 to 34.7 mg l(-1) h(-1) in bioreactor RB (from 23 +/- 1 to 14 degrees C). Fluorescence in situ hybridization (FISH) of the biofilm communities showed that, in all temperatures tested, the beta-proteobacteria were the major bacterial community (35-47%) followed by the gamma-proteobacteria (12.0-6.5%). When the temperature was decreased by 10 degrees C, the proportions of gamma-proteobacteria and Pseudomonas species increased significantly in both microbial communities.
  •  
45.
  • Liu, Beidong, 1972 (author)
  • Expression of the chitinase gene from Trichoderma aureoviride in Saccharomyces cerevisiae
  • 2005
  • In: Applied Microbiology and Biotechnology. - 0175-7598. ; 69:1, s. 39-43
  • Journal article (peer-reviewed)abstract
    • Chitinase gene ech42 was obtained from Trichoderma aureoviride M and amplified by PCR. The isolated DNA of ech42 was then sequenced. The results showed that the open reading frame of ech42 was 1,447 bp long, encoding 421 amino acids. Three introns were found in the sequence. The cloning vector pMD18-T and an E. coli DH5α host were used to yield clones as E. coli DH5α/ ech42. The ech42 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/ech42. Chitinase expressed by pYES2/ech42 was induced by galactose (maximal activity 0.50 units ml−1 ) and was produced in fermentation liquid cultured for 36 h.
  •  
46.
  • Ekström, Peter, et al. (author)
  • Immunohistochemical evidence for multiple photosystems in box jellyfish.
  • 2008
  • In: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 1432-0878 .- 0302-766X. ; 333:1, s. 115-124
  • Journal article (peer-reviewed)abstract
    • Cubomedusae (box jellyfish) possess a remarkable visual system with 24 eyes distributed in four sensory structures termed rhopalia. Each rhopalium is equipped with six eyes: two pairs of pigment cup eyes and two unpaired lens eyes. Each eye type probably captures specific features of the visual environment. To investigate whether multiple types of photoreceptor cells are present in the rhopalium, and whether the different eye types possess different types of photoreceptors, we have used immunohistochemistry with a range of vertebrate opsin antibodies to label the photoreceptors, and electroretinograms (ERG) to determine their spectral sensitivity. All photoreceptor cells of the two lens eyes of the box jellyfish Tripedalia cystophora and Carybdea marsupialis displayed immunoreactivity for an antibody directed against the zebrafish ultraviolet (UV) opsin, but not against any of eight other rhodopsin or cone opsin antibodies tested. In neither of the two species were the pigment cup eyes immunoreactive for any of the opsin antibodies. ERG analysis of the Carybdea lower lens eyes demonstrated a single spectral sensitivity maximum at 485 nm suggesting the presence of a single opsin type. Our data demonstrate that the lens eyes of box jellyfish utilize a single opsin and are thus color-blind, and that there is probably a different photopigment in the pigment cup eyes. The results support our hypothesis that the lens eyes and the pigment cup eyes of box jellyfish are involved in different and specific visual tasks.
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47.
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