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  • Biochemistry and Biotechnology  (3,415)
  • Animals  (2,869)
  • AERODYNAMICS
  • 1995-1999  (6,383)
  • 1925-1929  (1)
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  • 101
    Electronic Resource
    Electronic Resource
    On the use of the pH control reagent addition rate for fermentation monitoring (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 480-480 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260490402
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  • 102
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    Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 481-494 
    Details
    ISSN: 0006-3592
    Keywords: Atropa belladonna ; biotransformation ; Duboisia leichhardtii × D. myoporoides, root-shoot coculture ; shooty teratoma ; tropane alkaloids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii × D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6β-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g-1 dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 μM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d. © 1996 John Wiley & Sons, Inc.
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  • 103
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    Microencapsulation of yeast cells and their use as a biocatalyst in organic solvents (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 535-543 
    Details
    ISSN: 0006-3592
    Keywords: whole cell biotransformation ; biocatalyst ; baker's yeast ; immobilization ; microencapsulation ; organic solvents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Stable, semipermeable polyamide microcapsules were prepared by interfacial polymerization from a mixture of 1,6-hexanediamine and poly(allylamine) crosslinked with di-acid chlorides and were used to encapsulate baker's yeast. The size and distribution of cells within the capsules were investigated by a combination of laser confocal, electron scanning, and transmission electron microscopy. The encapsulated cells were studied as a biocatalyst for the model reduction of 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenyl-1-propanone in a number of organic solvents. The polymerization conditions were extensively investigated and were found to greatly influence the product yield. Microencapsulated yeast cells, prepared under optimized conditions, carried out the reduction more efficiently than free cells as well as those immobilized in alginate and κ-carrageenan beads. The developed methodology should be broadly applicable to other biotransformations of interest. © 1996 John Wiley & Sons, Inc.
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  • 104
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    Behavior of glucose oxidase immobilized in various electropolymerized thin films (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 544-552 
    Details
    ISSN: 0006-3592
    Keywords: glucose oxidase ; electropolymerization ; polypyrrole ; polyaniline ; polyindole ; polyphenylediamine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Glucose oxidase was immobilized by electropolymerization into films of polyaniline, polyindole, polypyrrole, poly(o-phenylediamine), and polyaniline crosslinked with p-phenylenediamine. The kinetics and the behavior of the entrapped enzyme toward elevated temperature, organic solvent denaturation, and pH were investigated, along with the response of the films to electroactive species such as acetaminophen, ascorbate, cysteine, and uric acid. For most of the films, linearity to glucose extended from 7 to 10 mM. The poly(o-phenylenediamine)/glucose oxidase film gave the best signal/noise ratio and polypyrrole/glucose oxidase film gave the most reproducible current responses. No significant shift of the optimum reaction pH (5.5), except for polypyrrole (5.0), was observed after immobilization of glucose oxidase in the various films. Enzymatic activity decreased rapidly when pH was raised above 7.5. Thermodeactivation studies at 55°, 60°, and 65°C have shown polypyrrole/and poly(o-phenylediamine)/glucose oxidase films to be the most resistant enzymatic films. Poly(o-phenylenediamine) films offered the best protection against glucose oxidase deactivation in hexane, chloroform, ether, THF, and acetonitrile when compared with the other electropolymerized films. All the enzymatic films exhibited permselection toward electroactive species. © 1996 John Wiley & Sons, Inc.
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  • 105
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    Nucleophile specificity of subtilisin in an organic solvent with low water content: Investigation via acyl transfer reactions (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 553-558 
    Details
    ISSN: 0006-3592
    Keywords: subtilisin ; acyl transfer reaction ; enzymatic peptide synthesis ; organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nucleophile specificity of subtilisin (subtilopeptidase A) was studied via acyl transfer reactions in acetonitrile containing piperidine and 10 vol% of water. Ac-Tyr-OEt was used as acyl donor and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) were used as nucleophiles. The nucleophilic efficiencies of these peptides were characterized by the values of the apparent partition constants, papp, determined from the HPLC analysis of the reactions. The order of preference for the P′1 position was estimated to be: Gly 〉 hydrophilic, positively charged 〉 hydrophobic, aromatic 〉 negatively charged 〉 Leu 〉〉〉 Pro side chain. For the P′2 position the order of preference was: Gly 〉 hydrophilic, charged 〉 hydrophobic, aromatic 〉 Pro side chain. The values of papp for Gly-Gly-Xaa tripeptides cover a range of only two orders of magnitude, with lower nucleophile efficiency for those with hydrophobic amino acid residues in the P′3 position. The dipeptide with Pro in P′1 did not react at all, but a tripeptide having Pro in P′3 was a very good nucleophile. The negatively charged amino acid residues in the P′1 position result in very weak nucleophilic behavior, whereas the peptides with Asp or Glu in P′2 and P′3 are well accepted. Generally, peptides of the Gly-Xaa or Gly-Gly-Xaa series were better nucleophiles than peptides of the Xaa-Gly series. The length of the peptide chain or amidation of α-carboxyl function had no influence on nucleophilic behavior. No significant difference in nucleophile specificity between subtilopeptidase A and nagarse was observed. © 1996 John Wiley & Sons, Inc.
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  • 106
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    Pretreatment of wheat straw using combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and hemicellulose (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 568-577 
    Details
    ISSN: 0006-3592
    Keywords: lignocellulose ; biomass ; lignin degradation ; enzymatic hydrolysis ; furfural ; Aspergillus niger fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The wet oxidation process of wheat straw has been studied as a pretreatment method to attain our main goal: To break down cellulose to glucose enzymatic, and secondly, to dissolve hemicellulose (e.g., for fermentation) without producing microbial inhibitors. Wet oxidation combined with base addition readily oxidizes lignin from wheat straw facilitating the polysaccharides for enzymatic hydrolysis. By using a specially constructed autoclave system, the wet oxidation process was optimized with respect to both reaction time and temperature. The best conditions (20 g/L straw, 170°C, 5 to 10 min) gave about 85% w/w yield of converting cellulose to glucose. The process water, containing dissolved hemicellulose and carboxylic acids, has proven to be a direct nutrient source for the fungus Aspergillus niger producing exo-β-xylosidase. Furfural and hydroxymethyl-furfural, known inhibitors of microbial growth when other pretreatment systems have been applied, were not observed following the wet oxidation treatment. © 1996 John Wiley & Sons, Inc.
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  • 107
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    Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 578-586 
    Details
    ISSN: 0006-3592
    Keywords: Saccharomyces cerevisiae ; hepatitis B surface antigen (HBsAg) ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24s monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24s monomer, is transcribed under the control of the GAL 10p on a chimeric 2-μm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h-1 to washout (0.143 h-1). Both p24s monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24s monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-μm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h-1. Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h-1), even though HBsAg expression was maximal. Total p24s monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h-1. © 1996 John Wiley & Sons, Inc.
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  • 108
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    The application of membrane biological reactors for the treatment of wastewaters (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 601-610 
    Details
    ISSN: 0006-3592
    Keywords: aerobic ; anaerobic ; biomass separation ; bioreactor ; bubbleless ; oxygen mass transfer ; extraction of organic pollutants ; membrane ; wastewaters ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Combining membrane technology with biological reactors for the treatment of municipal and industrial wastewaters has led to the development of three generic membrane processes within bioreactors: for separation and recycle of solids; for bubbleless aeration of the bioreactor; and for extraction of priority organic pollutants from hostile industrial wastewaters. Commercial aerobic and anaerobic membrane separation bioreactors already provide a small footprint alternative to conventional biological treatment methods, producing a high-quality effluent at high organic loading rates. Both the bubbleless aeration and extractive membrane bioreactors are in the development stages. The former uses gas-permeable membranes to improve the mass transfer of oxygen to the bioreactor by providing bubbleless oxygen. By using a silicone membrane process, extractive membrane bioreactors transfer organic pollutants from chemically hostile wastewaters to a nutrient medium for subsequent biodegradation. All three membrane bioreactor (MBR) processes are comparatively and critically reviewed. © 1996 John Wiley & Sons, Inc.
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  • 109
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    Efficient immobilization of lipases by entrapment in hydrophobic sol-gel materials (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 527-534 
    Details
    ISSN: 0006-3592
    Keywords: lipase ; immobilization ; sol-gel materials ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The commercial application of lipases as biocatalysts for organic synthesis requires simple but efficient methods to immobilize the enzyme, yielding highly stable and active biocatalysts which are easy to recover. In this study, we present a novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl-substituted silanes in the presence of the enzyme. A typical immobilization procedure uses: an aqueous solution of lipase; sodium fluoride as a catalyst; and additives like polyvinyl alcohol or proteins and alkoxysilane derivatives like RSi-(OMe)3 with R = alkyl, aryl, or alkoxy as gel precursors. The effect of various immobilization parameters like stoichiometric ratio of water, silane, type and amount of additive, type and amount of catalyst, and type of silane has been carefully studied. The new method is applicable for a wide variety of lipases, yielding immobilized lipases with esterification activities enhanced by a factor of up to 88, compared to the commercial enzyme powders under identical conditions. Studies on the stability of sol-gel immobilized lipases under reaction conditions or storage (dry, in aqueous or organic medium) revealed an excellent retention of enzymatic activity. The possible reasons for the increased enzyme activities are discussed. © 1996 John Wiley & Sons, Inc.
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  • 110
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    Development and bioreactor cultivation of a novel semidifferentiated tissue suspension derived from the marine plant Acrosiphonia coalita (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 559-567 
    Details
    ISSN: 0006-3592
    Keywords: Acrosiphonia ; macroalgae ; tissue culture ; stirred-tank bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A semidifferentiated tissue culture consisting of linear filaments in liquid suspension was established from Acrosiphonia coalita, a cold-water green macroalga known to express pharmacologically active oxylipins deriving from lipoxygenase metabolism of linolenic acid. The tissue was vegatively propagated by blending the filaments down to 1 to 5 mm in length prior to subculture. The filamentous A. coalita tissue suspension was successfully cultivated in an illuminated, 3-L stirred-tank bioreactor at 12°C, 0.46-vvm aeration rate, 250-rpm mixing speed, and incident illumination intensity of 77 μE m-2s-1. The mean specific growth rate over the exponential phase was 0.185 day-1 and a final cell density of 1083 mg dry cell weight (DCW) L-1 was achieved within 15 days of cultivation from an initial cell density of 200 mg DCW L-1. The addition of 3500 ppm CO2 to the aeration gas provided a maximum CO2 transfer rate of six times the maximum CO2 consumption rate and stabilized the pH to 8.0 during the light phase of growth, but did not improve biomass productivity. © 1996 John Wiley & Sons, Inc.
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  • 111
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    Transient-state behavior of a biofilter removing mixtures of vapors of MEK and MIBK from air (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 587-598 
    Details
    ISSN: 0006-3592
    Keywords: gas phase bioreactor ; dynamic behavior ; waste air ; MEK ; MIBK ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the work reported here, selected aspects of the dynamic behavior of biofilters for waste air treatment have been investigated. Emphasis was placed on transient state elimination of mixtures of methyl ethyl ketone (MEK) and methyl isobutyl ketone (MIBK) vapors and on explanation of the observed phenomena. The initial startup, the response of the biofilter to step changes in the pollutant loadings, responses to pollutant pulses, restarting after starvation, and the influence of step changes in gaseous phase oxygen partial pressure are presented and discussed. © 1996 John Wiley & Sons, Inc.
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  • 112
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    Masthead (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996) 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260490501
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  • 113
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    Long-term impact of dissolved O2 on the activity of anaerobic granules (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 611-620 
    Details
    ISSN: 0006-3592
    Keywords: anaerobic sludge ; coculture ; oxygen toxicity ; sludge activity ; wastewater treatment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The impact of influent dissolved O2 on the characteristics of anaerobic granular sludge was investigated at various dissolved O2 concentrations (0.5-8.1 ppm) in 1- and 5-L laboratory-scale upflow anaerobic sludge bed (UASB)-like anaerobic/aerobic coupled reactors with a synthetic wastewater (carbon sources containing 75% sucrose and 25% acetate). The rate of dissolved O2 supplied to the coupled reactor was as high as 0.40 g O2/Lrx·d, and the anaerobic/aerobic coupled reactors maintained excellent methanogenic performances at a COD loading rate of 3 g COD/Lrx·d even after the reactors had been operated with dissolved O2 for 3 months. The activities of granular sludge on various substrates (glucose, propionate, and hydrogen) were not impaired, and acetate activity was even improved over a short term. However, after 3 months of operation, slight declines on the acetoclastic activities of granules were observed in the coupled reactor receiving the recirculated fluid containing 8.1 ppm dissolved O2.Methane yield in the anaerobic control reactor and anaerobic/aerobic coupled reactors revealed that a significant aerobic elimination (up to 30%) of substrate occurred in the coupled reactors, as expected. The presence of dissolved O2 in the recirculated fluid resulted in the development of fluffy biolayers on the granule surface, which imposed a negative impact on the settleability of granular sludge and caused a slightly higher sludge washout. This research shows that the anaerobic/aerobic coupled reactor can be successfully operated under O2-limited conditions and is an ideal engineered ecosystem integrating oxic and anaerobic niches. © 1996 John Wiley & Sons, Inc.
