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  • Articles  (28,484)
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  • Process Engineering, Biotechnology, Nutrition Technology  (28,484)
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  • Articles  (28,484)
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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Cellulose 1 (1994), S. 1-25 
    ISSN: 1572-882X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Cellulose as a material has been widely used for centuries in all kinds of practical applications. However, its chemical composition, structure and morphology were also unknown for centuries. The modern history of cellulose chemistry actually began in 1837 when Anselme Payen chemically identified cellulose from plants. Since then, the establishment of its chemical and physical structures has undergone multitudinous periods of struggle. Until the early 1920s, many scientists believed that cellulose was made up of a few small molecules of glucose or cellobiose. Very few scientists accepted the premiss that it was a polymer. The controversial debates were continued for over ten years. Eventually, substantial experimental data provided proof that cellulose is a covalently linked, high-molecular-weight macromolecule. This fact also provided the foundation for the establishment of polymer science. Some of the historical development of chemistry and structures are briefly reviewed, and recent approaches to studying cellulose structures with new instrumentation are discussed.
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  • 2
    ISSN: 1572-882X
    Keywords: cellulose esters ; thermoplastics ; block-copolymers ; biopolymers ; degree of polymerization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Mono-functional cellulose propionate segments for use in ter- or star-block polymers have been prepared by the depolymerization (step 1) of cellulose propionate in homogeneous phase using a mixture of HBr and propionic anhydride in methylene chloride solution. The anomeric mixture of glycosyl bromide has subsequently (step 2) been hydrolyzed in aqueous acetone. Functionality was determined by H-NMR spectroscopy of triethyl silane derivatives in combination with gel permeation chromatography. The cellulose ester segments were semi-rigid, highly crystalline materials with melting points between 180° and 250°C. The lowest useful segment size, based on crystallinity and Mark-Houwink-Sakurada exponential factor, appeared to be DP 20, with an optimum around DP 40 to 50.
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  • 3
    ISSN: 1572-882X
    Keywords: kraft pulp surface properties ; ESCA ; lignin ; extractives ; carboxyl groups
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effect of digestion conditions (amount of effective alkali, digestion time) on the surface compositions of unbleached softwood (Pinus sylvestris) kraft pulp has been investigated by electron spectroscopy for chemical analysis (ESCA). The quantities monitored were the angular dependence of the total O/C ratio, the relative amounts of carbons in different states of oxidation and the adsorption of Al and Ca ions to the carboxyl groups in the surface. Examination of the angular dependence of ESCA intensities shows that the concentration of alkyl carbon is high in a very thin surface layer and that it decreases linearly with decreasing kappa number. The concentration of alkyl carbon is decreased by extraction of the fibres with dichloromethane, but the amount remaining in the surface after extraction still decreases linearly with decreasing kappa number (i.e. it decreases with increasing digestion time). It is suggested that the observed enrichment of alkyl carbon in the outermost surface layers most probably is due to reprecipitation of lignin. In pulp that has not been extracted, there is also strong enrichment of extractives in the surface. This amount increases with increasing effective alkali but is relatively independent of digestion time. ESCA analysis of the Al and Ca bound to the carboxyl groups shows that the amount depends on digestion time; the results are consistent with the notion that the reprecipitated lignin contains carboxyl groups.
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  • 4
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    Cellulose 1 (1994), S. 215-219 
    ISSN: 1572-882X
    Keywords: cellulose fillers ; epoxy resins ; lignocellulosic materials
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Cellulosic fillers have been chemically modified by attaching pendant primary amine groups (diamino propane). The modified cellulose fillers were used for curing an epoxy resin (diglycidyl ether of bisphenol A). Gel times thus obtained are much less than systems containing unmodified cellulose. The advantages from the use of such modified organic fillers are discussed. Our investigations could encourage greater utilization of renewable lignocellulosic materials as reactionincorporated fillers for polymer composites.
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  • 5
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    Cellulose 1 (1994), S. 259-280 
    ISSN: 1572-882X
    Keywords: semi-dilute solution ; cellulose urethane ; light scattering (static, dynamic) ; aggregation phenomena
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The polymer-solvent interaction was studied for two similar cellulose derivatives in the semi-dilute concentration range by static and dynamic light scattering. The trisubstituted 3-chlorophenyl carbamate (3Cl-CTC) and a mixed trisubstituted derivative with methyl groups (degree of methyl substituents: DS Me = 1.6–1.7)combined with the abovementioned 3-chlorophenyl carbamate groups filling the still open positions at the cellulose backbone were synthesized, fractionated and characterized according to standard methods. Different kinds of associations, entangled clusters with a rod-like shape on one side and entanglement networks on the other side, exist in semi-dilute dioxane solutions caused by different polymer-solvent interactions. These quite different associations lead to either a liquid crystalline or a gel-like state upon increase of concentration.
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  • 6
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    Cellulose 1 (1994), S. 26-56 
    ISSN: 1572-882X
    Keywords: low-temperature degradation ; kinetics ; mechanisms ; electrical insulation ; transformers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A critical review is given of the degradation of cellulose in the low-temperature region (below about 300°C) of power transformer operation. The large number of kinetic studies, under a variety of environmental conditions from Kraft paper in insulating oil, to cotton and paper in oxygen, are considered in terms of a first-order polymer chain scission model. In many cases, the data are replotted to suit the model. A common activation energy of 111±6 kjmol−1 is calculated and it is shown that the pre-exponential factor, rather than the activation energy, is sensitive to the oxidizing nature of the environment and the susceptibility to degradation of the material. The chemical mechanisms of degradation are reviewed, and conclusions and recommendations are made regarding chemical condition monitoring and life prediction of electrical insulation.
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  • 7
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    Cellulose 1 (1994), S. 87-106 
    ISSN: 1572-882X
    Keywords: plasma treatment ; bonding ; polypropylene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Plasma treatments can be utilized to upgrade the value of lignocellulosic materials for applications such as biobased composites. Poor adhesion in biobased composites is caused by incompatibility between polar cellulosics and non-polar thermoplastics. Plasma modification of both cellulose and polypropylene was evaluated by a T-peel test for improved compatibility and adhesion between these materials. Oxygen and argon plasmas were used to modify the surface of polypropylene films, while a cyclohexane plasma was used to modify the cellulose surface through deposition of a hydrophobic polymer layer. For plasma treatment of polypropylene, changes in power input had a greater effect on adhesion than changes in pressure. Surface oxidation and increased acid/base characteristics were found on both argon- and oxygen-plasma-treated polypropylene based on ESCA and wetting measurements. With the non-reactive argon plasma the persistence of reactive species, such as free radicals, was very important for enhanced adhesion. The amount of polar carbonyl groups introduced onto the surface was also an important factor for adhesion improvement. Modification of the cellulose (filter paper) surface to a hydrophobic character with a cyclohexane plasma did not improve adhesion to polypropylene.
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  • 8
    ISSN: 1572-882X
    Keywords: cellulose hetero-esters ; single crystals ; electron micrography ; electron diffraction crystallography ; polymer morphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Lamellar single crystals of some regio-selectively substituted cellulose hetero-esters: cellulose propionate diacetate (CPDA, 2,3-di-O-acetyl-6-O-propionyl cellulose), cellulose acetate dipropionate (CADP, 6-O-acetyl-2,3-di-O-propionyl cellulose), cellulose butyrate diacetate (CBDA, 2,3-di-O-acetyl-6-O-butyryl cellulose) and cellulose acetate dibutyrate (CADB, 6-O-acetyl-2,3-di-O-butyryl cellulose), have been prepared at high temperature in a mixture of dibenzyl ether andn-tetradecane. The CPDA crystals were lozenge-shaped whereas those of CADP, CBDA and CADB had a ribbon morphology. CPDA crystals gave well-resolved electron diffractograms from which the reciprocal lattice parameters a*=0.807 nm−1,b *=0.400 nm−1 andγ *=90° could be determined. Systematic absences occurred at every odd reflection along the two orthogonal axesa *andb *. Thus, the CPDA diffraction pattern is consistent with a pgg symmetry. For CADP, the electron diffraction pattern is consistent with a pmg two-dimensional space group withb the unique axis along the ribbon direction. The diagram yields the reciprocal lattice parameters a* = 0.902 nm−1,b *=0.651 nm−1 andγ *=90°. The CBDA electron diffractogram yields the following cell parameters and two-dimensional space group:a *=0.482 nm−1,b *=0.659 nm−1 andγ *=90°, and a pgg symmetry; and that of CADB:a *=0.834 nm−1,b *=0.645 nm−1 andγ *=90°, and a pmg symmetry.
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  • 9
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    Cellulose 1 (1994), S. 197-203 
    ISSN: 1572-882X
    Keywords: viscose fibres ; ferric chloride treatment ; dielectric properties
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Measurements of the dielectric constant and the dielectric loss, at frequencies of 0.05-10 kHz over a temperature range of 10–60 °C, were carried out on viscose fibres, viscose with iron adsorbed and with iron removed. The results obtained show that: (i) a relaxation process is observed in the low-frequency region only in the case of the viscose-iron complex, and (ii) the variation of the dielectric constant with temperature showed a transition at about 30 °C with the untreated fibres, and the transition disappeared when the fibres were treated with ferric chloride. These results, together with changes in hydrogen bonding obtained from infrared spectra for these samples are discussed. Oxidation and adsorption of ferric ions can modify the dielectric properties of viscose fibres.
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  • 10
    ISSN: 1572-882X
    Keywords: cellulose diacetate ; wood pulp ; hemicelluloses ; microgels ; size exclusion chromatography ; calcium interaction ; prehumps
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Solutions of cellulose diacetate (CDA) from two sources (cotton linters and wood pulp Floranier) were analysed in various solvents by size exclusion chromatography (SEC). Without special precautions, the SEC chromatograms presented three peaks — or prehumps — before the main polymer peak. The first prehump which could be eliminated by ultracentrifugation corresponded to microgels whose sugar composition was determined. These microgels were also investigated by electron microscopy, X-ray and electron diffraction analysis. They corresponded mainly to cellulose triacetate (CTA-II) in the case of CDA from cotton linters and a mixture of CTA-II and xylan diacetate (XDA) in the case of CDA from the wood pulp Floranier. The second and third prehumps could be attributed to ionic effects corresponding to the association of remaining sulfate groups on the CDA molecules with residual calcium. It was found that these ionic effects could be eliminated by the addition of LiBr or LiCl to the elution solvents. This led to chromatograms devoid of prehumps.
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  • 11
    ISSN: 1572-882X
    Keywords: bacterial cellulose ; Acetobacter xylinum ; cellulose crystals ; CP/MAS13C NMR ; xyloglucan ; CMC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To obtain further information about the formation of cellulose Iα and Iβ, cross polarization/magic angle spinning (CP/MAS)13C NMR spectroscopy was used to study the effects of polymeric additives, stirring and culture temperature on the Iα When xyloglucan (XG) or carboxymethyl cellulose sodium salt (CMC) was added to the incubation medium, the amount of cellulose Iα decreased markedly, from a normal level of 64% to as low as 30%, with the most additive giving the lowest levels of Iα. Moreover, stirring causes mixtures containing even small amounts of XG to have a large effect. These results suggest that CMC or XG interferes with the aggregation of fibrillar units into the normal ribbon assemblies. It may be that there is a strain associated with this aggregation that results in the higher-energy Iα form. Thus, cellulose Iβ may grow preferentially when the strain caused by aggregation is not present. Lower temperatures (36–10 °C) gave an increase in Iα (from 56 to 72%).
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  • 12
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    Cellulose 1 (1994), S. 107-130 
    ISSN: 1572-882X
    Keywords: chemical pulps ; kraft pulp ; sulfite pulp ; organosolv pulp ; tear strength ; paper strength ; fiber properties ; hornification ; recycling ; swelling of pulp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The literature related to differences between chemical cellulose pulps produced by different pulping processes has been reviewed. Kraft pulps tend to be stronger, particularly in tear strength, while sulfite pulps hydrate and beat more readily. Organosolv pulps tend to mirror the properties of sulfite more than those of kraft pulps. A number of theories have been offered to explain the different properties of the chemical pulps; however, none has been universally accepted. It may be that acidic processes develop weak points in the fibers which are magnified in tear strength losses since, at a constant tensile strength, a 10% loss in fiber strength can lead to a 25–30% loss in tear strength. The effects of acidic pulping may also be magnified in greater fiber breakage and damage in the subsequent refining stages. However, strength improvements for inferior pulps can be realized through post-chemical treatments. Caustic treatments appear to give the greatest improvements, presumably due to increases in acidic group content which results in enhanced swelling properties, and possible subtle reorientation of cell wall polymers. The strength of hornified, recycled fibers can also be enhanced with such treatments, although simple beating will restore considerable strength, but at the expense of drainage rates. It is clear that the processes are complex and involve both the chemistry and physics of the fibers and how these attributes combine to affect the subsequent beating of the fibers for bonding and strength development.
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  • 13
    ISSN: 1572-882X
    Keywords: oil- and water-based inks ; printed paper ; solvent ; interface ; polymer ; surfactant ; flotation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effect of interaction between solvent and three different kinds of printed wastepaper has been studied. The solubility parameter of the solvent was found to be among the most important in order to obtain an optimum ink-fiber interfacial swelling, necessary for de-inking wastepaper. The degree of ink-fiber interfacial swelling was qualitatively estimated by examining the dispersity of ink particles using an optical microscope. FT-IR analyses were carried out to correlate the degree of dispersion and the ink composition in the printed wastepapers studied. The effect of a selective swelling solvent on the de-inking characteristics of a mixture of old wastepapers was investigated. The solvent treatment of these wastepapers prior to flotation de-inking failed to add any positive effect on the brightness of the de-inked pulp. On the other hand, the incorporation of a custom-designed polymer additive improved the pulp brightness without any solvent treatment. The same additive played a negative role in the presence of a swelling solvent. The function of the polymer additive in the flotation de-inking process is also described.
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  • 14
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    Cellulose 1 (1994), S. 169-196 
    ISSN: 1572-882X
    Keywords: cellulases ; hydrolysis ; adsorption ; multidomain structure ; synergy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Most effective cellulolytic enzymes are made of at least two constitutive domains, a catalytic domain and a non-catalytic cellulose-binding domain linked by a flexible peptide. There are several families of catalytic domains and of cellulose-binding domains resulting in a large number of their possible combinations. Removal of the cellulose-binding domain drastically reduces the binding capacity of cellulases to insoluble cellulose while the catalytic efficiency on soluble substrates is usually maintained. Isolated cellulose-binding domains bear most of the binding properties of cellulases (quasi-irreversibility and dispersive effect) but do not hydrolyse cellulose. The multiple types of synergy that cellulases display when acting in combination on cellulose appear to result from their different activities and selectivity, from the substrate microheterogeneity, and sometimes from both.
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  • 15
    ISSN: 1572-882X
    Keywords: insulation paper ; 13C CPMAS NMR ; quantitative intensities ; relaxation times
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A full investigation of the13C CPMAS relaxation times for samples of virgin and aged insulation paper material has revealed the quantitative aspects of the CPMAS technique. We observe, as have others, that the peak due to methyloxy carbon C6 in the solid-state spectrum is reduced in intensity, compared with the other peaks, by ca. 7%. This is a direct result of the difference in relaxation times for the different carbon nuclei. It is shown that simplifying assumptions concerning the relative magnitude of the relaxation times used in the analysis of cross-polarization dynamics are not valid in these materials. In particular, the13C spin-lattice relaxation time in the rotating frame (13C T1ρ
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  • 16
    ISSN: 1572-882X
    Keywords: cellulose I ; molecular mechanics ; crystal structure ; molecular ; modelling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Energies for various trial packing arrangements of unit cells for the Iα and Iβ phases of native cellulose discovered by Sugiyamaet al. were evaluated. Both a rigid-ring method, PLMR, and the full-optimization, molecular mechanics program, MM3(90), were used. For both phases the models that had the lowest PLMR energy also had the lowest MM3 energy. Both calculated models have the chains packed ‘up’, O6s intg positions, and the same sheets of hydrogen-bonded chains. The Iβ structure model is essentially identical to that proposed previously for ramie cellulose by Woodcock and Sarko. It is also the same as the best parallel model previously proposed that was based on the X-ray data of Mann, Gonzalez and Wellard, once the various unit cell conventions are considered. Also, the energies from both methods for all three celluloses, Iα, Iβ and II, are in the order that rationalizes their relative stabilites.
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  • 17
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    Cellulose 1 (1994), S. 205-214 
    ISSN: 1572-882X
    Keywords: heat treatment ; UV exposure ; breaking strength ; chemical changes ; color
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The changes in properties of cellulose brought about by ultraviolet (UV) irradiation, heat exposure and by a combination of both treatments were determined. The methods of characterizing the changes included breaking strength, color, yellowness index, thermal analysis, dye adsorption as a measure of changes in fine structure of the fibers, and Turnbull's Blue test as an empirical measure of carboxyl content. The exposures increased the color and yellowness of the samples as well as the carboxyl content and decreased dye adsorption and breaking strength. A shift of the decomposition endotherm of cellulose to lower temperatures was also noted. It appears that, under the exposure conditions used in this study, the changes in carboxyl content, color changes, yellowness index and breaking strength induced by heat are accelerated by an initial exposure to UV light but still simulate heat ageing alone. There also appears to be a correlation between breaking strength and dye uptake.
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  • 18
    ISSN: 1572-882X
    Keywords: soluble cellulose trifluoroacetates ; thermal and hydrolytic behaviour ; extent of depolymerization ; distribution of substituent ; application of chlorinated hydrocarbons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Cellulose trifluoroacetate (CTFA) with DS values of 1.5 and 2.1 and DP values ranging from 170 to 800 have been prepared free from impurities of the reaction mixture (weakly bound trifluoroacetic acid) and of the procedure of isolation (diethyl ether). The CTFAs are soluble in DMSO, DMF, pyridine, and THF and thermostable up to 250 °C. A convenient synthetic method for CTFAs with DS 1.5 involves the acylation of cellulose with mixtures of trifluoroacetic acid (TFA) and trifluoroacetic anhydride (TFAA, 33% v/v) at room temperature for 4 h and subsequent treatment of the crude polymer at 150 °C and 80 Pa for 40 min. The preparation of CTFAs with DS-values up to 2.1 requires the addition of chloroform and 16 h reaction time.13 C-NMR studies as well as HPLC analyses after methylation and chain degradation show a preferred trifluoroacetylation of the primary hydroxy groups of the cellulose. The extent of depolymerization during the trifluoroacetylation was investigated for various cellulose materials. The cleavage of the trifluoroacetyl groups is possible by treating the derivative with a protic medium like water. Total hydrolysis of CTFA dissolved in DMF with water (room temperature) takes 6 min.
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  • 19
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    Applied microbiology and biotechnology 40 (1994), S. 599-605 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract. In an extended screening using d,l-carnitine amide as carbon or nitrogen source about 1300 strains were obtained by enrichment culture. Of these, 65 strains possessed carnitine amidase activities. A single strain was identified as containing an enzyme able to hydrolyse only l-carnitine amide and yield carnitine of high enantiomeric purity (≥97) when incubated with the racemic substrate. During the initial optimisation of the culture conditions the volume activity could be improved 6.7-fold whereas the specific activity increased 3.6-fold. The enzyme is inducible by l-carnitine amide and carnitine and to a lesser degree also by γ-butyrobetaine and dehydrocarnitine. As judged by the fatty acids and quinone composition the strain belongs into the α-subgroup of purple bacteria but has not yet been classified by the German Culture Collection into a known genus of bacteria.
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  • 20
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    Applied microbiology and biotechnology 40 (1994), S. 595-598 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract. We document here that in those rare cases where disease has been related to Bacillus licheniformis, infection was associated with bypassing the normal biological protective barriers or severely debililated patients. No case suggests any invasive properties of this bacterium. B. licheniformis can therefore be considered non-pathogenic to humans in general. Food-borne illness caused by possible B. licheniformis toxins have been reported, but only in a very few cases and only in connection with consumption of inappropriately prepared food. Considerable experience concerning the industrial use of recombinant B. licheniformis strains has now accumulated and authorities in the United States, Europe and Japan have approved production with and products from recombinant B. licheniformis strains. We conclude that B. licheniformis is a safe host for the production of harmless, industrial products.
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  • 21
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract. Candida tropicalis catalase (CTC) genomic DNA was recombined on a plasmid with the galactose-inducible GAL7 promoter and expressed highly as a heme protein in Saccharomyces cerevisiae as host. The percentage of recombinant CTC (rCTC) in total extractable protein amounted to at least 25%. The rCTC was purified and characterized in terms of subunit mass, behavior in native polyacrylamide gel electrophoresis, absorption spectrum, amino-terminal amino acid sequence, peptide map, specific activity, and Michaelis constant (K m) value for hydrogen peroxide. These properties were similar or identical to those of the purified enzyme from C. tropicalis (CTC). From these results, this system appears suitable for high expression of functional catalase protein having heme.
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  • 22
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    Applied microbiology and biotechnology 40 (1994), S. 676-681 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract. The Thermus thermophilus HB8 mdh and ldh genes and the T. aquaticus EP00276 nox and mdh genes encoding the biotechnologically important enzymes NADH oxidase (EC 1.6.99.3), malate dehydrogenase (EC 1.1.1.37) and lactate dehydrogenase (EC 1.1.1.27) were cloned on the basis of known sequences from related species using the polymerase chain reaction. The nox and mdh genes were directly placed under the control of regulatory expression elements from Escherichia coli. When the 5′-portions of the re-cloned nox gene and the mdh gene of T. thermophilus HB8 were simultaneously altered, enzyme yields of 18–42% of the total soluble cellular protein were obtained as compared to 2–6% obtained from the unchanged genes. The high overproduction level upon the alterations can be explained by the occurrence of additional potential base pairs between nucleotides in the mRNA downstream of the start codon (‘downstream box’) and the 16S rRNA. An ‘universal translation initiation sequence’ providing such strong interactions may be of general use for high overproduction levels.