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  • 114
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    High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 621-628 
    Details
    ISSN: 0006-3592
    Keywords: Kluyveromyces ; Candida utilis ; Kluyver effect ; chemostat ; biomass ; whey ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Many facultatively fermentative yeast species exhibit a “Kluyver effect”: even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L-1 from 200 g L-1 maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. © 1996 John Wiley & Sons, Inc.
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  • 115
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    Lysine production from methanol at 50°C using Bacillus methanolicus: Modeling volume control, lysine concentration, and productivity using a three-phase continuous simulation (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 639-653 
    Details
    ISSN: 0006-3592
    Keywords: lysine ; thermotolerant ; methylotroph ; Bacillus methanolicus ; kinetic model ; three-phase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simulation was developed based on experimental data obtained in a 14-L reactor to predict the growth and L-lysine accumulation kinetics, and change in volume of a large-scale (250-m3) Bacillus methanolicus methanol-based process. Homoserine auxotrophs of B. methanolicus MGA3 are unique methylotrophs because of the ability to secrete lysine during aerobic growth and threonine starvation at 50°C. Dissolved methanol (100 mM), pH, dissolved oxygen tension (0.063 atm), and threonine levels were controlled to obtain threonine-limited conditions and high-cell density (25 g dry cell weight/L) in a 14-L reactor. As a fed-batch process, the additions of neat methanol (fed on demand), threonine, and other nutrients cause the volume of the fermentation to increase and the final lysine concentration to decrease. In addition, water produced as a result of methanol metabolism contributes to the increase in the volume of the reactor. A three-phase approach was used to predict the rate of change of culture volume based on carbon dioxide production and methanol consumption. This model was used for the evaluation of volume control strategies to optimize lysine productivity. A constant volume reactor process with variable feeding and continuous removal of broth and cells (VFcstr) resulted in higher lysine productivity than a fed-batch process without volume control. This model predicts the variation in productivity of lysine with changes in growth and in specific lysine productivity. Simple modifications of the model allows one to investigate other high-lysine-secreting strains with different growth and lysine productivity characteristics. Strain NOA2#13A5-2 which secretes lysine and other end-products were modeled using both growth and non-growth-associated lysine productivity. A modified version of this model was used to simulate the change in culture volume of another L-lysine producing mutant (NOA2#13A52-8A66) with reduced secretion of end-products. The modified simulation indicated that growth-associated production dominates in strain NOA2#13A52-8A66. © 1996 John Wiley & Sons, Inc.
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    Role of glucose signaling in yeast metabolism (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 161-165 
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    ISSN: 0006-3592
    Keywords: yeast ; signaling ; regulation ; catabolite repression ; metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, knowledge concerning the relation between uptake of and signaling by glucose in the yeast Saccharomyces cerevisiae is reviewed and compared to the analogous process in prokaryotes. It is concluded that (much) more fundamental knowledge concerning these processes is required before rational redesign of metabolic fluxes from glucose in yeast can be achieved. © 1996 John Wiley & Sons, Inc.
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  • 117
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    Masthead (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996) 
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    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260520101
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  • 118
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    Uncoupling effect of nitrite during denitrification by Pseudomonas fluorescens: An in vivo 31P-NMR study (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 176-182 
    Details
    ISSN: 0006-3592
    Keywords: intracellular pH ; 31P-NMR ; nitrite toxicity ; denitrification ; Pseudomonas fluorescens ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In vivo 31P-NMR was used to investigate the basis for the inhibition of denitrification by nitrite accumulated endogenously by Pseudomonas fluorescens ATCC 17822 (biotype II) at pH 7.0. Cells were immobilized in κ-carrageenan to obtain high cell concentrations in the NMR tube. Acetate and nitrate in two concentration ratios were supplied as electron donor and acceptor, respectively, to achieve different levels of nitrite accumulation. During denitrification, cells were able to maintain a pH gradient of approximately 0.4 to 0.5 units, but when nitrite accumulation reached values approximating 27 mM the transmembrane ΔpH collapsed sharply. Nitrite stimulated the reduction rate of nitrate; furthermore, at nitrite concentrations below 1 mM, activation of oxygen respiratory rates was observed in cells grown under aerobic conditions. The results provide evidence for nitrite acting as a protonophore (an uncoupler that increases the proton permeability of membranes by a shuttling mechanism). © 1996 John Wiley & Sons, Inc.
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    Biochemical engineering IX - interdisciplinary foundations for creating new biotechnology: I (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 1-2 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260520102
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    Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2 (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 166-175 
    Details
    ISSN: 0006-3592
    Keywords: bcl-2 ; apoptosis ; cell culture ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. © 1996 John Wiley & Sons, Inc.
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    Novel bioseparations using two-phase aqueous micellar systems (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 185-192 
    Details
    ISSN: 0006-3592
    Keywords: aqueous micellar system ; hydrophilic protein ; excluded-volume interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We review our recent experimental and theoretical work aimed at investigating the potential use of two-phase aqueous micellar systems for the separation or concentration of hydrophilic biomaterials using the principle of liquid-liquid extraction. The systems studied include (1) a two-phase aqueous micellar system composed of the nonionic surfactant n-decyl tetra(ethylene oxide) (C10E4) and (2) a two-phase aqueous micellar system composed of the zwitterionic surfactant dioctanoyl phosphatidyl-choline (C8-lecithin). The experimental partitioning behavior of several hydrophilic proteins, including cytochrome c, soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in two-phase aqueous C10E4 and C8-lecithin micellar systems is reviewed. A theoretical formulation of the protein partitioning behavior, based on a description of excluded-volume interactions between the hydrophilic proteins and the micelles, is also reviewed. The theoretically predicted protein partitioning behavior is compared with that observed experimentally and is found to be in good agreement. The results of our investigation suggest that two-phase aqueous micellar systems of the type examined in this article are indeed potentially useful as extractant phases for the separation or concentration of proteins and other biomaterials. © 1996 John Wiley & Sons, Inc.
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    Optimization of affinity and ion-exchange chromatographic processes for the purification of proteins (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 204-222 
    Details
    ISSN: 0006-3592
    Keywords: protein purification ; ion exchange chromatography ; metal ion affinity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This study documents several alternative approaches for the optimization of the ion-exchange and affinity chromatographic purification of proteins. In these approaches, the chromatographic process has been treated as a four-stage (adsorption, washing, elution, and regeneration) operation. Central to these investigations has been the elaboration of practical iterative procedures based on the use of theoretical models describing each of these stages. Predictions derived from these models have then been evaluated in terms of experimental data obtained using batch adsorption measurements in finite bath configurations and frontal breakthrough measurements with packed beds of different dimensions, containing nonporous and porous adsorbents of different selectivities and capacities for proteins. Commencing with the kinetic and distribution parameters derived from batch equilibrium measurements, the effect of the initial concentration of the target protein, the solid-liquid volume ratio, the superficial velocity and the column dimensions on the pressure drop, production rate, concentration profile, column utilization, and yield have been determined with packed beds. The potential of these iterative approaches to simplify the determination of key mass transfer and interaction parameters required for scale-up and economic optimization of chromatographic purifications of proteins has been examined using ion exchange, immobilized metal ion affinity, and triazine dye pseudo-affinity adsorbents of different selectivity and adsorption capacities. © 1996 John Wiley & Sons, Inc.
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    Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 193-203 
    Details
    ISSN: 0006-3592
    Keywords: protein aggregation ; protein formulation ; specific interaction sites ; excipient screening ; affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We describe a new protein characterization technique called self-interaction chromatography (SIC), which exploits the specificity of protein-protein interactions that is common to protein aggregates and enables the rapid screening of protein formulation additives as physical stabilizers against aggregation. This technique also enables the identification of specific interaction sites and the determination of their relative importance for self-association. Mannitol, glycine, and dextran 40 were tested for their stabilizing effect toward the model protein lysozyme. Dextran 40 exhibited a poor stabilizing effect. While mannitol stabilized both the native and acid-denatured forms of lysozyme, glycine stabilized the native form with respect to the denatured species. These results are in good agreement with findings in the formulation literature. The SIC shows tremendous potential as a rapid formulation development tool. We also screened two putative interaction sites for involvement in the self-association of lysozyme and estimated the associated binding energies using a binding isotherm model that we developed. The sites screened consisted of residues 41-48 and 125-128 and were selected based on their apparent importance in forming crystal contacts in several different crystal forms of lysozyme. Of the two sites, only residues 125-128 were found to influence self-association under the conditions we employed. Because the success of this technique depends on the exploitation of self-interactions between native species, several important applications are also suggested such as separating native from misfolded or variant species and probing site utilization in aggregation versus crystallization phenomena. © 1996 John Wiley & Sons, Inc.
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    An advanced image analysis system for evaluation of somatic embryo development (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 65-72 
    Details
    ISSN: 0006-3592
    Keywords: somatic embryo ; plant cell culture ; image analysis ; pattern recognition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Somatic embryogenesis is among the most promising means of large scale plant micropropagation. The development of somatic embryos is characterized by their morphological changes. Embryos in culture usually exhibit high heterogeneity and abnormality. As conventional microscopic observation is laborious and subjective, an objective and quantitative morphokinetic description is important for further advancement of this important process technology. We developed an image analysis system capable of measuring morphological and size features of embryos. Subtle environmental effects on embryo development, which are often masked by the subjectivity of microscopic observation, are now discernible by statistically comparing the distributions of these morphological and size features. This image analysis and pattern recognition system was applied to examine the kinetics of a fed-batch culture. © 1996 John Wiley & Sons, Inc.
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    Prevention of clogging in a biological trickle-bed reactor removing toluene from contaminated air (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 91-97 
    Details
    ISSN: 0006-3592
    Keywords: waste-gas treatment ; trickle-bed reactor ; fungi ; biofilm ; toluene ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Removal of organic compounds like toluene from waste gases with a trickle-bed reactor can result in clogging of the reactor due to the formation of an excessive amount of biomass. We therefore limited the amount of nutrients available for growth, to prevent clogging of the reactor. As a consequence of this nutrient limitation a lower removal rate was observed. However, when a fungal culture was used to inoculate the reactor, the toluene removal rate under nutrient limiting conditions was higher. Over a period of 375 days, an average removal rate of 27 g C/(m3 h) was obtained with the reactor inoculated with the fungal culture. From the carbon balance over the reactor and the nitrogen availability it was concluded that, under these nutrient-limited conditions, large amounts of carbohydrates are probably formed. We also studied the application of a NaOH wash to remove excess biomass, as a method to prevent clogging. Under these conditions an average toluene removal rate of 35 g C/(m3 h) was obtained. After about 50 days there was no net increase in the biomass content of the reactor. The amount of biomass which was formed in the reactor equaled the amount removed by the NaOH wash. © 1996 John Wiley & Sons, Inc.
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    Application of light-emitting diodes in bioreactors: Flashing light effects and energy economy in algal culture (Chlorella pyrenoidosa) (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 98-107 
    Details
    ISSN: 0006-3592
    Keywords: algal culture ; bioreactor ; bioregenerative system ; energy economy ; light-emitting diode (LED) ; microsecond pulse modulation ; Chlorella pyrenoidosa ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Light-emitting diodes (LEDs) were used as the sole light source in continuous culture of the green alga Chlorella pyrenoidosa. The LEDs applied show a peak emission at 659 nm with a half-power bandwidth of 30 nm. Selection of this wavelength range, which is optimal for excitation of chlorophylls a and b in their “red” absorption bands makes all photons emitted potentially suitable for photosynthesis. No need for additional supply of blue light was found. A standardized panel with 2 LEDs cm-2 fully covered one side of the culture vessel. At standard voltage in continuous operation the light output of the diode panel appeared more than sufficient to reach maximal growth. Flash operation (5-μs pulse duration) enables potential use of higher operating voltages which may render up to three times more light output. Flat airlift fermentor-type continuous culture devices were used to estimate steady state growth rates of Chlorella pyrenoidosa as a function of the light flux (μmol photons · m-2 · s-1) and the flashing frequency of the light-emitting diodes (which determines the duration of the dark “off” time between the 5-μs “on” pulses). At the fixed voltage and turbidostat setting applied a 20-kHz frequency, which equals dark periods of 45 μs, still permitted the maximum growth rate to become nearly reached. Lower frequencies fell short of sustaining the maximal growth rate. However, the light flux decrease resulting from lowering of the flash frequency appeared to reduce the observed growth rates less than in the case of a similar flux decrease with light originating from LEDs in continuous operation. Flash application also showed reduction of the quantum requirement for oxygen evolution at defined frequencies. The frequency domain of interest was between 2 and 14 kHz. LEDs may open interesting new perspectives for studies on optimization of mixing in mass algal culture via the possibility of separation of interests in the role of modulation on light energy conversion and saturation of nutrient supply. Use of flashing LEDs in indoor algal culture yielded a major gain in energy economy in comparison to luminescent light sources. © 1996 John Wiley & Sons, Inc.
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    Masthead (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996) 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260500201
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    Production and isolation of chitosan by submerged and solid-state fermentation from Lentinus edodes (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 207-210 
    Details
    ISSN: 0006-3592
    Keywords: chitosan ; solid-state fermentation ; Lentinus edodes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method for the laboratory-scale production and isolation of chitosan (polyglucosamine) by liquid and solidstate fermentation from Lentinus edodes was developed. The yields of isolated chitosan were 120 mg/L of fermentation medium under liquid fermentation conditions and 6.18 g/kg of fermentation medium under solid-state fermentation conditions. The latter method, which gives up to 50 times yields than other chitosan production methods from fungi, provides a new flexible and easily scaledup procedure for the production of low acetylation degree chitosan. © 1996 John Wiley & Sons, Inc.