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  • 23
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract. We purified an extracellular thermostable β-galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s° 20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted β-d-galactopyranosides such as lactose [a Michaelis constant K m=0.75 mm and molecular activity (k cat)=63.1 s−1 at pH 7.2 and 55° C] and p-nitrophenyl β-d-galactopyranoside (K m=0.04 mm and k cat=55.8 s−1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70° C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)n-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula β-galactosidase was stable at pH 7.2 up to 60° C (for 4 h in the presence of 10 μm MnCl2) or 70° C (for 22 h in the presence of 1.75 m lactose and 10 μm MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60° C〈) for efficient production of the oligosaccharides from lactose.
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  • 24
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    Applied microbiology and biotechnology 40 (1994), S. 756-759 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract. Batch and continuous cultures of Pseudomonas fluorescens LB300 were shown to reduce hexavalent chromium, Cr(VI), aerobically at neutral pH (pH 7.0) with citrate as carbon and energy source. The product of Cr(VI) reduction was previously shown and confirmed in this work to be trivalent chromium, Cr(III), by quantitative reoxidation to Cr(VI) with KMnO4. In separate batch cultures (100 ml) containing initial Cr(VI) concentrations of 314.0, 200.0 and 112.5 mg Cr(VI) L−1, the organism reduced 61%, 69% and 99.7% of the Cr(VI), respectively. In a comparison of stationary and shaken cultures, the organism reduced 81% of Cr(VI) in 147 h in stationary culture and 80% in 122 h in shaken culture. In continuous culture, the organism lowered the influent Cr(VI) concentration by 28% with an 11.7-h residence time, by 39% with a 20.8-h residence time and by 57% with a 38.5-h residence time. A mass balance of chromium in a continuous culture at steady state showed an insignificant uptake of chromium by cells of P. fluorescens LB300.
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  • 25
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    Applied microbiology and biotechnology 40 (1994), S. 634-637 
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    Notes: Abstract. Xanthomonas campestris NRRL B-1459 and a variant E2, when preserved on agar slopes (transferred monthly) over 11 months did not deteriorate in their ability to produce xanthan in quantity and quality, as determined by culture in 500-ml baffled flasks. Variations between 8 and 14% (with respect to the average) in the final xanthan concentration were observed for the E2 and B-1459 strains, respectively. A wide range of final viscosities was obtained; these were consistent with the changes in gum concentration. Differences were more likely associated with differences in fermentation kinetics rather than being inherent to the strains. The rheological quality of both polysaccharides was relatively constant throughout the time of culture maintenance. Preservation of these bacteria on agar slopes was an adequate method, in contrast to previous reports. In the period studied, strain E2 produced higher gum titres and slightly lower gum quality compared to strain B-1459.
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  • 26
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    Applied microbiology and biotechnology 40 (1994), S. 606-610 
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    Notes: Abstract. A novel enzyme, l-carnitine amidase, was purified about 140-fold from a newly screened microorganism (DSM 6320) to yield a homogeneous protein. The native enzyme has a molecular mass of 125 kDa (gel filtration) and consists of two identical subunits as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Edman degradation. The pH optimum was found around pH 8.5. Out of 60 chemicals tested as substrates (amides of various aliphatic and aromatic acids, nitriles, amino acid amides and dipeptide amides) the amidase hydrolysed only l-carnitine amide. The Michaelis constant (Km) was found to be 11.6 mm, and the pure protein had a specific activity of 328 units/mg. Complex kinetics were observed with the racemic mixture of d,l-carnitine amide as starting material during enzymatic hydrolysis.
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  • 27
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    Notes: Abstract The effect of nitrogen and fatty-acid-rich substrates on the production of 1-octen-3-ol by the edible fungus Pleurotus pulmonarius, during growth in both shaken flask and fermentor cultures, and in-vitro, in post-harvested mycelium, was studied. Addition of soybean flour and soybean oil to the growth medium enhanced 1-octen-3-ol production about sevenfold and doubled the fungal biomass, as compared to that obtained from P. pulmonarius cultured on a defined synthetic medium. A clear relationship between the production of 1-octen-3-ol and lipoxygenase activity was found during the growth of mushroom pellets. The highest in-vitro generation of 1-octen-3-ol was obtained upon addition of exogenous linoleic acid and pure O2 to pellets grown with soybean fluor and soybean oil. This generation was even higher than that of fruiting bodies exposed to the same conditions. These results suggest that lipoxygenase activity and, subsequently, 1-octen-3-ol biosynthesis in P. pulmonarius are enhanced by the presence of substrates containing fatty acids in the growth medium.
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  • 28
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    Notes: Abstract Batch fermentation performances are usually optimized on the basis of an overall criterion, the mean volumetric productivity. For lack of more suitable criteria, a great number of experiments have to be carried out under various conditions, in order to identify the factors acting on product formation rate. With the help of a mathematical model, every batch fermentation is quantitatively described by a set of parameters, so the reason of every improvement observed for the fermentation productivity is easy to recognize. Therefore, such a model appears to be an invaluable tool for finding quickly and at lower expense the optimal conditions. Nitrogen supplementation and inoculum preparation for lactobacilli growing on whey and whey permeate have been assessed with the help of a new mathematical model.
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  • 29
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    Applied microbiology and biotechnology 40 (1994), S. 650-652 
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    Notes: Abstract A batch fermentation process for lipase production with the recombinant strain Staphylococcus carnosus (pLipMut2) was studied in a bubble column. The rates of growth and lipase production in this type of fermentor were compared with results from shakeflasks. It was seen that cultivation in the bubble column resulted in a prolonged lag time and a reduced lipase activity in comparison to flask cultures. However, by addition of catalase during the fermentation in the bubble column this different behaviour could be avoided.
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  • 30
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    Applied microbiology and biotechnology 40 (1994), S. 653-656 
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    Notes: Abstract Thermolysin was able to catalyze enantioselective peptide synthesis with non-natural amino acids, halophenylalanines. However, the reactivity of thermolysin was considerably influenced by the kind and position of halogen substituents on these analogues. The manner of the recognition of the amino component by the enzyme was different from that of the carboxyl component in the synthesis of peptides with non-natural phenylalanine analogues. The phenomena observed are discussed, based on the kinetic parameters obtained.
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  • 31
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    Notes: Abstract Alginate is used as a matrix for immunoisolation of cells and tissues in vivo. We have demonstrated previously that commercial alginates contain various fractions of mitogenic impurities and that they can be removed by free flow electrophoresis. The use of purified material is a necessity in order to reveal the parameters that control biocompatibility of the implanted material (such as stability, size, surface charge and curvature, etc.). In this study, we present a protocol for the chemical purification of alginates on a large-scale. Beads made from alginates purified by this multi-step chemical extraction procedure did not induce a significant foreign body reaction when implanted for 3 weeks either intraperitoneally or beneath the kidney capsule of Lewis or non-diabetic BB/Gi rats.
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  • 32
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    Notes: Abstract We purified an extracellular thermostable β-galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted β-d-galactopyranosides such as lactose [a Michaelis constant K m=0.75 mm and molecular activity (k cat)= 63.1 s−1 at pH 7.2 and 55° C] and p-nitrophenyl β-d-galactopyranoside (K m=0.04 mm k cat= 55.8 s−1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula β-galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 μm MnCl2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 μm MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C 〈) for efficient production of the oligosaccharides from lactose.
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  • 33
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    Notes: Abstract Bacillus stearothermophilus esterase contains two free cysteine residues at positions of 45 and 115, which react with sulfhydryl reagents resulting in a significant decrease in the enzymatic activity. To understand the role of the cysteine residues in catalytic regions of the esterase, the residues were replaced with serine or alanine by site-directed mutagenesis to construct four single-mutated enzymes (C45A, C45S, C115A, C115S) and two double-mutated ones (C45/115A and C45/115S). Wild-type and mutant enzymes were produced in Escherichia coli cells and purified to homogeneity to examine their chemical and kinetic properties. These mutant enzymes had esterase activity, which suggested that none of the cysteines were required for its activity. Moreover, replacement of both two-cysteine residues made the enzyme insensitive to p-chloromercuribenzoic acid and extensively stabilized it at high temperatures of around 70°C. These results demonstrate that replacement of free cysteine residues by site-directed mutagenesis can improve the thermostability of thermophilic enzymes.
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  • 34
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    Notes: Abstract Candida tropicalis catalase (CTC) genomic DNA was recombined on a plasmid with the galactose-inducible GAL7 promoter and expressed highly as a heme protein in Saccharomyces cerevisiae as host. The percentage of recombinant CTC (rCTC) in total extractable protein amounted to at least 25%. The rCTC was purified and characterized in terms of subunit mass, behavior in native polyacrylamide gel electrophoresis, absorption spectrum, amino-terminal amino acid sequence, peptide map, specific activity, and Michaelis constant (K m) value for hydrogen peroxide. These properties were similar or identical to those of the purified enzyme from C. tropicalis (CTC). From these results, this system appears suitable for high expression of functional catalase protein having heme.
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  • 35
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    Notes: Abstract Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P. mendocina, Pseudomonas sp. DSM 1650 and Pseudomonas sp. GP4BH1 were cloned from genomic libraries in the cosmid pHC79. A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydroxyalkanoic acids (HA mcl ) from octanoate as well as from gluconate to the PHA-negative mutant P. putida GPp104. An 11.0-kbp EcoRI fragment was cloned from P. mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated that strain GP4BH1 possesses three different functionally active PHA synthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HA scl ), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB−4, respectively. The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HA mcl and was expressed in GPp104 but not in PHB−4.
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  • 36
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    Notes: Abstract In Rhodobacter capsulatus, the hupL gene encoding the large subunit of the uptake-hydrogenase (Hup) enzyme complex was mutated by insertion of an interposon. The mutant neither synthesized an active hydrogenase nor grew photoautotrophically. Under conditions of nitrogen (N) limitation, photoheterotrophic cultures of the wild type and the mutant evolved H2 by activity of the nitrogenase enzyme complex. When grown with glutamate as an N source and either d,l-malate or l-lactate as carbon sources, the efficiency of H2 production by the HupL mutant was higher than 90%, whereas wild-type cultures exhibited efficiencies of 54% (with d,l-malate) and 64% (with l-lactate), respectively. With NH inf4 sup+ as the N source, efficiencies of H2 production were 70% (mutant) and 52% (wild type).
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  • 37
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    Applied microbiology and biotechnology 40 (1994), S. 691-698 
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    Notes: Abstract A variety of approaches to maximizing the production of recombinant human α1-antitrypsin (AAT) in Chinese hamster ovary (CHO) cells have been investigated. The highly active and inducible human cytomegalovirus immediate early (IE) promoter/ enhancer was used to drive transcription of a recombinant AAT gene in transiently transfected and stably transformed CHO cells. The AAT gene was modified to incorporate highly efficient 3′RNA processing signals from the herpes simplex virus type 2 IE gene 5, and optimal translational initiation signals were created by site-directed mutagenesis. The effect of flanking the recombinant gene with matrix attachment regions was investigated. Combinations of these modifications allowed secretion of up to 44 μg AAT/ml per day by cell lines growing in serum-rich medium. This could be increased to up to 100 μg AAT/ml per day upon chemical induction of expression by propionate, butyrate or hexamethylene bisacetamide. Cell lines adapted to grow in protein-free medium produced less AAT but still responded to chemical induction to secrete up to 14 μg/ml per day of readily purified AAT.
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  • 38
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    Notes: Abstract Various aerobic Gram-negative bacteria were analysed for utilizing 4-hydroxyhexanoic acid (4HHx) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA). Although many wild types grew on 4HHx, only recombinant strains of the PHA-negative mutants Pseudomonas putida GPp104 and Alcaligenes eutrophus PHB−4, which harboured plasmid pHP1014::E156 with the PHA-biosynthesis genes of Thiocapsa pfennigii, incorporated 4HHx up to a molar fraction of 47 or 1.4%, respectively, into PHA if the cells were cultivated in the presence of 4HHx as sole carbon source and under nitrogen starvation. A terpolyester consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyhexanoic acid (3HHx) and 4HHx was synthesized, as revealed by gas chromatographic analysis of the accumulated polyester and as confirmed by nuclear magnetic resonance spectroscopic analysis of the isolated polyester. 4HHx was also detected in PHA accumulated by Rhodococcus ruber if 4HHx was used as a carbon source. However, it occurred at a molar fraction of maximally 1.3 mol% only beside 3HB, 3-hydroxyvaleric acid and 3HHx.
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  • 39
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    Notes: Abstract Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-14C]d-(−)-hydroxybutyric acid, [1-14C]d-lactic acid, [1-14C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(−)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5′-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-14C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-14C]lactyl-CoA was used as a substrate at a rate that was only less than 10−4 of the rate than with [3-14C]d-(−)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-14C]d-lactyl-CoA as substrate at a relatively high rate.
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  • 40
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    Applied microbiology and biotechnology 40 (1994), S. 729-734 
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    Notes: Abstract In the transition phase of Candida apicola IMET 43747 from logarithmic to stationary growth a pyridine-nucleotide-independent alcohol oxidase was induced coinciding with the beginning of sophorose lipid production. This enzyme was not repressed by glucose and was measurable in stationary cells grown on glucose or on a mixture of n-hexadecane and glucose. An NAD+-dependent aldehyde dehydrogenase behaved in the same way. Both enzymes were localized in the microsomal fraction. The alcohol oxidase accepted long-chain (fatty) aliphatic alcohols (C8 to at least C16) and diols starting from decanediol. Trace activities were found with ω-hydroxy fatty acids. Aromatic, secondary and tertiary alcohols were not oxidized. In the stationary growth phase the substrate specificity of the alcohol oxidase tends to be changed to more hydrophobic substrates. The physiological role of both enzymes, the alcohol oxidase and aldehyde dehydrogenase, is discussed including their possible involvement in the synthesis of sophorose lipid.
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  • 41
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    Notes: Abstract The cellulosome multienzyme complex was dissociated into 12–14 components when incubated at 30° C in a reaction mixture that was buffered at pH 5.0 and was 50 mm with respect to sodium dodecyl sulphate and 10 mm with respect to both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT). The dissociated components reassociated into a complex when dialysed against 20 mm TRIS/HCl buffer, pH 7.7, containing 2.5 mm DTT. When incubated in the presence of Ca2+ and DTT the reassociated complex had the same activity to hydrogen-bond-ordered cellulose as the undissociated cellulosome. However, when Ca2+ ions were incorporated into the TRIS/HCl-DTT dialysis medium the reconstituted complex had very little activity towards cellulose. Other divalent cations such as Mg2+ and Ba2+ had the same effect, but the monovalent cation Na+ resulted in a complex that was very active on crystalline cellulose. The results are interpreted as indicating that the divalent cations bind to one or more of the dissociated polypeptide components and induce changes in conformation that prevent their reassociation into a complex with activity towards crystalline cellulose.
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  • 42
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    Applied microbiology and biotechnology 40 (1994), S. 951-952 
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  • 43
    ISSN: 1432-0614
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    Notes: Abstract A new procedure for the production of ultrafine calcite particles by the marine coccolithophorid alga Pleurochrysis carterae is reported. During continuous culture, calcite particles (coccoliths) were detached from the cell surface by optimized air-bubbling, which greatly reduced the damage associated with previous sonication methods. Detached calcite particles could be continuously recovered directly from the culture medium using a nylon mesh membrane filtration module. Cells remained viable and continued to produce coccoliths during culture. The optimum productivity of ultrafine calcite particles was 18 mg/l per day. These results demonstrate the potential for a continuous system for the photosynthetically driven removal of CO2 and its fixation into ultrafine inorganic calcite particles.
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  • 44
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    Applied microbiology and biotechnology 40 (1994), S. 812-817 
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    Notes: Abstract. A novel membrane bioreactor, previously assessed for its gas transfer characteristics, was used in various size and membrane configurations for the growth of the strictly aerobic bacterium Pseudomonas aeruginosa. The bioreactor was found to readily support growth, and the initial growth rates showed the previously demonstrated enhanced effect in gas O2 mass transfer of the dimpled membrane bioreactor over flat membrane bioreactors. The production of a secondary metabolite by a Pseudomonas sp. following growth was demonstrated, as was the biotransformation of a nitrile by Nocardia rhodochrous with the removal of the biotransformation products across a membrane. The potential of the bioreactor, in terms of other applications in the field of biotechnology, is discussed.
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  • 45
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    Notes: Abstract. Two mutant strains of Bacillus licheniformis insensitive to catabolite repression were selected by classical mutagenesis in connection with the development of a fed-batch procedure for protease production. B. licheniformis 4a produced up to 20 U (Anson-Units) subtilisin Carlsberg/ml in fed-batch experiments in the presence of up to 1.5 m glycerol, but was inhibited by excess ammonium. Formation of spores, excretion of α-amylase and the biosynthesis of citrate synthase and isocitrate dehydrogenase were likewise not repressed by glycerol. The strain was characterized by unusually low activity of the α-oxoglutarate dehydrogenase complex and increased biosynthesis of polyglutamic acid in the presence and excretion of α-oxoglutarate in the absence of ammonium, respectively. The results are discussed in view of a possible connection between the defect in the α-oxoglutarate dehydrogenase complex and insensitivity to catabolite repression. The second strain B. licheniformis 114 was able to synthesize 11.5 U protease/ml independently of the glycerol and ammonium concentration in the medium.
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  • 46
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    Notes: Abstract. The gene from Xanthomonas campestris pv. phaseoli that is involved in the C5 pathway of δ-aminolevulinic acid (ALA) of Escherichia coli. Subcloning of deletion fragments from the initial 2.5-kilobase (kb) chromosomal fragment allowed the isolation of a 1.6-kb fragment that could complement the hemM mutation. Nucleotide sequence analysis of the 1.6-kb DNA fragment revealed an open reading frame that encodes a polypeptide of 426 amino acid residues, and the deduced molecular mass of this polypeptide is 46 768 Da. The amino acid sequence shows a high degree of homology of the HemA protein, which is glutamyl-tRNA reductase, to other organisms. Thus, we examined the complementation test of the cloned gene from Xanthomonas with a hemA mutation of E. coli and found that the gene complemented the hemA mutation. These results suggest that the cloned gene is hemA and the gene from Xanthomonas also complements both hemA and hemM mutations, as in the case of the E. coli hemA.
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  • 47
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    Notes: Abstract. A novel enzyme, N-acylamino acid racemase, was purified to homogeneity from Streptomyces atratus Y-53 and characterized. This enzyme catalyzes the interconversion of optically active N-acylamino acids. The relative molecular mass (Mr) of the enzyme was estimated to be about 41 000 and 244 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of six subunits with an equal Mr. The enzyme showed a broad substrate specificity toward N-acylamino acids, such as N-acetylmethionine, N-chloroacetylphenylalanine and N-chloroacetylvaline. The apparent Michaelis constant (Km) values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 15.2 and 5.6 mm, respectively. Enzyme activity was markedly enhanced by divalent metal ions, such as Co2+, Mg2+ and Mn2+, and was inhibited by metal-chelating reagent, indicating that the enzyme is a metalloenzyme. We propose to name the enzyme N-acylamino acid racemase (acylamino acid racemase).
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  • 48
    ISSN: 1432-0614
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    Notes: Abstract. A novel catechol-substituted cephalosporin, S-9096, showed potent antibacterial activity against Pseudomonas aeruginosa under both iron-deficient and aerobic conditions. S-9096 and ferric iron formed a chelate complex at the molar ratio of 3 to 1, which could be incorporated into P. aeruginosa cells grown under such conditions. Incorporation decreased when the cells were grown under either iron-sufficient or anaerobic conditions, with a concomitant disappearance of iron-regulated outer membrane proteins that were considered to function as receptors for ferric siderophores. These results indicated that the ferric chelate of S-9096 was incorporated into P. aeruginosa cells via a ferric iron transport pathway, which caused the high antibacterial potency of S-9096. All of the S-9096-resistant mutants that were able to grow even under iron-deficient conditions lacked an iron-regulated outer membrane protein having an apparent molecular mass of 66 kDa, suggesting the role of this protein as a receptor for the ferric chelate of S-9096.
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  • 49
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    Applied microbiology and biotechnology 40 (1994), S. 920-925 
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    Notes: Abstract. The occurrence of high concentrations (up to 4900 mg/l) of anions of acetic, propionic and butyric acids in co-produced water from oil reservoirs represents a large pool of potential electron donors for bacterial sulphate reduction. Enrichment cultures in defined media, isolated from a variety of oil-field environments demonstrated the wide distribution of acetate- and propionate-utilising sulphate-reducing bacteria (SRB). A propionate-utilising enrichment culture consisting predominantly of SRB, tentatively identified as species of Desulfobulbus, was used to inoculate a pressurised porous rock bioreactor operating under simulated reservoir conditions. Using a flood velocity of 6.3 cm/h with an inlet propionate concentration of 168 mg/l, a sulphide generation rate of 2.5 μg/ml of rock per hour was achieved at 30° C and 20 MPa. This rate indicates that a sphere of reservoir rock 9.3 m in radius, colonised with propionate-utilising SRB, could produce 50 kg sulphide day. The rates of propionate-driven bacterial sulphate reduction observed in the porous rock bioreactor could sustain the H2S production rates observed from wells in souring reservoirs.