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    Growth kinetics of a bacteriophage in continuous culture (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 217-221 
    Details
    ISSN: 0006-3592
    Keywords: Qβ phage ; molecular evolution ; phage display ; continuous culture ; cellstat ; wall growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lytic coliphage Qβ was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qβ infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed. © 1996 John Wiley & Sons, Inc.
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    Mathematical modeling and analysis of monoclonal antibody production by hybridoma cells (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 238-247 
    Details
    ISSN: 0006-3592
    Keywords: hybridomas ; monoclonal antibody ; kinetic model ; instability ; nutrient and product effects ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An attempt has been made to mathematically describe and analyze monoclonal antibody (MAB) productivity of hybridoma cells, with particular emphasis on continuous cultures under unsteady-state conditions. A simple and unstructured general kinetic model that takes account of productivity loss during long-term cultivation, cell proliferation, and the effects of nutrients and toxic products is proposed. The model is verified with data of continuous culture from five different cell lines under a wide range of experimental conditions. Analysis of these results showed that for a reliable assessment of effects of different factors and for comparison of kinetic data on MAB production it is important to consider possible loss of MAB productivity, the time dependence of which can be modeled by an exponential function plus a constant term. Variations of nutrient concentration, particularly that of glucose, glutamine, and serum, can significantly alter MAB production under certain conditions. These effects can be described in terms of saturation kinetic and/or noncompetitive inhibition kinetics. © 1996 John Wiley & Sons, Inc.
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    Cell cycle analysis of foreign gene (β-galactosidase) expression in recombinant mouse cells under control of mouse mammary tumor virus promoter (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 229-237 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle analysis ; foreign gene expression ; MMTV promoter control ; recombinant mouse cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The cell cycle dependency of foreign gene expression in recombinant mouse L cells was investigated. Two different recombinant mouse L cell lines having the glucocorticoid receptor-encoding gene and the lacZ reporter gene were used in this study. The lacZ gene expression was controlled by the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter in both cell lines. In “M4” cells the gr gene was under the control of another MMTV promoter, but in “R2” cells it was under the control of the constitutive Rous sarcoma virus promoter. These normally attachment-grown cells were adapted to suspension culture, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (β-galactosidase) content of single living cells. Expression of β-galactosidase as a function of cell cycle phase was evaluated for cells in exponential growth without any addition of the glucocorticoid inducer, dexamethasone. Cell cycle positions in the S phase were estimated on the basis of DNA content per cell, and position in the G1 phase was estimated on the basis of cell size as measured by pulse-width time of flight. The results showed that β-galactosidase synthesis occurred through all cell cycle phases, but the expression rate in the G1 phase was much lower than that in the S and G2/M phases in both cell lines. On the basis of cell size analysis, β-galactosidase expression in M4 cells (with autoinducible promoter) was found to be higher than that in R2 cells (with inducible promoter) during the G1 phase. © 1996 John Wiley & Sons, Inc.
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    Optimization of the hydroxylamine cleavage of an expressed fusion protein to produce recombinant human insulin-like growth factor (IGF)-I (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 265-272 
    Details
    ISSN: 0006-3592
    Keywords: recombinant fusion protein ; chemical cleavage ; hydroxylamine ; insulin-like growth factor-I ; protein modification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The application of gene fusion technology for the production of heterologous proteins in Escherichia coli has required the development of specific cleavage methods to separate the coexpressed fusion protein partner from the protein of interest. When hydroxylamine is used to cleave Asn-Gly fusion protein linkages, undesirable chemical modification of asparagine and glutamine amino acids can also occur. In this study, hydroxylamine cleavage conditions were modified to minimize unwanted chemical heterogeneity that occurred during the cleavage of the fusion protein [Met1]-pGH(1-11)-Val-Asn-IGF-I (Long-IGF-I). The cleavage reaction was shown to be dependent on the hydroxylamine concentration, temperature, and pH. Optimal cleavage conditions were identified that resulted in very low levels of chemical heterogeneity, but under these mild conditions that cleavage of the labile Asn-Gly bond was reduced. Therefore, the reaction was further modified to improve the yield of IGF-I while minimizing chemical heterogeneity. The yield of unmodified IGF-I was improved from less than 25% to greater than 70%. Analysis of the heterogeneity produced using the modified cleavage technique showed that Asn26 was converted to a hydroxamate. This variant was characterized in refolding and biological assays where it was equivalent to IGF-I. To further assess the effectiveness of the modified cleavage technique and to evaluate the potential for process scale-up, a gram-scale cleavage reaction of Long-IGF-I was carried out. The process yielded IGF-I with a low level of chemical heterogeneity that was easily removed by ion-exchange chromatography. Moreover, this work shows that the production of unmodified IGFs using hydroxylamine cleavage of fusion proteins is facilitated using the mild cleavage reaction. © 1996 John Wiley & Sons, Inc.
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    Extractive lactic acid fermentation in poly (ethyleneimine)-based aqueous two-phase system (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 280-290 
    Details
    ISSN: 0006-3592
    Keywords: extractive fermentation ; poly(ethyleneimine) ; aqueous two-phase system ; lactic acid ; Lactococcus lactis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The potential of an aqueous two-phase system composed of a polycation, poly(ethyleneimine) (PEI), and an uncharged polymer, (hydroxyethyl) cellulose (HEC), for extractive lactic acid fermentation was tested. Batch fermentation with 20 g/L glucose in two-phase medium using Lactococcus lactis without external pH control resulted in 3-4 times higher amount of lactate and biomass produced as compared to that in a conventional one-phase medium. Lactic acid was preferentially partitioned to the PEI-rich bottom phase. However, the cells which favored the HEC-rich top phase in a fresh two-phase medium were partitioned to a significant extent to the bottom phase after fermentation. Addition of phosphate buffer or pH adjustment to 6.5 after fermentation caused fewer cells to move to the bottom phase. With external pH control, fermentation in normal and two-phase medium showed no marked differences in glucose consumption and lactic acid yield, except that about 1.3 times higher cell density was obtained in the two-phase broth, especially at initial glucose concentrations of 50-100 g/L. Use of higher concentration of phosphate during batch fermentation in the two-phase medium with 50 g/L sugar provided a 15% higher yield of lactic acid, but the growth rate of cells was nearly half of the normal, thus affecting the productivity. Continuous fermentation with twice the normal phosphate concentration resulted in higher cell density, product yield, and productivity in two-phase medium than in monophasic medium. © 1996 John Wiley & Sons, Inc.
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    Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 273-279 
    Details
    ISSN: 0006-3592
    Keywords: metabolic engineering ; CHO cell ; E2F-1 ; serum-free cell culture ; two-dimensional electrophoresis of proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation. © 1996 John Wiley & Sons, Inc.
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    Active-site titration of serine proteases in organic solvents (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 329-335 
    Details
    ISSN: 0006-3592
    Keywords: active-site titration ; serine proteases ; organic solvents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Calculation of kinetic constants of an enzymatic reaction in organic solvents requires knowledge of the functional active-site concentration in organic solvents, and this can be significantly different than that in water. An experimental method for active-site titration of serine proteases in organic media has been developed based on the kinetics of inhibition by phenylmethanesulfonyl fluoride (PMSF), a serine-specific inhibitor (or suicide substrate). This kinetic approach is fundamentally different from other techniques that require complete titration of all accessible enzyme active sites. This active site titration method was applied to subtilisins BPN′ and Carlsberg and α-chymotrypsin and resulted in fractions of active sites that ranged from 8 to 62% (of the fraction active in water) depending on the enzyme, the method of enzyme preparation, and the organic solvent used. The active-site concentration of subtilisin BPN′ and Carlsberg increased with increasing hydrophobicity of the solvent and with increasing solvent hydration in tetrahydrofuran. The dependence of the fraction of active sites on the nature of the organic solvent appears to be governed largely by solvent-induced inactivation caused by direct interaction of a hydrophilic solvent with the enzyme. © 1996 John Wiley & Sons, Inc.
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    Metabolic flux analysis of hybridoma cells in different culture media using mass balances (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 299-318 
    Details
    ISSN: 0006-3592
    Keywords: metabolic flux ; hybridoma cells ; mass balances ; biosynthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The estimation of the intracellular fluxes of mammalian cells using only the mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. Either additional experimental flux data or additional theoretical constraints are required to find one unique flux distribution out of the solution space that is bound by the mass balances. Here, a method is developed using the latter approach. The uptake and production rates of amino acids, glucose, lactate, O2, CO2, NH4, MAB, and the intracellular amino acid pools have been determined for two different steady-states. The cellular composition {total protein and protein composition, total lipids and fatty acid distribution, total carbohydrates, DNA and RNA} has been measured to calculate the requirements for biosynthesis. It is shown to be essential to determine the uptake/production rates of ammonia and either carbon dioxide or oxygen. In mammalian cells these are cometabolites of cyclic metabolic pathways. The flux distribution that is found using the Euclidean minimum norm as the additional theoretical constraint and taking either the CO2 or the NAD(P)H mass balance into account is shown to be in agreement with the measured O2 and CO2 metabolic rates.The metabolic fluxes in hybridoma cells in continuous culture at a specific growth rate of 0.83 day-1 are estimated for a medium with (optimal medium) and without (suboptimal medium) Primatone RL, an enzymatic hydrolysate of animal tissue that causes a more than twofold increase in cell density. It is concluded that (i)The majority of the consumed glucose (〉90%) is channeled through the pentose-phosphate pathway in rapidly proliferating cells.(ii)Pyruvate oxidation and tricarboxylic acid (TCA) cycle activity are relatively low, i.e., 8% of the glucose uptake in suboptimal and 14% in optimal medium, respectively. Under both conditions, only a small fraction of pyruvate is further oxidized to CO2.(iii)The flux from glutamate to α-ketoglutarate (catalyzed by glutamate dehydrogenase) is almost zero in medium with and even slightly reversed in medium without Primatone RL. Almost all glutamate enters the TCA cycle due to the action of transaminases.(iv)Transhydrogenation plays a significant role in hybridoma cells under our experimental conditions. NADPH is produced at relatively high rates (11 × 10-12 to 13 × 10-12 mol · cell-1 · day-1) compared to other fluxes in both culture media. © 1996 John Wiley & Sons, Inc.
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    Kinetics of chromate reduction during naphthalene degradation in a mixed culture (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 357-363 
    Details
    ISSN: 0006-3592
    Keywords: chromium ; chromate ; naphthalene ; reduction ; kinetics ; model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mixed culture of Bacillus sp. K1 and Sphingomonas paucimobilis EPA 505 was exposed to chromate and naphthalene. Batch experiments showed that chromate was reduced and naphthalene was degraded by the mixed culture. Chromate reduction occurred initially at a high rate followed by a decrease in rate until chromate reduction ceased. Chromate reduction decreased in the mixed culture when a lower ratio of S. paucimobilis EPA 505 to Bacillus sp. K1 was utilized. A kinetic model incoporating a term for the cell density ratio is proposed to describe chromate reduction in the mixed culture under both chromate limited and electron donor limited conditions. The validity of the model, and its parameter values, was verified by experimental data generated under a variety of initial population compositions and a broad range of chromate concentrations. The consistent result of experimental data with model predictions implies that the model is useful for evaluating the interactions and the use of mixed culture for chromate removal. © 1996 John Wiley & Sons, Inc.
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    On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 364-372 
    Details
    ISSN: 0006-3592
    Keywords: fiber optic ; firefly luciferase ; in situ ; luminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method was developed to provide a real-time measurement of intracellular adenosine 5′-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for KM. Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. © 1996 John Wiley & Sons, Inc.
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    Monitoring and control of the physiological state of cell cultures (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 271-289 
    Details
    ISSN: 0006-3592
    Keywords: bioprocess control ; physiological state ; Escherichia coli ; plant cell culture ; mammalian cell culture perfusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Advances in bioprocess engineering depends ultimately on the level of understanding and control of the physiological state of the cell population. Process efficiency is strongly influenced by changes in the cellular state which should be monitored, interpreted, and, if possible, properly manipulated. In most control systems this function is not explicitly considered, which hampers process development and optimization. Conventional control logic is based on direct mapping of the growth environment into process efficiency, thereby bypassing the cell state as an intermediate control objective. Today, this limitation is well realized, and explicit monitoring and control of cellular physiology are considered to be among the most challenging tasks of modern bioprocess engineering. We present here a generic methodology for the design of systems capable of performing these advanced monitoring and control functions.The term “physiological state” is quantified by a vector composed of several process variables that convey significant information about cellular state. These variables can be selected among different classes, including specific metabolic rates, metabolic rate ratios, degees of limitation, and others. The real-time monitoring of many of these is possible using commercial sensors. The definition and calculation of representative sets of physiological state variables is demonstrated with examples from several fermentor cultures: recombinant Escherichia coli for phenylalanine production, bioluminescent E. coli (harboring lux genes driven by a heat shock protein promoter) for detection of environmental pollutants, plant cell culture of Perilla frutescensfor anthocyanin production, and perfusion cultures of recombinant mammalian cells (NS0 and CHO) for therapeutic protein production.If the physiological state vector is on-line calculated, the fermentation process can be described by its trajectory in a space defined by the vector components. Then, the goal of the control system is to maintain the physiological state of the cell as close as possible to the trajectory, providing maximum efficiency. A control structure meant to perform this function is proposed, along with the mechanism for its design. In contrast to conventional systems which work in a closed loop in respect to the cell environment, this scheme operates in a closed loop in respect to the cell state. The discussed control concept has been successfully applied to the recombinant phenylalanine production, resulting in physiologically consistent operation, total computer control, and high process efficiency. Initial results from the application of the method to perfusion mammalian cell cultures are also presented. © 1996 John Wiley & Sons, Inc.