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  • 50
    ISSN: 1432-0614
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    Notes: Abstract. Utilization of the side-chain precursors phenoxyacetic acid (POA) and phenylacetic acid (PA) for penicillin biosynthesis by Penicillium chrysogenum was studied in shake flasks. Precursor uptake and penicillin production were followed by HPLC analysis of precursors and products in the medium and in the cells. P. chrysogenum used both POA and PA as precursors, producing phenoxymethylpenicillin (penicillin V) and benzylpenicillin (penicillin G), respectively. If both precursors were present simultaneously, the formation of penicillin V was blocked and only penicillin G was produced. When PA was added at different times to cells that were induced initially for POA utilization and were producing penicillin V, the POA utilization and penicillin V formation were blocked, whereas the cells started utilizing PA and produced penicillin G. The blocking of the POA turnover lasted for as long as PA was present in the medium. If POA was added to cultures induced initially for PA utilization and producing penicillin G, this continued irrespective of the presence of POA. Utilization of POA increased concomitant with depletion of PA from the medium. Analysis of cellular pools from a growing cell system with POA as precursor to which PA was added after 48 h showed that the cellular concentration of POA was kept high without production of penicillin V and at a concentration comparable to the concentration in the medium. The cellular concentration of POA was higher than the concentration of PA that was utilized for penicillin G production.
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  • 51
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    Notes: Abstract. The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (QsrAb) increased in parallel with increases in the specific growth rate (μ). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qsproduct increased as μ decreased. This report shows that the relationship between cell growth and Qsproduct for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregrate, Qsproduct increased with increases in μ. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers, Qsproduct decreased as μ increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qsproduct to μ are correlated with changes in cell morphology. The relationship between Qsproduct and μ is also affected by the choice of cell line.
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  • 52
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    Notes: Abstract. Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb-CEP) that is biochemically and genetically related to that of the lactococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysis and remains associated with the membrane during lysozyme treatment of cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that preserves the complete in-situ proteinase (mature proteinase) assumed to be the N-terminally processed translation product including the membrane anchor: its monomer molecular mass is approximately 180 kDa. The purified enzyme appeared to be more stable towards heat than hitherto known related, but C-terminally truncated cell-envelope proteinases of lactobacilli and lactococci, which were released from the cells by autoproteolysis. On the basis of its specificity towards caseins, towards the α sl-casein-(1-23)-fragment and towards two differently charged chromophoric peptides, the proteinase was recognized as an (Lb-)CEPI/III mixed-type variant different from those identified so far among the lactococcal proteinases.
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  • 53
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    Applied microbiology and biotechnology 41 (1994), S. 400-406 
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    Notes: Abstract Thermus sp. Rt41A produced a single extracellular proteinase, as determined by fast protein liquid chromatography and isoelectric focusing. Proteinase activity was expressed from very early in the log phase, and halted when the growth substrate was exhausted. There was no continued proteinase production in the stationary phase. Proteinase production was not stimulated by O2 limitation, not repressed by amino acid growth substrates, and its production could not be correlated to the type or oxidation state of the carbon and energy source or the growth rate on different carbon and energy sources. Growth on certain substrates, e.g. glutamate and glucose, resulted in production of high levels of proteinase, whereas others, such as acetate, resulted in low proteinase levels. Acetate repressed proteinase production in cultures growing on L-glutamate. In continuous culture on L-glutamate, acetate or pyruvate, proteinase production was highest at higher growth (dilution) rates. The kinetics of proteinase production in continuous culture on L-glutamate can be interpreted as evidence for the constitutive nature of proteinase expression byThermus sp. Rt41A. The data obtained show that the control of proteinase production is different to that postulated forThermus sp. Ok6.A1.
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  • 54
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    Applied microbiology and biotechnology 41 (1994), S. 384-387 
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    Notes: Abstract The time course of 3-acetyldeoxynivalenol (AcDON) formation was analysed over a 11 day culture period of Fusarium graminearum (DSMO 4258) on rice. The maximum of 2840 mg/kg AcDON was detected after 9 days of culture. To overcome the complicated clean up of the solid substrate extract, 10% methanol in water was applied as extract solvent. After liquid-liquid partition with ethyl acetate, more than 75% of the toxin could be detected. After one simple clean up step (column chromatography over florisil) AcDON could be crystallised. After ion exchange chromatography deoxynivalenol (DON) could be crystallised and an overall yield of 1,44 g DON per kg of culture was obtained.
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  • 55
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    Applied microbiology and biotechnology 41 (1994), S. 495-499 
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    Notes: Pseudomonas putida was grown in a 2.5-l fermentor on a mineral salts medium. Glucose was fed normally over an 18-h period. Gluconate reached about 5 g l-1 in the medium and then fell to zero as it was utilised. The maximum toluene dioxygenase specific activity (2 g g-1 h-1) was obtained over the last 6 h of the fermentation when the pH was fully controlled. In fermentations done at low dissolved O2 tension (DOT) values there was an overall reduction in the cellular enzyme level. When stored at 4°C in phosphate buffer, pH 7.0, harvested bacteria lost half their activity in about 90 h.
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  • 56
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    Notes: Abstract In this work the volumetric O2 transfer coefficient (OTC) through a membrane of a miniaturized hollow-fibre bioreactor was measured by the use of modified O2 microaxial needle electrodes. Before measurment, available electrodes were modified by inserting and gluering them in thin galss capillaries to avoid damage. No differences in the behaviour of the electrodes occurred in comparison to the non-modified electrodes. These modified electrodes allowed O2 partial pressure measurement in the 0.8-mm-high extracapillary space (ECS) of the bioreactor with high sensitivity and reliability. O2 measurements were carried out the two ports of the ECS at different insertion depths. The results of the measurements showed a homogenous O2 supply during variation of the radial co-coordinate of the electrode. In addition to these results, an increase in the local supply in the direction of medium flow was observed . The calculated mean OTC (47–63 h−1) gave extremely improved O2 transfer due to membrane aeration compared to conventional hallow-fibre systems and other bioreactors used in animal-cell culture technology. The improved OTC and the small ECS volume (4.3 ml) makes this culture system suitable for the cultivation of primary cells with tissue-like densities.
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  • 57
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    Applied microbiology and biotechnology 41 (1994), S. 495-499 
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    Notes: Abstract A constitutive blocked mutant (UV4) of Pseudomonas putida was grown in a 2.5-l fermentor on a mineral salts medium. Glucose was fed normally over an 18-h period. Gluconate reached about 5 gl−1 in the medium and then fell to zero as it was utilised. The maximum toluene dioxygenase specific activity (2 g g−1 h−1) was obtained over the last 6 h of the fermentation when the pH was fully controlled. In fermentations done at low dissolved O2 tension (DOT) values there was an overall reduction in the cellular enzyme level. When stored at 4°C in phosphate buffer, pH 7.0, harvested bacteria lost half their activity in about 90 h.
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  • 58
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    Applied microbiology and biotechnology 41 (1994), S. 505-509 
    ISSN: 1432-0614
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    Notes: Abstract Bacillus subtilis strain 316 M was found to produce extracellular alkaline serine proteinase and lectin. The characteristics of proteinase and lectin accumulation during the growth of the producer organism were found to be similar. The maxima of proteolytic and lectin activities were close and observed at 16 h and 14 h of B. subtilis 316 M batch cultivation, respectively. Alkaline serine proteinase was purified by ion exchange chromatography directly from the culture fluid. Proteinase (eluate) purified 40-fold possessed 60–90 units/ml of caseinolytic activity and 240–320 units/ml of elastolytic activity. Eluate obtained after enzyme sorption on the ion exchanger was used for lectin isolation followed by ammonium sulphate precipiration. Lectin purified 12.3-fold was shown to have a high carbohydrate specificity to N-glycolylneuraminic, N-acetylneuraminic, N-acetylmuramic and d-galacturonic acids with minimal inhibiting concentrations of 2.5–7.5 mm. *** DIRECT SUPPORT *** AG903053 00002
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  • 59
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    Notes: Abstract The purpose of this work was to optimize the growth conditions for scale-up of mass production of the main phospholipid (MPL) fraction from the archaebacterium Thermoplasma acidophilum. T. acidophilum was cultivated in flasks in the presence of 32P-ortho-phosphate under varying conditions. The lipids were extracted from the lyophilized cells and analysed by means of two-dimensional thin-layer chromatography. Autoradiography detected up to 11 different fractions. The monoglycosyl derivative of the MPL amounts to 60–80% and thus constitutes the main fraction of the total phospholipid. Variations in growth temperature, pH, yeast extract (YE), glucose and substitution of glucose by citrate were tested. The cells were examined by conventional and electron microscopy. During growth, the pO2 decreased considerably, indicative of rapid O2 consumption by the Thermoplasma cells. Growth under low O2 tension, without addition of O2, and under gassing with N2 or CO2 revealed that T. acidophilum is obligatorily aerobic. For large cell mass production the addition of YE was varied quantitatively and at time intervals. The apparently most economic procedure in the fermentors is: growth at 59°C and pH 2.0, with 10 g glucose/l and total amount of YE 6 g/l, applied in three portions, 2 g/l each, initially, after 18 and 24 h. Harvesting after 40 h yielded 0.66 g/l of cell dry mass. Scaling-up was successful from 1-l flasks to 10-l and 50-l fermentors under aeration of 0.02–0.04 vvm.
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  • 60
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    Applied microbiology and biotechnology 40 (1994), S. 756-759 
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    Notes: Abstract Batch and continuous cultures of Pseudomonas fluorescens LB300 were shown to reduce hexavalent chromium, Cr(VI), aerobically at neutral pH (pH 7.0) with citrate as carbon and energy source. The product of Cr(VI) reduction was previously shown and confirmed in this work to be trivalent chromium, Cr(III), by quantitative reoxidation to Cr(VI) with KMnO4. In separate batch cultures (100 ml) containing initial Cr(VI) concentrations of 314.0, 200.0 and 112.5 mg Cr(VI) L−1, the organism reduced 61%, 69% and 99.7% of the Cr(VI), respectively. In a comparison of stationary and shaken cultures, the organism reduced 81% of Cr(VI) in 147 h in stationary culture and 80% in 122 h in shaken culture. In continuous culture, the organism lowered the influent Cr(VI) concentration by 28% with an 11.7-h residence time, by 39% with a 20.8-h residence time and by 57% with a 38.5-h residence time. A mass balance of chromium in a continuous culture at steady state showed an insignificant uptake of chromium by cells of P. fluorescens LB300.
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  • 61
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    Notes: Abstract A method for enumerating micro-organisms degrading polycyclic aromatic hydrocarbons (PAHs) was developed. The micro-organisms present in water samples are incubated in 96-well microplates, in which the desired PAHs are available as sole carbon source in a liquid mineral-salts medium (MSM). Walls and bottoms of the wells in the microplates are covered with PAHs by dissolving them in a non-polar solvent and pipetting this solution into the wells. After solvent elimination under vacuum, the PAHs remain on the surface of the wells. The formation of coloured products during microbial degradation of PAHs causes colouring of the MSM, thus allowing evaluation of the cell titre by determining the most probable number. Usage of an electronic multichannel pipette makes the work faster and more effective. This allows the inoculation of several microplates pre-treated with different PAHs out of one serial dilution. On the one hand, this method is very effective in screening the usability spectrum of different PAHs microorganisms; on the other hand it allows the additional employment of other sources of hydrocarbons.