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    Prediction of optimal biofilm thickness for membrane-attached biofilms growing in an extractive membrane bioreactor (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 373-386 
    Details
    ISSN: 0006-3592
    Keywords: membrane-attached biofilms ; modeling ; extractive membrane bioreactor ; toxic VOC ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents a mathematical model of membrane-attached biofilm (MAB) behavior in a single-tube extractive membrane bioreactor (STEMB). MABs can be used for treatment of wastewaters containing VOCs, treatment of saline wastewaters, and nitrification processes. Extractive membrane bioreactors (EMBs) are employed to prevent the direct contact between a toxic volatile pollutant and the aerated gas by allowing counterdiffusion of substrates; i.e., pollutant diffuses from the tube side into the biofilm, whereas oxygen diffuses from the shell side into the biofilm. This reduces the air stripping problems usually found in conventional bioreactors. In this study, the biodegradation of a toxic VOC (1,2-dichloroethane, DCE) present in a synthetic wastewater has been investigated. An unstructured model is used to describe cell growth and cell decay in the MAB. The model has been verified by comparing model predicted trends with experimental data collected over 5 to 20-day periods, and has subsequently been used to model steady states in biofilm behavior over longer time scales. The model is capable of predicting the correct trends in system variables such as biofilm thickness, DCE flux across the membrane, carbon dioxide evolution, and suspended biomass. Steady states (constant biofilm thickness and DCE flux) are predicted, and factors that affect these steady states, i.e., cell endogeneous decay rate, and biofilm attrition, are investigated. Biofilm attrition does not have a great influence on biofilm behavior at low values of detachment coefficient close to those typically reported in the literature. Steady-state biofilm thickness is found to be an important variable; a thin biofilm results in a high DCE flux across the membrane, but with the penalty of a high loss of DCE via air stripping. The optimal biofilm thickness at steady state can be determined by trading off the decrease in air stripping (desirable) and the decrease in DCE flux (undesirable) which occur simultaneously as the thickness increases. © 1996 John Wiley & Sons, Inc.
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    Continuous enzymatic production of xylitol with simultaneous coenzyme regeneration in a charged membrane reactor (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 387-396 
    Details
    ISSN: 0006-3592
    Keywords: xylitol ; NADH regeneration ; charged membrane ; continuous production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed a new process for the production of xylitol from D-xylose by enzyme technology. An NADH-dependent xylose reductase (XR) from Candida tenuis catalyzes the reduction of xylose, which is coupled to enzymatic oxidations of D-glucose or D-xylose by glucose dehydrogenase from Bacillus cereus to make achievable an up to 10,000-fold regeneration of NADH per cycle of discontinuous conversion. Using a simple kinetic model as a tool for process optimization, suitable conditions with regard to performance and stability of the multi-component reaction system were established, and 300 g/L of substrate could be converted in yields above 96% in one single batch reaction. Due to selective and over 98% complete retention of the native coenzyme by negatively charged nanofiltration membranes used in a continuously operated enzyme reactor, a specific productivity of 80 g xylitol per liter, day, and kilounit of XR was maintained over the 150-h reaction time with only a single dosage of NADH. © 1996 John Wiley & Sons, Inc.
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    Cell harvesting by cross-flow microfiltration using a shear-enhanced module (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 397-403 
    Details
    ISSN: 0006-3592
    Keywords: cell harvesting ; protein separation ; Vibrio cholerae ; cross-flow microfiltration ; shear-enhanced module ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein, produced by a bacterial culture of recombinant Vibrio cholerae, was separated from cells in a fermentation broth by cross-flow microfiltration. A new, mechanically agitated (rotational) shear filter, the DMFTM filter from Pall, was used to perform the separation. Higher protein recovery and permeate flux than commonly obtained during cell harvesting were demonstrated using sixfold concentration followed by twofold diafiltration. The transmembrane pressure only increased by 10 kPa when the flux was kept constant at 150 L/m2 h during both concentration and diafiltration. The protein transmission was about 100% initially, and over 90% at the end of the concentration process. The protein transmission during the diafiltration was over 80%. The total recovery of protein was 97%. When using an enzymatic cleaning agent, no significant pure water flux decrease was detected during the course of the experiments. © 1996 John Wiley & Sons, Inc.
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    Perturbation of syntrophic isobutyrate and butyrate degradation with formate and hydrogen (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 404-411 
    Details
    ISSN: 0006-3592
    Keywords: anaerobic digestion ; isobutyrate ; butyrate ; isomerization ; hydrogen ; formate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and isobutyrate was investigated using a defined methanogenic culture, consisting of syntrophic isobutyrate-butyrate degrader strain IB, Methanobacterium formicicum strain T1N, and Methanosarcina mazeii strain T18. Formate and hydrogen were used to perturb syntrophic butyrate and isobutyrate degradation by the culture. The reversible isomerization between isobutyrate and butyrate was inhibited by the addition of either formate or hydrogen, indicating that the isomerization was coupled with syntrophic butyrate degradation for the culture studied. Energetic analysis indicates that the direction of isomerization between isobutyrate and butyrate is controlled by the ratio between the two acids, and the most thermodynamically favorable condition for the degradation of butyrate or isobutyrate in conjunction with the isomerization is at almost equal concentrations of isobutyrate and butyrate. The degradation of isobutyrate and butyrate was completely inhibited in the presence of a high hydrogen partial pressure (〉2000 Pa) or a measurable level of formate (10 μM or higher). Significant formate (more than 1 mM) was detected during the perturbation with hydrogen (17 to 40 kPa). Resumption of butyrate and isobutyrate degradation was related to the removal of formate. Energetic analysis supported that formate was another electron carrier, besides hydrogen, during syntrophic isobutyrate-butyrate degradation by this culture. © 1996 John Wiley & Sons, Inc.
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    Two-dimensional versus three-dimensional culture systems: Effects on growth and productivity of BHK cells (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 429-432 
    Details
    ISSN: 0006-3592
    Keywords: BHK ; aggregates ; porous microcarriers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of surface growth (two-dimensional microcarriers) and three-dimensional growth (aggregates and macroporous supports) in agitated, suspended batch culture systems upon growth and productivity of BHK was compared. Cultures using three porous microcarriers (CultiSpher G, Cellsnow EX, and Cytocell), one nonporous microcarrier (Cytodex 3) and natural aggregates were performed in stirred tanks using two different agitation rates (60 and 100 RPM). With the exception of Cytocell, cell growth, viability, and productivity were similar when three-dimensional structures (porous microcarriers and aggregates) were used. Nonporous microcarriers only compared well at 60 RPM as growth ceased under overagitation. These results suggest that cultures less susceptible to fluid shear are advantageous for scale-up. © 1996 John Wiley & Sons, Inc.
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    Growth and respiration of Petunia hybrida cells in chemostat cultures: A comparison of glucose-limited and nitrate-limited cultures (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 412-422 
    Details
    ISSN: 0006-3592
    Keywords: Petunia hybrida ; chemostat cultures ; glucose limitation ; nitrate limitation ; growth ; respiration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nitrate-limited and glucose-limited chemostat cultures of Petunia hybrida cells were compared at a specific biomass (+extracellular product) formation rate of 0.0042 C·mol/C·mol h. The composition of the biomass differed considerably in both culture types. The N/C (mol/mol) ratio in the biomass was almost four times lower in the nitrate-limited than in the glucose-limited cultures. On a dry weight basis (g/g DW) the biomass in the nitrate-limited cultures contained about 2.5 times less ions and protein N and about 2.5 times more carbohydrates than the biomass in the glucose-limited cultures. On a fresh weight basis (mmol/g FW) the biomass in nitrate-limited and glucose-limited cultures differed mainly in carbohydrate content. The yields of biomass on glucose and oxygen were generally higher in the nitrate-limited than in the glucose-limited cultures. Average values for these parameters were 0.27 C · mol biomass/C · mol glucose and 0.42 C · mol biomass/mol O2 in the glucose-limited cultures and 0.34 C · mol biomass/C · mol glucose and 0.55 C · mol biomass/mol O2 in the nitrate-limited cultures. On a C · mol basis the total respiration was about 25% and the maximally attainable cytochrome pathway activity (measured in the presence of hydroxamate) about 30% higher in the glucose-limited than in the nitrate-limited cultures. The maximally attainable activity of the alternative pathway (measured in the presence of KCN) was significantly lower in the glucose-limited cultures. On an organic N (≈protein) basis all respiratory parameters were significantly higher in the nitrate-limited cultures. In the presence of the respiratory uncoupler carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and excess glucose, cellular respiratory activity shows its maximal activity; under these conditions the total respiration increased more than 150% in the glucose-limited and only 30% in the nitrate-limited cultures. It is suggested that glucose-limited cultures are able to react more flexibly to changes in the environmental conditions than nitrate-limited cultures. © 1996 John Wiley & Sons, Inc.
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    Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 423-428 
    Details
    ISSN: 0006-3592
    Keywords: hydrogenase ; organic biocatalysis ; polyethylene glycol ; modified enzymes ; sulfur ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H2S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen nor PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H2S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bio-processing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology. © 1996 John Wiley & Sons, Inc.
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    Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 529-540 
    Details
    ISSN: 0006-3592
    Keywords: T-lymphocytes ; immunotherapy ; HTLV-1 ; CTL ; dendritic cell ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: CD8+ cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8+ T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 × 106 cells following 5 weeks of culture. Expanded cells contained primarily CD3+ T-cells, of which CD8+ T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro 51Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8+ T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. © 1996 John Wiley & Sons, Inc.
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    Effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 555-561 
    Details
    ISSN: 0006-3592
    Keywords: human aortic smooth muscle cells ; shear stress ; restenosis ; growth rate ; PCNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: After cardiovascular intervention, smooth muscle cells (SMC) are directly exposed to blood flow and thus their behavior might be affected by fluid hemodynamic forces. The aim of this study was to determine the effect of fluid shear stress on the growth rate of SMC. Human aortic smooth muscle cells (hASMC) were seeded on fibronectin-coated glass slides and were exposed to different levels of shear stress using parallel plate flow chambers. After 24 h, cell numbers in the stationary and sheared cultures were measured by a Coulter counter. Results demonstrated that increasing shear stress significantly reduces the proliferation rate of hASMC (P 〈 0.05). Comparable lactate dehydrogenase levels in the media of stationary and flow cultures provided evidence that the reduction of cell number was not due to cell injury. Proliferating cell nuclear antigen (PCNA) immunofluorescence studies indicated that the cell cultures were not growth arrested 24 h after exposure to shear stress, and that the differences in PCNA staining between stationary control and flow cultures were comparable to the cell counts. © 1996 John Wiley & Sons, Inc.
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    Tissue engineering lamb heart valve leaflets (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 562-567 
    Details
    ISSN: 0006-3592
    Keywords: tissue engineering ; synthetic biodegradable matrix ; polyglycolic acid ; polylactic acid ; endothelial cell ; heart valve ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Tissue engineered lamb heart valve leaflets (N - 3) were constructed by repeatedly seeding a concentrated suspension of autologous myofibroblasts onto a biodegradable synthetic polymeric scaffold composed of fibers made from polyglycolic acid and polylactic acid. Over a 2-week period the cells attached to the polymer fibers, multiplied, and formed a tissue core in the shape of the matrix. The tissue core was seeded with autologous large-vessel endothelial cells that formed a monolayer which coated the outer surface of the leaflet. The tissue engineered leaflets were surgically implanted in place of the right posterior pulmonary valve leaflet of the donor lamb while on cardiopulmonary bypass. Pulmonary valve function was evaluated by two-dimensional echocardiography with color Doppler which demonstrated valve function without evidence of stenosis and with only trivial regurgitation under normal physiologic conditions. Histologically, the tissue engineered heart valve leaflets resembled native valve leaflet tissue. © 1996 John Wiley & Sons, Inc.