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  • 62
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    Applied microbiology and biotechnology 40 (1994), S. 768-771 
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    Notes: Abstract During the anaerobic biodegradation of effluent from a dimethyl terephthalate (DMT) manufacturing plant, reduction in chemical oxygen demand (COD) degradation and biogas formation was observed after the waste-water concentration exceeded 25% of added feed COD. This condition reverted back to normal after 25–30 days when the DMT waste-water concentration in the feed was brought down to a non-toxic level. However, the above effects were observed only after the concentration of DMT waste-water reached more than 75% of added feed COD when biomass support particles (BSP) were augmented to the system. In the BSP system, a biomass concentration of up to 7000 mg/l was retained and the sludge retention time increased to 〉 200 days compared to 2200 mg/l and 8–10 days, respectively, in the system without BSP (control). Formaldehyde in the waste-water was found to be responsible for the observed toxicity. The BSP system was found to resist formaldehyde toxicity of up to 375 mg/l as against 125 mg/l in the control system. Moreover, the BSP system recovered from the toxicity much faster (15 days) than the control (25–30 days). The advantages of the BSP system in anaerobic treatment of DMT waste-water are discussed.
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  • 63
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    Notes: Abstract A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neo r gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 102 to 5 × 103 copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neo r gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.
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  • 64
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    Applied microbiology and biotechnology 41 (1994), S. 615-619 
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    Notes: Abstract Several pieces of evidence indicate that Microcoleu chthonoplastes and Phormidium corium, the predominant cyanobacteria in microbial mats on crude oil polluting the Arabian Gulf coasts, contribute to oil degradation by consuming individual n-alkanes. Both cyanobacteria grew phototrophically better in the presence of crude oil or individual n-alkanes than in their absence, indicating that hydrocarbons may have been utilized. This result was true when growth was measured in terms of dry biomass, as well as in terms of the content of biliprotein, the accessory pigment characteristic of cyanobacteria. The phototrophic biomass production by P. corium was directly proportional to the concentration of n-nonadecance (C19) in the medium. The chlorophyll to carotene ratio of hydrocarbon-grown cyanobacteria did not decrease compared to the ratio in the absence of hydrocarbons, indicating that on hydrocarbons the organisms were not stressed. Comparing the fatty acid patterns of total lipids from hydrocarbon-grown cyanobacteria to those of the same organisms grown without hydrocarbons confirms that n-alkanes were taken up and oxidized to fatty acids by both cyanobacteria.
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  • 65
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    Applied microbiology and biotechnology 41 (1994), S. 620-625 
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    Notes: Abstract Initial hydrolysis rates were examined for mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220 °C (PTW220) and Avicel. Linear rates were observed for fractional conversion relative to the theoretical up to 0.2 for PTW220 and 0.4 for Avicel. Initial rates were essentially unaffected by the presence of growth medium components over a range of pH values. Avicel-hydrolyzing activity was inhibited linearly by ethanol, with a 50% rate reduction at 8 wt.% ethanol. Rate saturation with either substrate or enzyme was observed in a manner qualitatively consistent with previously reported adsorption data. Although somewhat less reactive than Avicel at very low enzyme loadings, much higher reaction rates were observed for PTW220 at moderate and high enzyme loading because of its higher capacity to bind cellulase. At equal subtrate concentrations (as potential glucose) and fractional substrate coverage of 0.09, the initial rate of pretreated wood hydrolysis exceeded that of Avicel by 15-fold. For fractional substrate coverage values up to 0.09 (the maximum value achieved for PTW220), the initial rate was proportional to adsorbed enzyme for PTW220. However, the rate per adsorbed enzyme declined sharply with increasing fractional coverage for Avicel hydrolysis.
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  • 66
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    Applied microbiology and biotechnology 40 (1994), S. 780-785 
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    Notes: Abstract Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h−1. The cell yield reached a maximum of 0.48 g cells·g−1 glucose utilized between dilution rates of 0.05 h−1 and 0.07 h−1 and decreased markedly at higher dilution rates.
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  • 67
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    Notes: Abstract  Immobilized cells of Bacillus licheniformis 44MB82-G were used for the production of thermostable α-amylase. The immobilization was carried out by entrapment in agar gel or by binding to formaldehyde-activated acrylonitrile/acrylamide membranes. The α-amylase production after 144 h of cultivation of membrane immobilized cells was 40% higher in comparison with the free cells. The respective value for the agar-entrapped cells was 22%. Similar trends were observed in the repeated batch fermentations performed with the immobilized cells. The scanning electron micrographs (SEM) of the immobilized cells gave additional information about their binding to the respective carriers.
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  • 68
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    Applied microbiology and biotechnology 41 (1994), S. 505-509 
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    Notes: Abstract  Bacillus subtilis strain 316 M was found to produce extracellular alkaline serine proteinase and lectin. The characteristics of proteinase and lectin accumulation during the growth of the producer organism were found to be similar. The maxima of proteolytic and lectin activities were close and observed at 16 h and 14 h of B. subtilis 316 M batch cultivation, respectively. Alkaline serine proteinase was purified by ion exchange chromatography directly from the culture fluid. Proteinase (eluate) purified 40-fold possessed 60–90 units/ml of caseinolytic activity and 240–320 units/ml of elastolytic activity. Eluate obtained after enzyme sorption on the ion exchanger was used for lectin isolation followed by ammonium sulphate precipitation. Lectin purified 12.3-fold was shown to have a high carbohydrate specificity to N-glycolylneuraminic, N-acetylneuraminic, N-acetylmuramic and D-galacturonic acids with minimal inhibiting concentrations of 2.5–7.5 mM.
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  • 69
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    Notes: Abstract. Alginate is used as a matrix for immunoisolation of cells and tissues in vivo. We have demonstrated previously that commercial alginates contain various fractions of mitogenic impurities and that they can be removed by free flow electrophoresis. The use of purified material is a necessity in order to reveal the parameters that control biocompatibility of the implanted material (such as stability, size, surface charge and curvature, etc.). In this study, we present a protocol for the chemical purification of alginates on a large-scale. Beads made from alginates purified by this multi-step chemical extraction procedure did not induce a significant foreign body reaction when implanted for 3 weeks either intraperitoneally or beneath the kidney capsule of Lewis or non-diabetic BB/Gi rats.
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    Applied microbiology and biotechnology 41 (1994), S. 117-123 
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    Notes: Abstract. A Pseudomonas sp. strain, designated CPE1, was found to be capable of completely mineralizing 4-chlorobiphenyl via 4-chlorobenzoate and of partially dechlorinating 3,4′-dichlorobiphenyl in the presence of biphenyl. A three-membered bacterial consortium, designated ECO3, prepared by combining CPE1 with two chlorobenzoate (CBA)-degrading strains, was capable of extensively degrading and dechlorinating all the monochlorinated biphenyls and several dichlorinated biphenyls in the presence of biphenyl. Both CPE1 and ECO3 were capable of co-metabolizing several low-chlorinated biphenyl congeners of Fenclor 42 in the presence of biphenyl; however, only in ECO3 cultures were high degradation rates and chloride release observed. The higher rate of degradation and mineralization of some polychlorinated biphenyls (PCBs) of Fenclor 42 due to the concerted action of ECO3 members both on PCBs and CBAs suggested that the removal of CBAs from the culture medium may favour PCB degradation, and, therefore, that CBAs may be involved in the regulation of the degradation process of several chlorinated biphenyl congeners.
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  • 71
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    Notes: Abstract. Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O2 limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O2 supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O2 supply.
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  • 72
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    Applied microbiology and biotechnology 41 (1994), S. 90-94 
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    Notes: Abstract. The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60° C) occurs by a different mechanism.
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  • 73
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    Notes: Abstract. Saccharomyces cerevisiae LBG H620 and DSM 2155 strains were continuously cultivated under carbon (C)-limited, phosphorus (P)-limited and nitrogen (N)-limited growth conditions. Cell and protein concentrations in feed, foam, and residue as well as the degree of cell recovery and the rate of foaming were measured, and the concentration and enrichment factors were evaluated at different dilution rates (D). The LBG H620 cells were reduced, while the DSM 2155 cells were enriched in the foam. The highest concentration factors in DSM 2155 cells were attained if they were cultivated under strong P-limitation at a low D. Fairly high concentration factors were also found under C-limitation. Under N-limitation, low concentration factors were found with low Ds. At the beginning of the continuous cultivations, all of the cells were recovered, but with advancing time the degree of recovery and cell concentration and the enrichment factor ratio diminished. The cellular properties of the yeast were characterized by flow cytometry, and the surface properties by measurements of their hydrophobicity, electrophoretic mobility, and chemical composition (using X-ray photoelectron spectroscopy, XPS). These investigations indicated that the large difference in flotation between the two strains is due to different surface properties. Strain DSM 2155 has higher surface hydrophobicity and lower electrokinetic potential. Cell wall properties and the cell flotation depend on medium composition and age of the culture.
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  • 74
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    Notes: Isochrysis galbana rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been grown as a chemostat culture at 20° C and pH 8.00 controlled by CO2 injection. From a low dilution rate (D) of 0.0024 h−1 to 0.0377 h−1, close to maximum growth, a decrease in EPA content from 5.21% dry weight (d.w.) to 2.80% d.w. was observed, although the percentage of EPA in the total fatty acids increased. Lipids were fractionated, EPA being the major fatty acid found in the glycolipid fraction, whereas in the neutral lipid fraction were mainly palmitic and palmitoleic acids. At the same time, the biomass concentration also decreased from 1015 mg·l−1 to 202 mg·l−1 over the range of Ds mentioned. Nonetheless, EPA productivity had a maximum value of 15.26 mg·l−1·day−1 at D=0.0208 h−1.
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  • 75
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    Notes: Abstract. Pure and mixed controlled-pH batch cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 have been conducted. The characteristics of growth and acidification and the productivity of the cultures were compared. During the mixed cultures, the growth characteristics revealed a pronounced stimulation of S. thermophilus whereas L. bulgaricus metabolism was not significantly improved. The final total population was 1.4 to 4.9 higher than in pure cultures. The acidification characteristics were not enhanced by the mixed culture conditions. The productivity of mixed cultures was 1.7 to 2.4 times higher as compared to an equivalent mixing of pure cultures.