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    Effect of culture age on the susceptibility of differentiating neuroblastoma cells to retinoid cytotoxicity (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 580-586 
    Details
    ISSN: 0006-3592
    Keywords: tissue engineering ; N1E-115 neuroblastoma cells ; electrophysiological differentiation ; retinoid cytotoxicity ; teratogenesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The cytotoxic effects of retinoids on neuroblastoma cells at various times during electrophysiological differentiation were evaluated. We used N1E-115, a clone of the murine neuroblastoma C1300 derived from the neural crest, and three retinoids: vitamin A (retinol), all-trans retinoic acid (tretinoin), and 13-cis-retinoic acid (isotretinoin). Differentiating N1E-115 cells exposed to retinoids at an isotretinoin EC50 of 16 μM exhibited the greatest vulnerability in terms of cell death during a period (8 to 10 days) that was previously found to be the most sensitive for induction of gross malformations in rodents. This finding suggested possible similarities between the in vivo and in vitro retinoid mechanism(s) of action. The greatest period of vulnerability to retinoid cytotoxicity was also found to coincide with the rapid resting membrane potential (Vm) development period, suggesting a linkage between neuronal Vm and/or electrical excitability development and vulnerability to retinoid cytotoxicity. © 1996 John Wiley & Sons, Inc.
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    Masthead (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996) 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260500501
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    Tissue engineering, bioartificial organs, and cell therapies: II (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 463-463 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260500502
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    Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 609-616 
    Details
    ISSN: 0006-3592
    Keywords: enzyme inactivation ; substrate modulation ; product modulation ; penicillin acylase ; 6-aminopenicillanic acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inactivation of immobilized penicillin acylase has been studied in the presence of substrate (penicillin G) and products (phenylacetic acid and 6-aminopenicillanic acid), under the hypothesis that substances which interact with the enzyme molecule during catalysis will have an effect on enzyme stability. The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free-enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage. 6-Aminopenicillanic acid and penicillin G stabilized the enzyme in the first stage of decay. Modulation factors in that stage were 0.96 for penicillin G and 0.98 for 6-aminopenicillanic acid. Phenylacetic acid increased the rate of inactivation in both stages, modulating factors being -2.31 and -2.23, respectively. Modulation factors influence enzyme performance in a reactor and are useful parameters for a proper evaluation. © 1996 John Wiley & Sons, Inc.
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    Characterization of acetone-washed yeast biomass functional groups involved in lead biosorption (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 1-10 
    Details
    ISSN: 0006-3592
    Keywords: metal biosorption ; acetone-washed yeast biomass ; Saccharomyces uvarum ; lead binding mechanisms ; X-ray photoelectron spectroscopy ; Fourier transform infrared photoacoustic spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The mechanism of lead cation biosorption by acetone-washed biomass of Saccharomyces uvarum was investigated by chemical modifications and spectroscopic monitoring of the cell components. Reacting the carboxyl groups with propylamine, which neutralizes these anions, considerably decreased the metallic ion uptake, indicating that negatively charged carboxyl groups play an important role in lead bisorption due to electrostatic attraction. After lead biosorption the photoacoustic Fourier transform infrared spectroscopy showed a change in the symmetrical stretch of the carboxylate groups of the acetone-washed yeast biomass, and the X-ray photoelectron spectroscopy oxygen peak was also found to be shifted. These findings support the hypothesis that lead uptake occurs mainly through binding to the carboxyl group. In X-ray photoelectron spectroscopy the nitrogen peak decreased after the biosorption of lead, suggesting that nitrogen-containing groups are also involved in the biosorption process. Acylation of amino groups was shown to increase the lead biosorption capacity. The acylation reaction converts the positively charged amino group to an amide capable of coordination to lead cations. Deproteination by boiling the biosorbent with NaOH increased the lead uptake. The acetone-washed biomass uptake of lead from an aqueous solution at ph 5.5 was 48.9 mg/g dry weight. Pure chitin adsorbed 48.8 mg lead/g dry weight. Mannan isolated from S. uvarum did not adsorb lead at all. Electrostatic attraction of the carboxyl groups and other anions present in the acetone-washed biomass, and complexation with nitrogen atoms, especially in chitin, appear to be the main mechanisms involved in lead cation biosorption. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 1-10, 1997.
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    Mechanism of aluminum interference on uranium biosorption by Rhizopus arrhizus (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 16-27 
    Details
    ISSN: 0006-3592
    Keywords: uranium ; aluminum ; biosorption ; Rhizopus arrhizus ; mechanism ; competing ions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Uranium competitive uptake experiments by Rhizopus arrhizus were carried out at three different solution pH levels and in the presence of different concentrations of competing aluminum ions in order to examine the competing ion effect. The coion effect became more pronounced as the coion concentration in solution and pH level increased. A preliminary examination of the effect of aluminum on the rate of uranium uptake was also completed. Results showed that the presence of aluminum does not interfere with the kinetics of uranium uptake by R. arrhizus. Electron microscopic and energy dispersive X-ray analyses were also performed on samples of the biomass. The combination of spectral data and the information from the equilibrium studies and the kinetic studies suggested that aluminum interferes with the uranium biosorptive uptake capacity of R. arrhizus by the precipitation of a metastable amorphous hydroxy polymeric precipitate through a mechanism we refer to as steric competition. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 16-27, 1997.
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    Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 28-32 
    Details
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; poly(3-hydroxybutyrate) ; fed-batch fermentation ; phosphate limitation ; dissolved oxygen concentration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH4OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L · h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.
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    Steady-state solution of a two-species biofilm problem (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 675-686 
    Details
    ISSN: 0006-3592
    Keywords: biofilm ; steady state ; heterotrophs ; nitrosomonas ; nitrobacter ; model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Through a thorough investigation of the boundary conditions for a general two-species biofilm model, a simple and fast method for solving the steady-state case is developed and presented. The methods used may be extended to biofilm models in which more than two species are considered. Four different sets of boundary conditions are possible for the two-species biofilm model. Each set is shown to be asymptotically stable. A biofilm model describing the competition between autotrophic and heterotrophic bacteria and a biofilm model considering only Nitrosomonas and Nitrobacter are used for illustration. A parameter Lcrit, critical film thickness for bacterial coexistence, is introduced from which criteria on the bulk concentrations for coexistence are derived. From these criteria it is seen that the thinner the biofilm, the more restrictive the conditions are for steady-state coexistence. For thin biofilms there may, in many cases, be no point in considering more than one species in the biofilm model. Furthermore, the gradients of the bacterial concentrations are in many cases negligible in thin biofilms, and the biofilm may then be assumed to be homogeneous. The criteria on the bulk concentrations together with the four sets of boundary conditions provide the necessary information for a direct solution of the steady-state two-species biofilm model by means of an ordinary differential and algebraic equation solver. © 1996 John Wiley & Sons, Inc.
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    Masthead (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996) 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260500601
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    Enzymatic synthesis of a CCK-8 tripeptide fragment in organic media (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 700-708 
    Details
    ISSN: 0006-3592
    Keywords: α-chymotrypsin ; organic media ; tripeptide synthesis ; CCK-8 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the α-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R1 and Met-R2 have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-α protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of α-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. © 1996 John Wiley & Sons, Inc.
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    Microscale-based modeling of polynuclear aromatic hydrocarbon transport and biodegradation in soil (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 1-14 
    Details
    ISSN: 0006-3592
    Keywords: biodegradation ; desorption ; mathematical model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model to describe polynuclear aromatic hydrocarbon (PAH) desorption, transport, and biodegradation in saturated soil was constructed by describing kinetics at a microscopic level and incorporating this description into macroscale transport equations. This approach is novel in that the macroscale predictions are made independently from a knowledge of microscale kinetics and macroscopic fluid dynamics and no adjustable parameters are used to fit the macroscopic response. It was assumed that soil organic matter, the principal site of PAH sorption, was composed of a continuum of compartments with a gamma distribution of desorption rate coefficients. The mass transport of substrates and microorganisms in a mesopore was described by diffusion and that in a macropore by one-dimensional advection and dispersion. Naphthalene was considered as a test PAH compound for initial model simulations. Three mechanisms of naphthalene biodegradation were considered: growth-associated degradation as a carbon and energy source for microbial growth; degradation for maintenance energy; and growth-independent degradation. The Haldane modification of the Monod equation was used to describe microbial growth rates and to account for possible growth inhibition by naphthalene. Multisubstrate interactions were considered and described with a noninteractive model for specific growth rates. The sensitivity of selected model parameters was analyzed under conditions when naphthalene was the sole growth-rate-limiting substrate. The time necessary to achieve a specific degree of naphthalene biodegradation was found to be proportional to the initial concentration of naphthalene in soil organic matter. The biodegradation rate of naphthalene increased when the sorption equilibrium constant of naphthalene was reduced. The presence of an alternative carbon source inhibited naphthalene biodegradation in spite of the calculated increase in biomass. © 1996 John Wiley & Sons, Inc.
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    Selective extraction using preferential transport through adsorptive membranes (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 539-548 
    Details
    ISSN: 0006-3592
    Keywords: adsorptive membranes ; facilitated diffusion ; parametric pumping ; uphill transport ; integrated processes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Selective extraction of a protein from a mixture can be accomplished using an adsorptive membrane and low displacement recuperative parametric pumping. Low displacement recuperative parametric pumping can lead to the preferential transport of an adsorbing solute and the rejection of nonadsorbing solutes by the adsorptive membrane. Using a protein mixture consisting of lysozyme and myoglobin, we have found the conditions under which lysozyme is preferentially transported through an ion-exchange membrane cartridge while myoglobin is rejected by the membrane. Trends observed when parameters such as the desorbent concentration, feed concentration, and flow rate are varied agree with the predictions of a mathematical model. Comparison with facilitated diffusion shows that preferential transport can lead to higher solute fluxes, albeit at lower selectivity. Additionally, preferential transport can be used to transport a solute up a concentration gradient and to selectively extract a solute from a feed that contains suspended solids. © 1996 John Wiley & Sons, Inc.
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    Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: I. The phenomena and characterization of oscillation and hysteresis (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 549-560 
    Details
    ISSN: 0006-3592
    Keywords: glycerol fermentation ; Klebsiella pneumoniae ; oscillation ; hysteresis ; growth and metabolism ; substrate excess ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Oscillation and hysteresis phenomena are observed in the anaerobic continuous fermentation of glycerol by Klebsiella pneumoniae in long-term cultivations under a variety of conditions. In this work, the conditions for the occurrence of these phenomena are reported and the patterns of cell growth and metabolism under oscillation are characterized. During an oscillation period, the formation rates of CO2, H2, and formate and the consumption rate of alkali periodically pass values of maxima and minima, the latter being close to zero. The formation of biomass and fermentation products such as 1,3-propanediol, acetate, and ethanol also undergo periodic changes which shift maxima and minima. Sustained oscillation occurs only under conditions of substrate excess within a distinct regime. At pH 7.0, it is only found at dilution rates above 0.15 h-1 under the experimental conditions. At lower pH values, oscillations are more likely to happen, even at a relatively low dilution rate and low substrate excess. Whereas the amplitude of oscillations at pH 7.0 depends on both the dilution rate and the residual glycerol concentration (CGlyc) the interval of oscillations appears to be only a function of CGlyc. An increase of CGlyc in culture damps the oscillation and leads to its disappearance at CGlyc = 1100 to 1200 mmol/L (pH 7.0). The operation mode was also found to be an important parameter in determining the stability and actual state of the culture, resulting in hysteresis under certain conditions, particularly at low pH values. Generally, a large perturbation of cultivation conditions tends to cause oscillation and hysteresis. The results unambiguously demonstrate that the oscillation and hysteresis phenomena shown in this work are bound to genuine metabolic fluctuations of the microorganism. They reveal several differences and new features compared with those reported in the literature and cannot be readily explained by the mechanisms known so far. © 1996 John Wiley & Sons, Inc.
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    Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 572-578 
    Details
    ISSN: 0006-3592
    Keywords: nar promoter ; inducible promoter ; nitrate reductase ; anaerobic conditions ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing β-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed β-galactosidase, and induction ratio (specific β-galactosidase activity after maximal induction/specific β-galactosidase activity before induction.)The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of β-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD600 is congruent to 1.3) before being transferred to the fermentor; the amount of β-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD600 = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD600 became 3.2 and specific β-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. © 1996 John Wiley & Sons, Inc.
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    Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: II. Analysis of metabolic rates and pathways under oscillation and steady-state conditions (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 561-571 
    Details
    ISSN: 0006-3592
    Keywords: oscillation ; Klebsiella pneumoniae ; glycerol metabolism ; metabolic fluxes ; pathway analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The oscillation phenomena reported in the preceding article for the anaerobic continuous fermentation of glycerol by Klebsiella pneumoniae are analyzed in terms of metabolic fluxes (metabolic rates and yields) and stoichiometry of pathways. Significant oscillations in the fluxes of CO2, H2, formic acid, ethanol, and reducing equivalents are observed which show obvious relationships to each other. Changes in the consumption or production rates of glycerol, acetic acid, 1,3-propanediol, and ATP are irregular and have relatively small amplitudes compared with their absolute values. By comparing the metabolic fluxes under oscillation and steady state that have nearly the same environmental conditions it could be shown that pyruvate metabolism is the main step affected under oscillation conditions. The specific formation rates of all the products originating from pyruvate metabolism (CO2, H2, formic acid, ethanol, acetic acid, lactic acid, and 2,3-butanediol) show significant differences under conditions of oscillation and steady state. In contrast, the specific rates of substrate uptake, ATP generation, and formation of products deriving either directly from glycerol (1,3-propanediol) or from the upstream of pyruvate metabolism (e.g., succinic acid) are not, or at least not significantly, affected during oscillation. Stoichiometric analysis of metabolic pathways confirms that other enzyme systems, in addition to pyruvate: formate-lyase, must be simultaneously involved in the pyruvate decarboxylation under both oscillation and steady-state conditions. The results strongly suggest oscillations of activities of these enzymes under oscillation conditions. It appears that the reason for the occurrence of oscillation and hysteresis lies in an unstable regulation of pyruvate metabolism of different enzymes triggered by substrate excess and drastic change(s) of environmental conditions. © 1996 John Wiley & Sons, Inc.
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    Material balance studies on animal cell metabolism using a stoichiometrically based reaction network (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 579-590 
    Details
    ISSN: 0006-3592
    Keywords: fed batch ; hybridoma ; material balance ; reaction network ; stoichiometric analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A detailed reaction network of mammalian cell metabolism contains hundreds of enzymatic reactions. By grouping serial reactions into single overall reactions and separating overlapped pathways into independent reactions, the total number of reactions of the network is significantly reduced. This strategy of manipulating the reaction network avoids the manipulations of a large number of reactions otherwise needed to determine the reaction extents. A stoichiometric material balance model is developed based on the stoichiometry of the simplified reaction network. Closures of material balances on glucose and each of the 20 amino acids are achieved using experimental data from three controlled fed-batch and one-batch hybridoma cultures. Results show that the critical role of essential amino acids, except glutamine, is to provide precursors for protein synthesis. The catabolism of some of the essential amino acids, particularly isoleucine and leucine, is observed when an excess amount of these amino acids is available in the culture medium. It was found that the reduction of glutamine utilization (for reducing ammonia production) is accompanied by an increase in the uptake of nonessential amino acids (NAAs) from the culture medium. This suggests that NAAs are necessary even though they are not essential for cell growth. A glutamine balance shows that less than 20% of the glutamine nitrogen is utilized for essential roles, such as protein and nucleotide syntheses. A relatively constant percentage (about 45%) of the glutamine nitrogen is utilized for NAA biosynthesis, despite the fact that the absolute amount varies among the four experiments. As to the carbon skeleton of glutamine, a significant portion enters the tricarboxylic acid (TCA) cycle. A material balance on glucose shows that most of the glucose (81%) is converted into lactate when glucose is in excess. On the other hand, when glucose is limited, lactate production is considerably reduced, while a major portion of glucose (48%) enters the TCA cycle. The fraction of glucose used for the synthesis of cellular components ranges from 9 to 28%. © 1996 John Wiley & Sons, Inc.
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    Modeling substrate inhibition of microbial growth (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 602-608 
    Details
    ISSN: 0006-3592
    Keywords: modeling ; microbial growth ; substrate inhibition ; inhibition sites ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents a general equation for substrate inhibition of microbial growth using a statistical thermodynamic approach. Existing empirical models adapted from enzyme kinetics, for example, the Haldane-Andrews equation, often criticized for not being physically based for microbial growth, are shown to derive from the general equation in this article, and their empirical parameters are shown to be well defined physically. Three sets of experimental data from the literature are used to test the modeling abilities of the general equation to represent experimental data. The results are compared with those obtained by fitting the same data set to a widely used empirical model existing in the literature. The general equation is found to represent all three experimental data sets better than the alternative model tested. In addition, a graphical method existing in enzyme kinetics is successfully adapted and further developed to determine the number of inhibition sites of a basic functional unit of a bacterial cell. © 1996 John Wiley & Sons, Inc.
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    Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 591-601 
    Details
    ISSN: 0006-3592
    Keywords: animal cell metabolism ; ATP balance ; energy metabolism ; reaction network ; stoichiometric analysis ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH2. A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH2 accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (〈27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. © 1996 John Wiley & Sons, Inc.
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    Masthead (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996) 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260520501
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    Pathway kinetics and metabolic control analysis of a high-yielding strain of Penicillium chrysogenum during fed batch cultivations (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 631-631 
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    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260520502
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    A Monte Carlo simulation of plasmid replication during the bacterial division cycle (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 633-647 
    Details
    ISSN: 0006-3592
    Keywords: Monte Carlo simulation ; plasmid replication ; bacterial division cycle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Plasmids have cell cycle replication patterns that need to be considered in models of their replication dynamics. To compare current theories for control of plasmid replication with experimental data for timing of plasmid replication with the cell cycle, a Monte Carlo simulation of plasmid replication and partition was developed. High-copy plasmid replication was simulated by incorporating equations previously developed from the known molecular biology of ColE1-type plasmids into the cell-cycle simulation. Two types of molecular mechanisms for low-copy plasmid replication were tested: accumulation of an initiator protein in proportion to cell mass and binding of the plasmid origin to the cell membrane. The low-copy plasmids were partitioned actively, with a specific mechanism to mediate the transfer from mother to daughter cells, whereas the high-copy plasmids were partitioned passively with cell mass.The simulation results and experimental data demonstrate cell-cycle-specific replication for the low-copy F plasmid and cell-cycle-independent replication for the high-copy pBR322, ColBM, and R6K plasmids. The simulation results indicate that synchronous replication at multiple plasmid origins is critical for the cell-cycle-specific pattern observed in rapidly growing cells. Variability in the synchrony of initiation of multiple plasmid origins give rise to a cell-cycle-independent pattern and is offered as a plausible explanation for the controversy surrounding the replication pattern of the low-copy plasmids. A comparison of experimental data and simulation results for the low-copy F plasmid at several growth rates indicates that either initiation mechanism would be sufficient to explain the timing of replication with the cell cycle. The simulation results also demonstrate that, although cell-cycle-specific and cell-cycle independent replication patterns give rise to very different gene-expression patterns during short induction periods in age-selected populations, long-term expression of genes encoded on low-copy and high-copy plasmids in exponentially growing cells have nearly the same patterns. These results may be important for the future use of low-copy plasmids as expression vectors and validate the use of simpler models for high-copy plasmids that do not consider cell-cycle phenomena. © 1996 John Wiley & Sons, Inc.
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    A method for measuring bacterial chemotaxis parameters in a microcapillary (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 120-125 
    Details
    ISSN: 0006-3592
    Keywords: chemotaxis ; single cell ; capillary ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new method was developed which enables chemotaxis parameters to be measured at a single-cell level inside a capillary for the first time. The chemotaxis chamber consists of two reservoirs communicating through a capillary tube 50 μm in diameter. Chemotaxis parameters are measured inside the capillary using image analysis, after a nearly linear attractant concentration gradient has been generated along the capillary by diffusion. Compared to previously published techniques, this method provides a well-characterized chemoattractant concentration profile in addition to allowing single-cell parameters to be measured inside a fine capillary. This procedure was used to measure the single-cell chemotaxis parameters for Escherichia coli K12, and the results are compared to published data on single E. coli cells chemotaxing in bulk. © 1996 John Wiley & Sons, Inc.
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    Removal of chlorophenols from wastewater by immobilized horseradish peroxidase (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 126-130 
    Details
    ISSN: 0006-3592
    Keywords: chlorophenol ; peroxidase ; immobilization ; magnetite ; immobilized enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Immobilization of horseradish peroxidase on magnetite and removal of chlorophenols using immobilized enzyme were investigated. Immobilization by physical adsorption on magnetite was much more effective than that by the crosslinking method, and the enzyme was found to be immobilized at 100% of retained activity. In addition, it was discovered that horseradish peroxidase was selectively adsorbed on magnetite, and the immobilization resulted in a 20-fold purification rate for crude enzyme. When immobilized peroxidase was used to treat a solution containing various chlorophenols, p-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, each chlorophenol was almost 100% removed, and also the removal of total organic carbon (TOC) and adsorbable organic halogen (AOX) reached more than 90%, respectively. However, in the case of soluble peroxidase, complete removal of each chlorophenol could not be attained, and in particular, the removal of 2,4,5-trichlorophenol was the lowest, with a removal rate of only 36%. © 1996 John Wiley & Sons, Inc.
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    L-DOPA production from tyrosinase immobilized on nylon 6,6 (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 141-147 
    Details
    ISSN: 0006-3592
    Keywords: L-DOPA ; tyrosinase ; enzyme immobilization ; nylon 6,6 ; inactivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production of L-DOPA immobilized on chemically modified nylon 6,6 membranes was studied in a batch reactor. Tyrosinase was immobilized on nylon using glutaraldehyde as a crosslinking agent. The effects of membrane pore size and glutaraldehyde concentration upon enzyme uptake and L-DOPA production were investigated. Enzyme uptake was unaffected by glutaraldehyde concentration; approximately 70% uptake was observed when 25% w/v (group 1), 5% (group 2), and 3% (group 3) glutaraldehyde were used, indicating that glutaraldehyde was in excess. Similarly, uptake was the same for membranes with 0.20 and 10 μm pore sizes.Membranes produced using different levels of glutaraldehyde exhibited dramatically different capacities for L-DOPA production, despite the fact that enzyme uptake was equivalent. Membranes from groups 2 and 3 (5% and 3% glutaraldehyde) produced L-DOPA at a rate of 1.70 mg L-1 h-1 over 170 h in a 500-mL batch reactor. However, no free L-DOPA was detected when group 1 membranes were used. Experimental evidence suggests that L-DOPA was produced, but remained bound to these membranes via excess glutaraldehyde left over from the immobilization process. Membrane pore size also effected L-DOPA production; less production was observed when 10-μm membranes were used, despite equivalent enzyme uptake. The observed difference in production may be due to differences in the pore density on the two types of membranes which could affect the access of the substrate to the immobilized enzyme.The results of these studies indicate that tyrosinase can be effectively immobilized on nylon 6,6. L-DOPA production was optimal when 0.20-μm-pore-size membranes were activated with 3-5% glutaraldehyde. Stability studies indicated a 20% reduction in activity over 14 days when the immobilized enzyme was used under turnover conditions. © 1996 John Wiley & Sons, Inc.
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    Secretion of recombinant proteins from hydroxyethyl methacrylate-methyl methacrylate capsules (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 271-280 
    Details
    ISSN: 0006-3592
    Keywords: immuno-isolation ; fibroblasts ; gene therapy ; β-glucuronidase ; β-hexosaminidase ; Matrigel ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Current human gene therapy relies on genetic modification of the patient's own cells. An alternate nonautologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immunoisolation devices before implantation would allow the use of the same recombinant cell line for treating different patients, thus potentially lowering the cost of treatment. To study the properties of a mechanically stable synthetic biomaterial, hydroxyethyl methyacrylate-methyl methacrylate (HEMA-MMA) as the immuno-isolation device, we encapsulated recombinant mouse fibroblast cells engineered to secrete products ranging from 27 to 300 kDa in size (human growth hormone, mouse β-hexosaminidase and β-glucuronidase) in the presence or absence of the extracellular matrix Matrigel. Both viability and cell number in the microcapsules increased with time after encapsulation and cell morphology indicated viable cell growth, thus showing that the capsule membrane barrier was compatible with nutrient/waste exchange necessary for normal metabolic activity.The intracellular levels of these recombinant gene products were constant throughout the experimental period of 22 days in the presence or absence of Matrigel, thus demonstrating that the microenvironment did not lead to downregulation of the transgenes. However, the extracellular levels of the gene products secreted from the cells and trapped within the microcapsules were dependent on the molecular size of the product and presence of Matrigel. With the 27-kDa human growth hormone, the presence of Matrigel caused its retention within this intracapsular space, but its release from the microcapsules to the culture medium was not impeded. With the 120-kDa β-hexosaminidase or the 300-kDa β-glucuronidase, they were retained within the microcapsule space regardless of the presence or absence of Matrigel, and their passage from the microcapsules to the media was totally blocked. In conclusion, the HEMA-MMA microcapsules are supportive of recombinant cell growth and maintained their molecular cutoff at ∼100 kDa. Inclusion of extracellular matrix was unable to improve cell growth and may impede the exit of some gene products. © 1996 John Wiley & Sons, Inc.
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    ATP regeneration by thermostable ATP synthase (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 305-316 
    Details
    ISSN: 0006-3592
    Keywords: ATP ; regeneration ; ATPase ; ATP synthase ; electrodialysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We investigated the possibility of using thermostable ATP synthase (TF0F1) for a new ATP regeneration method. TF0F1 was purified from a thermophilic bacterium, PS3, and reconstituted into liposomes. ATP synthesis experiments showed that TF0F1 liposomes could synthesize ATP in micromole concentrations by acid-base change. The acid-base change was repeated six times over an 11-day period with no detectable loss of activity at the reaction temperature (45°C). Given these encouraging results, we conceptualized and modeled a system to synthesize ATP using ATP synthase with energy supplied by acid-base change. In this system, liposomes containing ATP synthase are immobilized on small glass spheres that facilitate separation of buffers from the liposomes after the acid-base change. Compared to an alternate system that uses membranes to separate the buffers from the liposomes, the glass spheres reduce inefficient mixing of acidic and basic buffers during the acid-base change. To increase the ATP synthesis yield, this system uses electrodialysis to regenerate a potassium gradient after the acid-base change. It also employs water-splitting electrodialysis to regenerate KOH and HCl required to adjust the pH of acidic and basic buffers. All reagents are recycled, so electrical energy is the only required input. © 1996 John Wiley & Sons, Inc.