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  • 76
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    Notes: Abstract. The growth of four strains of the shiitake mushroom Lentinus edodes in solid substrate fermentation in synthetic oak sawdust logs was studied over a 14-week period. Total extracellular phenol oxidase activity and soluble protein were monitored and biomass estimated as the ergosterol content of the fermented sawdust. It was observed that two of the strains had a similar pattern of phenol oxidase activity with two cycles with maxima at 2 and 8 weeks of mycelial growth prior to fruiting. With the other two strains there was a maximum at week 4. For each strain, phenol oxidase activity increased with the cold shock used to induce fruiting. Phenol oxidase activity was not found to be correlated with either soluble protein or total fungal biomass in the fermented sawdust, which were correlated for each strain. Quantification of biomass from submerged liquid culture on the basis of dry weight and ergosterol contents showed that the strains fell into the same two groups with respect to the ergosterol to biomass ratio, which was markedly lower than that for a strain of L. lepideus.
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  • 77
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    Applied microbiology and biotechnology 41 (1994), S. 130-139 
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    Notes: Abstract. Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improve pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90° C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80° C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.
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  • 78
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    Notes: Abstract. Aerobic biodegradation of a xenobiotic recalcitrant compound sodium anthraquinone-2-sulphonate (SAS), was investigated using as an inoculum a mixed microbial culture, which was activated sludge from industrial and domestic waste-water treatment plants. The difference in SAS degradation was examined using two main systems: (1) suspended cells and (2) immobilized cells, both in batch and in continuous culture. In the suspended cell system, under continuous culture conditions using SAS as a unique source of carbon and energy, it was possible to degrade about 95% of this substrate after 6 days. Maximal SAS removal rates in the suspended-cell system were 59.3 mg SAS l−1 h−1 and 88.7 mg SAS l−1 h−1 for dilution rates (D) of 0.05 h−1 and 0.075 h−1, respectively. In the immobilized-cell system, almost all SAS was degraded in 6 days and the maximal removal rate reached 88.7 mg SAS l−1 h−1 at D=0.05 h−1. Application of a continuous-flow enrichment procedure resulted in selection of several kinds of micro-organisms and led to a progressive elimination of some species of Aeromonas. A stable microbial community of 11 strains has been established and characterized at D=0.075 h−1. Most of them were Gram-negative and belonged to the genus Pseudomonas.
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  • 79
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    Applied microbiology and biotechnology 41 (1994), S. 4-7 
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    Notes: Abstract. Biosurfactant production of eight Streptococcus thermophilus strains, isolated from heat exchanger plates in the downstream side of the regenerator section of pasteurizers in the dairy industry has been measured using axisymmetric drop shape analysis by profile (ADSA-P). Strains were grown in M17 broth with either lactose, saccharose or glucose added. After harvesting, cells were suspended in water or in 10 mm potassium phosphate buffer, pH 7.0, and suspension droplets were put on a piece of FEP-Teflon. Changes in droplet profile were analysed by ADSA-P to yield the surface tension decrease due to biosurfactant production as a function of time. Surface tension decreases larger than 8 mJ·m−2 were taken as indicative of biosurfactant production. Only five strains produced biosurfactants in water, solely when saccharose was added to the growth medium. In buffer, all strains produced biosurfactants and production was generally greater than in water. Also, most strains suspended in buffer produced maximally when saccharose was added to the growth medium, whereas one strain produced maximally in buffer upon the addition of glucose. Four strains suspended in buffer produced biosurfactants when glucose was added and only two strains when lactose was added. The possible role of these biosurfactants as anti-adhesives in the dairy industry and for the survival of these strains in natural systems is discussed.
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  • 80
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    Applied microbiology and biotechnology 41 (1994), S. 58-61 
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    Notes: Abstract. The goal of this work was to select higher fungi for the production of enzymes able to produce plant protoplasts. The hydrolytic capacity of various species of wood-destroying fungi was examined to select those with the maximal hydrolytic capacity. The selection criteria included attack on cellulose, and oligo- and polysaccharides by each fungus species. Lenzites elegans, Ganoderma applanatum and Pycnoporus sanguineus produced the richest enzymic mixtures, which were evaluated for their protoplasting ability. L. elegans has the best enzymic mixture able to produce plant protoplasts with the shortest time for maceration of plant tissues, followed by G. applanatum and P. sanguineus. The potential of these fungi and the simplicity of enzyme production suggest the possibility of commercial applications.
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  • 81
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    Applied microbiology and biotechnology 41 (1994), S. 90-94 
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    Notes: Abstract The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60°C) occurs by a different mechanism.
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  • 82
    ISSN: 1432-0614
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    Notes: Abstract Pure and mixed controlled-pH batch cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 have been conducted. The characteristics of growth and acidification and the productivity of the cultures were compared. During the mixed cultures, the growth characteristics revealed a pronounced stimulation of S. thermophilus whereas L. bulgaricus metabolism was not significantly improved. The final total population was 1.4 to 4.9 higher than in pure cultures. The acidification characteristics were not enhanced by the mixed culture conditions. The productivity of mixed cultures was 1.7 to 2.4 times higher as compared to an equivalent mixing of pure cultures.
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  • 83
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    Notes: Abstract A rapid and sensitive photometric method was devised to assay naphthalene dioxygenase in whole cells of Pseudomonas fluorescens NCIMB 40531, a strain derived from a naphthalene-metabolizing isolate by means of N–methyl-N′-nitro-N–nitrosoguanidine mutagenesis. The naphthalene-assimilating pathway of NCIMB 40531 is functionally blocked at the level of cis–1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase and therefore cis–1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene dihydrodiol) is accumulated when cultures are supplied with naphthalene. This modified metabolism allowed dioxygenase to be assayed by monitoring product formation. Optimal conditions were selected to give linear optical density vs time curves and reaction rates proportional to dry cell weight (DCW): specific activities of 0.125(±0.008) μmol·min–1·mgDCW–1 were consistently obtained in cultures grown on succinate in the presence of naphthalene as inducer. By means of the developed assay, 62 compounds (mainly mono- and bi-cyclic aromatics) were screened as potential inducers of the dioxygenase activity, when added to the growth medium at the concentration of 100 mg·l–1: besides naphthalene, the highest activities were induced by 3-methylsalicylic acid (2-hydroxy-3-methylbenzoic acid), O–acetylsalicylic acid and 5-chlorosalicylic acid with 0.198, 0.167 and 0.076 μmol·min–1.mg DCW–1, respectively. Under the conditions used, no detectable dioxygenase activity was induced by salicylic acid, which is recognized as the natural inducer of the enzyme in Pseudomonas.
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  • 84
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    Applied microbiology and biotechnology 41 (1994), S. 130-133 
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    Notes: Abstract Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.
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  • 85
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    Applied microbiology and biotechnology 41 (1994), S. 134-136 
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    Notes: Abstract The relative toxicity of seven major ground-water pollutants (benzene, chlorobenzene, propylbenzene, ethylbenzene, trichloroethylene, toluene, and styrene) and their metabolites to a soil mycobacterium (Mycobacterium vaccae strain JOB-5) that can catabolize all of these pollutants was determined. The metabolites of chlorobenzene, styrene and trichloroethylene degradation (4-chlorophenol, styrene oxide, and 2,2,2-trichloroethanol, respectively) were less toxic to M. vaccae than was their parent compound. The pollutants propylbenzene, ethylbenzene and benzene were less toxic than their metabolites (4-propylphenol, 4-ethylphenol, and phenol). Metabolites were also examined for their ability to interfere with the biodegradation of selected groundwater pollutants. The metabolites of ethylbenzene, propylbenzene and chlorobenzene biotransformation by M. vaccae were found to adversely affect biodegradation by M. vaccae. Toluene degradation by M. vaccae was inhibited by 4-chlorophenol, 4-ethylphenol and 4-propylphenol at 0.2 mm, 0.4 mm, and 0.4 mm, respectively.
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  • 86
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    Applied microbiology and biotechnology 41 (1994), S. 137-141 
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    Notes: Abstract When an initial cell loading of about 30–40 µg chlorophyll (Chl)·g−1 gel and alginate suspension of 3% (w/v) were used for immobilization of Chlamydomonas reinhardtii, the resulting cell beads showed optimum nitrite uptake rate, at 30° C and pH 7.5, of 14 µmol NO inf2 sup− ·mg−1 Chl·h−1, the photosynthetic and respiratory activities being about 120 µmol O2 produced·mg−1 Chl·h−1, and 40 µmol O2 consumed ·mg−1 Chl·h−1, respectively. The nitrite uptake activity required CO2 in the culture and persisted after 8 days of cells immobilization, or in the presence of 0.2 mm ammonium in the medium. Our data indicate that alginate-entrapped C, reinhardtii cells may provide a stable and functional system for removing nitrogenous contaminants from waste-waters.
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  • 87
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    Applied microbiology and biotechnology 41 (1994), S. 149-154 
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    Notes: Abstract Yeast cells are capable of accumulation of various heavy metals, preferentially accumulating those of potential toxicity and also those of value. They retain their ability to accumulate heavy metals under a wide range of ambient conditions. In the present study it was shown that yeast cells in suspension accumulate heavy metal cations such as Cu2+, Co2+. The level of copper accumulation was dependent on the ambient metal concentration and was markedly inhibited by extremes of ambient pH. Temperature (5–40°C) and the presence of the alkali metal sodium had much smaller effects on the level of copper accumulation. This suggests that in waste-waters of pH 5.0–9.0, yeast biomass could provide an effective bioaccumlator for removal and/or recovery of the metal. During bioaccumulation and subsequent processes it is necessary to retain the biomass. It was shown in the present study that this could be achieved by cell immobilization. Immobilization allowed for complete removal of Cu2+, Co2+, and Cd2+ from synthetic metal solutions. The immobilized material could be freed of metals by use of the chelating agent ethylenediamine tetraacetic acid (EDTA) and recycled for further bioaccumulation events with little loss of accumulation capacity.
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  • 88
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    Notes: Abstract. Thermoanaerobacter thermohydrosulfuricus Rt8.B1 exhibited hyperbolic growth (i.e. a continuous rate of growth, without diauxie, during growth and utilization of two carbon sources) on mixed carbohydrate substrates when grown in pH-controlled batch culture. Hyperbolic growth was observed with xylose in combination with either glucose or cellobiose. Diauxic growth ways observed when T. thermohydrosulfuricus Rt8.B1 was grown on a glucose plus cellobiose substrate mix. The major fermentation end-products under all substrate conditions were ethanol and acetate. Ethanol production varied depending on the substrate supplied and was always greatest on mixtures that included xylose (i.e. hyperbolic growth). High ethanol-to-acetate ratios could not be explained on the basis of a greater substrate uptake and thus more ethanol production under these conditions, or by variations in the levels of acetate kinase and NADP-linked alcohol dehydrogenase synthesis. The high ethanol-to-acetate ratio could not be increased by growing T. thermohydrosulfuricus Rt8.B1 under a partial pressure of hydrogen (1 atm) or by growth at different pH. Growth under these conditions decreased the ethanol-to-acetate ratio.
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  • 89
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    Notes: Abstract The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine κ-casein was studied. A 4-h digest (pH 6.2, 15°C) of κ-casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1–160, 1–151, 1–95 and 1–79 of κ-casein, whereas the main LMM products found were the 161–169 and 152–160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160–161 and 151–152 peptide bonds. Two minor LMM products were identified as the fragments 96–104 and 103–106, indicating additional cleavage at positions 102–103, 104–105 and 106–107 of the sequence. Also several peptide bonds within the 161–169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococaal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79–80 and 95–96. However, the C-terminal region of the κ-casein sequence is the exclusive target of the CEPIII-aand, to variable extents, of the mixed-type enzymes.
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  • 90
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    Applied microbiology and biotechnology 42 (1994), S. 36-39 
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    Notes: Abstract Two isozymes of rice α-amylases expressed and secreted by recombinant yeast were purified by immunoaffinity chromatography by using cross-reactive antibody. Antibodies raised against partially purified barley α-amylase adsorbed rice α-amylases in fermentation broth by a cross-reaction. By use of these antibodies as ligands, rice α-amylases were concentrated and purified to a high degree in one-step immunoaffinity chromatography. Because of the differences in the contaminating impurities between the barley α-amylase (antigen) from barley malt and rice α-amylases (target protein) secreted from yeast, the high purity of eluted α-amylases was attained without the use of highly purified antigen for immunization. Utilization of cross-reactive antibodies in immunoaffinity chromatography is useful for the purification of recombinant proteins in the absence of a sufficient amount and high enough purity of the target proteins to be purified.
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  • 91
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    Applied microbiology and biotechnology 42 (1994), S. 40-45 
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    Notes: Abstract An NADP+ —dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40–43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate. Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH. 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid. The specific activity of the enzyme was about 1400 μmol min−1 mg−1 protein for the reduction of 3-oxobutyrate at pH 5.0. The Michaelis constant (K m) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively. The K m values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively. The identity of 43 of the first 45 N-terminal amino acid residues has been determined. So far such enzyme activities have been described in eucaryotes only.
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  • 92
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    Applied microbiology and biotechnology 42 (1994), S. 57-66 
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    Notes: Abstract Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC encode the ACV (α-aminoadipyl-cysteinyl-valine) synthetase and isopenicillin N-synthetase, respectively. The two adjacent genes are orientated in opposite directions on the chromosomal DNA, separated by a 1.2-kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences from homologous sequences from other strains. To assess the putative promoter strength, we constructed an expression vector carrying the β-glucuronidase (gusA) and β-galactosidase (lacZ) genes in opposite orientation. Fusion of the pcbAB-pcbC promoter region resulted in recombinant vector molecules, which were used for in-vivo expression studies. Using the co-transformation procedure, the reporter gene fusions were transferred into A. chrysogenum recipient strains together with vector pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter genes. The data provide in-vivo evidence that the pcbC promoter is at least five times stronger than the pcbAB promoter. Our approach should prove useful in evaluating regulatory sequences that govern gene expression in A. chrysogenum.
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  • 93
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    Applied microbiology and biotechnology 42 (1994), S. 78-84 
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    Notes: Abstract The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively. On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec− strain as well as in a B. subtilis sacU(HY) strain.
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    Applied microbiology and biotechnology 42 (1994), S. 16-21 
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    Notes: Abstract To produce propionic acid and vitamin B12 from sucrose, the strain Propionibacterium acidipropionici NRRL B3569 was selected by screening a number of Propionibacterium strains. The nutrient composition and the fermentation conditions for this strain were optimized in continuous culture. The investigations show that within a concentration range of 30–170 g l−1 of sucrose in the fermentation medium, no significant substrate inhibition occurred. For the production of propionic acid and vitamin B12, concentrations of 1.5 mg FeSO4·7H2O g−1 dry biomass, 0.75 mg cobalt ions g−1 dry biomass, 0.3 mg 5,6-dimethylbenzimidazole g−1 dry biomass, and 12 g yeast extract 1−1 were necessary additions to the sources of nitrogen, phosphate, and magnesium ions. The extra addition of up to 2.8 g betaine g−1 dry biomass significantly increases the production of vitamin B12. In the optimization of the pH value, temperature, and aeration, it was established that the conditions for propionic acid production and vitamin B12 production are different. Whereas the optimal production of propionic acid took place under completely anaerobic conditions with a pH value of 6.5 and a temperature of 37°C, optimal vitamin B12 production required a temperature of 40°C and aerobic conditions (0.5 vvm aeration at 100 rpm) with a pH value of 6.5.
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    Applied microbiology and biotechnology 42 (1994), S. 22-27 
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    Notes: Abstract Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 109 cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h−1 could be used, which gave higher volumetric productivities (0.96 g l−1 h−1 for propionic acid and 0.26 g l−1 h−1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.
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    Applied microbiology and biotechnology 42 (1994), S. 128-133 
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    Notes: Abstract A lipid component was found in cellulosomes (multienzymatic cellulase complexes) of the thermophilic bacterium Clostridium thermocellum. Two major fractions of the cellulosomes have been studied, one with a relative molecular mass (Mr) of 10–50 million (polycellulosomes, fraction A) and the other with an Mr 0.5–10 million (fraction B) It was found that the larger cellulosomes contained higher relative amounts of lipids (8.1%) as well as Ca2+ ions (0.6%), and showed higher cellulolytic activity Among the lipids was cardiolipin, 1,2- and 1,3-diglycerides, triglycerides, and up to 11 free fatty acids, including both saturated (palmitic, lauric, myristic, pentadecanoic, stearic, arachinic) and unsaturated (myristoleic, palmitoleic, and oleic) moieties Cardiolipin was a major phospholipid component in cellulosomes and was also found to be a major phospholipid component of the cell membrane, palmitic acid was a major fatty acid Fraction B contained less fatty acids (0.5% vs 1.27% in fraction A), with fewer acids detected than in fraction A Removal of the extractable lipids led to fragmentation of the cellulosomes with a concurrent sharp drop in their enzymatic activity Total removal of the lipids from cellulosomes was possible only when the proteins were completely denatured The qualitative composition of the extractable and non-extractable fatty acids was the same The lipid component of the cellulosomes, containing a high content of the unsaturated fatty acids, was located mainly in the part of cellulosomes that is in tight contact with the cellulose surface, and it apparently plays an important role in the tight adsorption of the cellulosomes on cellulose.
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    Applied microbiology and biotechnology 42 (1994), S. 147-152 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The biological removal of ammonia and butanal in contaminated air was investigated by using, respectively, a laboratory-scale filter and a scrubber-filter combination. It was shown that ammonia can be removed with an elimination efficiency of 83% at a volumetric load of 100 m3·m–2·h–1 with 4–16 ppm of ammonia. During the experiment percolates were analysed for nitrate, nitrite, ammonium and pH. It was found that the nitrification in the biofilter could deteriorate due to an inhibition of Nitrobacter species, when the free ammonia concentration was rising in the percolate. It should be easy to control such inhibition through periodic analysis of the liquid phase by using a filter-scrubber combination. Such a combination was studied for butanal removal. Butanal was removed with an elimination efficiency of 80% by a scrubber-filter combination at a volumetric load of 100 m3·m–2·h–1 and a high butanal input concentration. Mixing the filter material with CaCO3 and pH control of the liquid in the scrubber resulted in an increase of the elimination efficiency. These results, combined with previous results on the biofiltration of butanal and butyric acid, allow us to discuss the influence of odour compounds on the removal efficiency of such systems and methods for control. The results were used to construct a full-size system, which is described.
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  • 98
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    Springer
    Applied microbiology and biotechnology 42 (1994), S. 158-166 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The biodegradation of chloroallyl alcohols by pure and mixed bacterial cultures was investigated. Only 2-chloroallyl alcohol and cis– and trans–3-chloroallyl alcohol served as growth substrate for pure cultures. The other chloroallyl alcohols could be cometabolically degraded during growth on 2-chloroallyl alcohol. Cometabolic degradation of trichloroallyl alcohol, which was the most recalcitrant congener, by a Pseudomonas strain isolated on 2-chloroallyl alcohol resulted in 60% dechlorination. Efficient degradation of a mixture of chloroallyl alcohols in continuous culture could only be achieved in the presence of a satellite population. The mixed culture degraded 99% of the total chloroallyl alcohols added with 71% chloride release. The culture contained strains with a new catabolic potential. The results indicate the importance of mixed cultures and genetic adaptation for efficient chloroallyl alcohol removal.
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  • 99
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    Springer
    Applied microbiology and biotechnology 42 (1994), S. 173-178 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract During the rapid mineralization in soil of sucralose (4-chloro-4-deoxy-α,D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-β,D-fructofuranoside), a metabolic product was formed that appears to be the corresponding unsaturated aldehyde. During the slow and incomplete mineralization of sucralose in lake water, which was not increased by the addition of nitrogen and phosphorus, the same compound was produced. That product was further metabolized by microorganisms in lake water and soil. Mineralization was also slow in sewage under aerobic conditions, but organic products were not detected. Little or no CO2 was formed from the disaccharide in flooded soil or anaerobic sewage. Bacteria in culture did not use sucralose as a carbon source but did convert it to the presumed unsaturated aldehyde, 1,6-dichloro-1,6-dideoxy-D-fructose and possibly the uronic acid of sucralose. Sucralose carbon was not incorporated into cells of two sucralosemetabolizing bacteria or the microbial biomass of sewage or lake water. The chlorinated disaccharide was slowly metabolized by a galactose oxidase preparation. It is concluded that the chlorinated sugar is acted on microbiologically by cometabolism.
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  • 100
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    Springer
    Applied microbiology and biotechnology 42 (1994), S. 167-172 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The removal of 5 mg l–1 1,2-dichloroethane [(CH2Cl)2] was studied in two granular activated carbon (GAC) reactors run with hydraulic retention times of below 1 h. One reactor was operated abiotically. The other one was inoculated with microorganisms able to degrade (CH2Cl)2. While the (CH2Cl)2-adsorption capacity of the non-inoculated GAC reactor was exhausted after 20 days, it apparently did not exhaust for at least 170 experimental days in the biologically activated system because (CH2Cl)2 was removed to over 95% as a result of the microbial degradation. The biodegradation was quantified: during the passage through the biologically activated GAC reactor, (CH2Cl)2 (5±1 mg l–1) disappeared, chloride ions (3.3±0.2 mg l–1) were produced, and oxygen (4 to 6 mg l–1) was consumed. Removal of 30% of GAC at the entrance of the reactor, which visibly carried most of the biomass, and its replacement by virgin GAC at the end of the column did not change the apparent (CH2Cl)2removal capacity of the GAC column, indicating that still enough biomass was available to degrade most of the chemical fed. After the addition of the virgin carbon, the effluent concentration fell for a short period of time from about 200 μg l–1 to below 100 μg l–1, indicating partial adsorption of the non-degraded (CH2Cl)2 at the end of the reactor by the virgin carbon. Thus, the modification of the adsorption process by inoculation and maintenance of bacteria with special degradation capabilities resulted in a lower consumption of GAC and thus led to an extended service life of the GAC columns.
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