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    Immobilization of Candida rugosa lipase to nylon fibers using its carbohydrate groups as the chemical link (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 327-341 
    Details
    ISSN: 0006-3592
    Keywords: Candida rugosa ; immobilization ; olive oil hydrolysis ; phenylglycidate ; glutaraldehyde ; adipic dihydrazide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The aim of this study was to evaluate the immobilization of lipase from Candida rugosa on a nylon support by methods used to attach biomolecules to solid supports through their carbohydrate moieties. The carbohydrate groups were converted to dialdehydes by treatment with sodium periodate. The length of exposure and the periodate amount were optimized to the point where almost total activity retention was obtained. Tests of the immobilized enzyme showed the expressed activity to be significantly higher than the activity obtained with the unimmobilized enzyme. The use of reverse micelles as a way of delivering water to the enzyme was tested and found to give significantly higher activities. The immobilized enzyme activity was also tested with other substrates, one of which was a chiral ester. The immobilized enzyme was found to have high stereoselective efficiency and activity toward racemic methyl methoxyphenyl glycidate, a chiral intermediate used in the manufacture of the drug diltiazem. © 1996 John Wiley & Sons, Inc.
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    Fouling effects of yeast culture with antifoam agents on microfilters (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 10-16 
    Details
    ISSN: 0006-3592
    Keywords: microfiltration ; fouling ; yeast ; antifoam agents ; depressurization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The fouling effects of yeast fermentation broths of Candida utilis in the presence of various commercial antifoam agents (PPG2000, B5600, and G832) up to 4.0 mL/L were studied, using Millipore polyvinylidene fluoride 0.22-μm hydrophilic membranes (GVWP), in a stirred-cell system at 50 kPa and 700 rpm. PPG2000, which has a low value of work of adhesion (Wa of 0.81 mN/m), gave a steady flux of broth of 29 L/(h m2) and was found to have no significant fouling effect on the microfiltration of broth. G832, which has a high Wa, (26.0 mN/m) reduced the flux of the broth to 17 L/(h m2); i.e., by 42% when only 1.0 mL/L was used. However, B5600, which has a Wa of 14.3 mN/m, was found to enhance the flux of broth to 54 L/(h m2); i.e., by 86%, due to the preferential adsorption of the B5600 components onto the hydrophobic cell contents released. These results were reinforced by the depressurization experiments performed with both hydrophilic (GVWP) and hydrophobic (GVHP) membranes, using both young and aged broths. B5600 was found to be the optimum antifoam agent in this study in terms of membrane performance and defoaming efficiency. © 1997 John Wiley & Sons, Inc.
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    Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 21-25 
    Details
    ISSN: 0006-3592
    Keywords: starch fermentation ; recombinant yeast ; ethanol production ; glucoamylase activity ; fed-batch culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. © 1997 John Wiley & Sons, Inc.
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    Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 375-383 
    Details
    ISSN: 0006-3592
    Keywords: cellulase ; enzyme recycling ; enzyme adsorption ; lignocellulosic hydrolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Past technoeconomic modeling work has identified the relatively large contribution that enzymatic hydrolysis adds to the total cost of producing ethanol from lignocellulosic substrates. This cost was primarily due to the high concentration of enzyme and long incubation time that was required to obtain complete hydrolysis. Although enzyme and substrate concentration and end-product inhibition influenced the rate of hydrolysis, the effect was less pronounced during the initial stages of hydrolysis. During this time most of the cellulases were adsorbed onto the unhydrolyzed residue. By recycling the cellulases adsorbed to the residual substrate remaining after an initial 24 h, a high rate of hydrolysis, with low overall residence time and minimal cellulase input, could be achieved for several rounds of enzyme recycle. A comparison of the front end (pretreatment, fractionation, and hydrolysis) of a softwood/hardwood to ethanol process indicated that the lignin associated with the softwood-derived cellulose stream limited the number of times the cellulose containing residue could be recycled. © 1996 John Wiley & Sons, Inc.
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    Thermal stability studies of a globular protein in aqueous poly(ethylene glycol) by 1H NMR (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 410-421 
    Details
    ISSN: 0006-3592
    Keywords: lysozyme ; thermal stability ; 1H NMR ; conformational flexibility ; melting temperature ; PEG ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The reversible folding destabilization of hen lysozyme has been confirmed by a melting temperature (Tm) decrease in aqueous poly(ethylene glycol) (PEG). The percent denatured, extracted from the histidine 15 C2H (H15 C2H) native and denatured peak areas from 500-MHz one-dimensional proton nuclear magnetic resonance (1D 1H NMR) spectra in D2O, was analyzed through denaturation temperatures at 0% and 20% (w/w) PEG 1000. The lysozyme (3.5 mM) Tm decreased by 4.2°C and 7.1°C in 20% (w/w) PEG 1000 at pH 3.8 and 3.0, respectively. The Tm decreased with increasing lysozyme concentration. Additionally, the temperature-induced resonance migrations of 17 protons from 8 residues indicate that the native lysozyme structure undergoes temperature-induced conformational changes. The changes were essentially identical in both 0% and 20% (w/w) PEG 1000 at both pH 3.0 and 3.8. This small, local restructuring of the hydrophobic box region may be a manifestation of temperature-dependent solution hydrophobicity, whereas active-site cleft fluctuations may be due to the inherent active-site flexibility. The lysozyme structure in PEG at 35°C was determined to be essentially native from the 1H nuclear Overhauser effect spectroscopy (NOESY) fingerprint regions. Additionally, lysozyme chemical shifts, from 1D spectra, in PEG 200, 300, and 1000 at 35°C and various concentrations were essentially identical, further confirming that the conformation remains native in various PEG solutions. © 1996 John Wiley & Sons, Inc.
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    Biosorption of uranium by Pseudomonas aeruginosa strain CSU: Characterization and comparison studies (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 237-247 
    Details
    ISSN: 0006-3592
    Keywords: biosorption ; sorption ; uranium ; iron ; Pseudomonas aeruginosa ; bacteria ; remediation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pseudomonas aeruginosa strain CSU, a nongenetically engineered bacterial strain known to bind dissolved hexavalent uranium (as UO22+ and/or its cationic hydroxo complexes), was characterized with respect to its sorptive activity (equilibrium and dynamics). Living, heat-killed, permeabilized, and unreconstituted lyophilized cells were all capable of binding uranium. The uranium biosorption equilibrium could be described by the Langmuir isotherm. The rate of uranium adsorption increased following permeabilization of the outer and/or cytoplasmic membrane by organic solvents such as acetone. P. aeruginosa CSU biomass was significantly more sorptive toward uranium than certain novel, patented biosorbents derived from algal or fungal biomass sources. P. aeruginosa CSU biomass was also competitive with commercial cation-exchange resins, particularly in the presence of dissolved transition metals. Uranium binding by P. aeruginosa CSU was clearly pH dependent. Uranium loading capacity increased with increasing pH under acidic conditions, presumably as a function of uranium speciation and due to the H+ competition at some binding sites. Nevertheless, preliminary evidence suggests that this microorganism is also capable of binding anionic hexavalent uranium complexes. Ferric iron was a strong inhibitor of uranium binding to P. aeruginosa CSU biomass, and the presence of uranium also decreased the Fe3+ loading when the biomass was not saturated with Fe3+, suggesting that Fe3+ and uranium may share the same binding sites on biomass. Although the equilibrium loading capacity of uranium was greater than that of Fe3+, this biomass showed preference of binding Fe3+ over uranium. Thus, a two-stage process in which iron and uranium are removed in consecutive steps was proposed for efficient use of the biomass as a biosorbent in uranium removal from mine wastewater, especially acidic leachates. © 1996 John Wiley & Sons, Inc.
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    Membrane adsorption characteristics determine the kinetics of flow-through transductions (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 260-270 
    Details
    ISSN: 0006-3592
    Keywords: retrovirus ; gene therapy ; gene transfer ; virus adsorption ; membranes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Retrovirus-mediated gene transfer is currently limited by random Brownian motion of the retrovirus. This limitation can be overcome by flowing the retrovirus solution through a porous membrane that supports the target cells, leading to a significant increase in the transduction efficiency. This procedure is termed “flow-through transduction.” In this study, we characterized the effects of the fluid flowrate and the influence that membrane characteristics have on the flow-through transduction procedure. The transduction efficiencies increased with flowrate until a plateau was reached at average flow velocities exceeding 0.3 cm/h for flow times of 3 to 4 h, using a collagen-coated depth (COL) membrane. A correlation between the optimal time for maximal gene transfer using flow-through transductions and the optimal time for maximal virus activity on the membrane was found, suggesting that the membrane adsorption capacity for virus determined the amount of gene transfer that could occur.Membrane adsorption characteristics were further investigated using two different membrane types: a tracketched polyester screen (PE) membrane and the COL membrane. Flow-through transductions using the PE and COL membranes showed that a high level of gene transfer could be attained using the COL membrane while the PE membrane gave much lower transduction efficiencies. The addition of the polycation polybrene (PB) changed these results markedly, making transductions achieved on the PE membrane similar in number to those obtained on the COL membrane. Since PB is believed to influence the virus adsorption to PE membrane, these results further support the conclusion that the increase in gene transfer achieved by the flow-through transduction procedure is due to virus adsorption to the membrane. The flow-through transduction procedure thus leads to co-localization of the viral vector and the target cell that in turn leads to a high transduction efficiency. © 1996 John Wiley & Sons, Inc.
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    Intracellular receptor/ligand sorting based on endosomal retention components (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 281-297 
    Details
    ISSN: 0006-3592
    Keywords: endosome ; sorting ; retention ; epidermal growth factor ; transforming growth factor α ; epidermal growth factor receptor ; intracellular trafficking ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Endocytosed molecules are sorted in endosomes to different cellular destinations (e.g., to lysosomes or to the plasma membrane). Diverse endosomal sorting results have been reported for different ligands and receptors in a variety of cell types, but the general principles governing these sorting outcomes are not well understood. For example, we observed a wide range of sorting outcomes with the epidermal growth factor (EGF)/receptor system in fibroblasts using several members of the EGF family and site-directed ligand and receptor mutants. In this article we describe a mechanistic mathematical model of endosomal sorting based on the hypothesis that receptors may be selectively retained by the endosomal sorting apparatus and that this process may be modulated by receptor occupancy. Our results show that this single mechanism can account for the wide variety of observed sorting outcomes. By providing a conceptual framework for understanding endosomal sorting, this model not only helps interpret our experimental results for the EGF/receptor system, but also provides some insight into the principles governing sorting. For example, the model predicts that the influence of selective endosomal retention of receptor/ligand complexes is seen in deviations of ligand sorting outcomes from pure fluid phase sorting behavior. Furthermore, the model suggests that selective endosomal retention of complexes within endosomes gives rise to three sorting regimes characterized by distinguishable qualitative trends in the dependence of ligand sorting fractions on intracellular ligand concentrations. © 1996 John Wiley & Sons, Inc.
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    NSO myeloma cell death: Influence of bcl-2 overexpression (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 298-304 
    Details
    ISSN: 0006-3592
    Keywords: NOS ; myeloma ; apoptosis ; Bcl-2 ; bax ; Bcl-xL ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The productivity of recombinant mammalian cell lines growth in batch culture is often limited by the rapidity with which cells die on entry into the decline phase (the period of culture after the maximum cell density has been reached and where cell viability begins to fall). We examined the decline phase characteristics of the NSO myeloma cell line with a view to modulating the cell death that ensues. Examination of nuclear morphology during culture revealed that the onset of the decline phase was marked by a time-dependent increase in the percentage of cells that exhibited condensed and fragmented nuclei. Furthermore, these changes coincided with a fall in DNA integrity. High molecular weight DNA appeared to be degraded into oligonucleosomal fragments. Taken together, these observations indicated that NSO cells die by the process of apoptosis. The protein encoded by the bcl-2 gene has been shown to counter apoptosis induced by a large variety of stimuli and in a number of different cell types, but is not expressed in NSO cells. We examined whether overexpression of this protein could prevent/delay the onset of cell death seen during batch culture and also in response to serum limitation. Bcl-2 failed to affect the decline phase characteristics and serum dependence of NSO cells. In our search to explain these findings, we found that the NSO cell line expresses bax and also a high level of another Bcl-2 related protein, Bcl-xL. Given that Bcl-XL is a sequence and functional homologue of Bcl-2, it is possible that Bcl-2 is redundant in the NSO cell background. These data therefore indicate that cells such as NSO, which are used in biotechnologically important processes such as generation of hybridomas and expression of recombinant proteins, may express only a subset of genes important in apoptotic regulation. Modulation of the death characteristics of such cells will need to take account of the expression profile of such genes and their regulatory interactions. © 1996 John Wiley & Sons, Inc.
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    Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed-bed bioreactor (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 317-326 
    Details
    ISSN: 0006-3592
    Keywords: xylitol ; recombinant yeast ; immobilization ; continuous packed-bed reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al3+ to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L · h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. © 1996 John Wiley & Sons, Inc.
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    Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 349-359 
    Details
    ISSN: 0006-3592
    Keywords: trichloroethylene ; methane monooxygenase ; Methylosinus trichosporium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: By complementing cell-free extracts of Pseudomonas putida F1/pSMMO20 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichosporium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMMO20 and pSMMO50 into various bacterial species including pseudomonads and α-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO20 (resulting in pSMMO40) and found to enhance plasmid stability in P. putida F1 and R. meliloti 102F34 (first report of hok/sok in Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(II), Ni(II), and Zn(II)] and chloramphenicol from the medium and by supplementing with an iron source (3.6 μM of ferrous ammonium sulfate). Using chemostat-grown P. putida F1/pSMMO40, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity. © 1996 John Wiley & Sons, Inc.
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    Activation and regeneration of whole cell biocatalysts: Initial and periodic induction behavior in starved Escherichia coli after immobilization in thin synthetic films (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 360-370 
    Details
    ISSN: 0006-3592
    Keywords: induction ; Escherichia coli ; biofilms ; immobilization ; protein synthesis in starved bacteria ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Activation and regeneration of whole cell biocatalytic activity via initial and subsequent induction of the lacZ gene was investigated in starved Escherichia coli using a novel synthetic biofilm. Stationary-phase bacteria were entrapped in 10-80 μm thick multi-layer films, where a copolymer of acrylic and vinyl acetate was the immobilization matrix. The E. coli were placed in a defined starvation medium containing essentially no nitrogen or carbon source and induced initially using lactose or isopropylthiogalactoside (IPTG). Subsequent inductions were performed with IPTG. Comparison studies with suspended bacteria showed that when IPTG was the initial inducing agent, induction kinetics are linear for both immobilized and suspended cells. After induction with lactose, however, a lag time is noted for suspended cells, but not for E. coli in the biofilm. Biocatalytic activity was successfully regenerated by re-inducing starved suspended cells 1-3 days after an initial induction with lactose. This regeneration was demonstrated in the synthesis of additional active β-galactosidase. However, immobilized cells could be re-induced for at least 17 days after the initial induction, and viability in the synthetic biofilms remained greater than 90%, demonstrating that periodic induction is a valuable method for extending the life of whole cell biocatalysts. © 1996 John Wiley & Sons, Inc.
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    Effects of kinetics of adsorption and coalescence on continuous foam concentration of proteins: Comparison of experimental results with model predictions (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 384-398 
    Details
    ISSN: 0006-3592
    Keywords: foam fractionation ; foam concentration ; protein separation ; protein adsorption ; preconcentration of proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein enrichment and recovery were measured in a continuous foam concentration column for bovine serum albumin (BSA) for different pool heights, foam heights, superficial gas velocities, bubble sizes, feed flow rates, pH, and ionic strengths. Protein enrichment was found to decrease with an increase in pool height for low pool heights, reach a minimum at an intermediate pool height, and subsequently increase with pool height for sufficiently large pool heights eventually approaching an asymptotic value. Such a behavior was due to the combined effects of kinetics of adsorption of protein and coalescence. The increase in protein enrichment with pool height was due to the predominant effect of kinetics of adsorption of protein, whereas the opposite behavior at low pool heights was due to the predominant effect of coalescence in the foam. Protein enrichment was found to be higher for smaller feed concentrations, smaller gas velocities, larger bubble sizes, and larger foam heights. Enrichment at pH values different from the isoelectric point was found to be higher because of more coalescence. A model for foam concentration of proteins was employed to predict enrichment and recovery. The model predictions agreed well with the experimental data. © 1996 John Wiley & Sons, Inc.
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    Sparging and agitation-induced injury of cultured animals cells: Do cell-to-bubble interactions in the bulk liquid injure cells? (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 399-409 
    Details
    ISSN: 0006-3592
    Keywords: cell damage ; cell culture ; bubble aeration ; agitation ; bubble coalescence and breakup ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. © 1996 John Wiley & Sons, Inc.
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    Production of tissue plasminogen activator (tPA) in two and three dimensionally growing cultures of Bowes melanoma cells (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 422-433 
    Details
    ISSN: 0006-3592
    Keywords: tissue plasminogen activator ; multicell spheroids ; microcarrier ; Bowes melanoma cells ; cell cycle ; cellular protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bowes melanoma cells synthesize more tissue plasminogen activator (tPA) in monolayer cultures than in multicell spheroids. Cellular production of tPA in these cells was measured during a cultivation period of 800 h. Without changing the cell culture assay, we were able to obtain monolayers, multilayers, and multicell spheroids (cell aggregates) by stirring microcarrier beads in 500-mL spinner flasks operated at 50 rpm. Thus, the medium conditions in the liquid were similar for cells in monolayers and in multicell spheroids. Probes for measurements of intracellular and extracellular parameters were taken from the same culture at distinct times; therefore, their variations during cultivation can directly be compared. Because cells were cultured in an unregulated (with regard to pH, glucose, etc.) spinner flask, their concentration was kept below 106 cells/mL, thus avoiding too fast and too severe depletion of oxygen and other medium factors. Nevertheless, the tPA productivity decreased from 8 ng/h/106 cells (monolayer) to 4 ng/h/106 cells (multicell spheroids with microcarrier nucleus, 800 μm diameter), matching the decrease of total cellular protein. Due to medium depletion, the cell cycle distribution changed from 45% to 68% G1 cells in a characteristic way during growth of multicell spheroids. This is accompanied by changes in amino acids, glucose, lactate, and pH, which may account for the reduction of tPA productivity. © 1996 John Wiley & Sons, Inc.
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    Continuous detoxification, transformation, and degradation of nitrophenols in upflow anaerobic sludge blanket (UASB) reactors (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 439-449 
    Details
    ISSN: 0006-3592
    Keywords: anaerobic treatment ; continuous reactors ; degradation ; detoxification ; granular sludge ; nitrophenol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The anaerobic transformation and degradation of nitrophenols by granular sludge was investigated in upflow anaerobic sludge blanket (UASB) reactors continuously fed with a volatile fatty acid (VFA) mixture as the primary substrate. During the start-up, subtoxic concentrations of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2, 4-dinitrophenol (2, 4-DNP) were utilized. 4-NP and 2, 4-DNP were readily converted to the corresponding aromatic amine; whereas 2-NP was converted to nonaromatic products via intermediate formation of 2-aminophenol (2-AP). These conversions led to a dramatic detoxification of the mononitrophenols because the reactors treated the nitrophenolics at the concentrations which were over 25 times higher than those that caused severe inhibition. VFA removal efficiencies greater than 99% were achieved in both reactors at loading rates greater than 11.4 g COD per liter of reactor volume per day even at volumetric loading of mononitrophenols up to 910 mg/L · d.The sludges obtained from each of the reactors at the end of the continuous experiments were assayed for their specific nitrophenol reducing activity in the presence of different primary substrates. Reduction rates of 45 and 26 mg/g volatile suspended solids per day were observed for 2-NP and 4-NP, respectively, when utilizing the VFA mixture as primary substrate. Hydrogen, an interspecies-reduced compound, and substrates that provide interspecies-reducing equivalents - such as butyrate, propionate, and ethanol stimulated nitrophenol reduction, whereas acetate and methanol did not. Anaerobic batch biodegradability tests with the 2-NP-adapted sludge revealed that its corresponding aromatic amine, 2-AP, was degraded to methane at a specific rate of 14.5 mg/g VSS · d. Acetate was observed to be the major intermediate during 2-AP degradation in the presence of a specific methanogenic inhibitor 2-bromoethanesulfonate. The results of this study indicate that UASB reactors can be applied to rapidly detoxify and, under certain circumstances, degrade nitroaromatic compounds. © 1996 John Wiley & Sons, Inc.
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    NMR on-line monitoring of esterification catalyzed by cutinase (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 636-644 
    Details
    ISSN: 0006-3592
    Keywords: NMR studies ; esterification ; cutinase ; lipase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A nuclear magnetic resonance (NMR) method has been developed to monitor on-line lipase-catalyzed esterification reactions without the need to sample the reaction medium. The technique, through 1H NMR, measures the concentrations of alcohol, ester, hydroxylic hydrogens in the organic phase, and hydroxylic hydrogens in the aqueous phase, if any. Also, the chemical shift evolution of the two types of hydroxylic hydrogens has been followed, providing information on water content of the organic phase and on the appearance of a distinct aqueous phase. As far as 13C NMR is concerned, it has been possible to measure, first the acid and the ester concentrations in the carbonyl region, and second, the alcohol and the ester concentrations in the methylene region. All 1H and 13C results are in agreement with one another. Furthermore, NMR allows for the choice of detection zone. Preliminary studies on the solid phase proved the presence of much more water in the solid phase than in the organic phase, and also gave evidence of the existence of two types of esters, one in the organic phase, mainly associated with the acid, and the other one not associated with the acid, most probably entrapped within the solid enzyme.
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    Structural basis for the molecular memory of imprinted proteins in anhydrous media (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 609-614 
    Details
    ISSN: 0006-3592
    Keywords: imprinted proteins ; molecular memory ; FTIR ; secondary structure ; lysozyme ; chymotrypsinogen ; bovine serum albumin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fourier-transform infrared (FTIR) spectroscopy has been used to quantitatively examine the secondary structure of imprinted (i.e., lyophilized in the presence of multifunctional ligands followed by removal of the latter) proteins in anhydrous media. Lysozyme, chymotrypsinogen, and bovine serum albumin, imprinted with L-malic acid, all exhibited significant differences in the secondary structure compared to that of their nonimprinted counterparts. A rise in the β-sheet content, which invariably occurs upon lyophilization, is substantially lower for imprinted proteins. Alterations in the α-helix contents of these proteins have also been observed upon imprinting, although these changes are specific to the protein. A structural explanation has been obtained herein for other previously observed aspects of the protein imprinting phenomenon, including the effects of the ligand and the solvent and the lack of enantioselectivity. Exposure to aqueous solution, but not to anhydrous solvents, results in the disappearance of imprinting-induced changes in the secondary structure of proteins. © 1996 John Wiley & Sons, Inc.
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    Properties of two insect cell lines useful for the baculovirus expression system in serum-free culture (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 494-499 
    Details
    ISSN: 0006-3592
    Keywords: cell metabolism ; baculovirus ; insect cells ; recombinant protein OSF-2 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The properties of Sf9 and Tn5 insect cells were analyzed comparatively under serum-free culture conditions. Sf9 cells in SF900II medium apparently utilized sucrose as a primary nutrient both before and after virus infection, yielding small amounts of lactate and ammonia. Tn5 cells in Excell 401 medium consumed all the nutrients examined, including sucrose. The productivity of a recombinant glycoprotein, OSF-2, by Tn5 cells, was moderate in both monolayer and spinner cultures, but the ability to secrete it was compromised in the former case. Relative to the Tn5 cultures, Sf9 produced 30-fold more OSF-2 in either culture mode. © 1996 John Wiley & Sons, Inc.
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    A novel centrifugal impeller bioreactor. I. Fluid circulation, mixing, and liquid velocity profiles (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 511-519 
    Details
    ISSN: 0006-3592
    Keywords: bioreactor development ; centrifugal impeller ; fluid circulation ; liquid velocity profile ; mixing ; shear stress ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel centrifugal impeller bioreactor for shear-sensitive biological systems was designed by installing a centrifugal-pumplike impeller in a stirred vessel. The fluid circulation, mixing, and liquid velocity profiles in the new bioreactor (5-L) were assessed as functions of the principal impeller designing and bioreactor operating parameters. The performances of the centrifugal impeller bioreactor were compared with those of a widely used cell-lift bioreactor. The newly developed bioreactor showed higher liquid lift capacity and shorter mixing time than the cell lift with comparable dimensions. Furthermore, the experiments of the liquid velocity profiles around an impeller region indicated that the centrifugal impeller bioreactor produced lower shear stress than the cell lift. This conclusion was also supported by evaluating the changes in size distributions of granulated agar particles that were sheared with those two types of impeller.
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    Degradation of chloro- and methyl-substituted benzoic acids by a genetically modified microorganism (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 528-537 
    Details
    ISSN: 0006-3592
    Keywords: chlorobenzoic acid ; methylbenzoic acid ; genetically modified strain ; Pseudomonas sp. B13 FR1 SN45P ; batch cultivation ; chemostat ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Degradation of 3-chlorobenzoic acid (3CB), 4-chlorobenzoic acid (4CB), and 4-methylbenzoic acid (4MB) as single substrates (carbon sources) and as a substrate mixture were studied in batch and continuous culture using the genetically modified microorganism Pseudomonas sp. B13 FR1 SN45P. The strain was able to mineralize the single compounds as well as the substrate mixture completely. Conversion of the three compounds in the substrate mixture proceeded simultaneously. Maximum specific substrate conversion rates were calculated to be 0.9 g g-1 h-1 for 3 CB and 4CB and 1.1 g g-1 h-1 for 4MB. Mass balances indicated the transient accumulation of pathway intermediates during batch cultivations. Hence, the rate limiting step in the degradative pathway is not the initial microbial attack of the original substrate or its transport through the cell membrane. Degradation rates on 3CB were comparable to those of the parent strain Pseudomonas sp. B13. The stability of the degradation pathways of strain Pseudomonas sp. B13 FR1 SN45P could be demonstrated in a continuous cultivation over 3.5 months (734 generation times) on 3CB, 4MB, and 4CB, which were used as single carbon sources one after the other.
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    Effects of nitrate and acetate availability on chloroform production during carbon tetrachloride destruction (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 551-557 
    Details
    ISSN: 0006-3592
    Keywords: denitrify ; carbon tetrachloride ; chloroform ; acetate ; nitrate ; bioremediation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fed batch experiments were performed to test the effects of electron donor and electron acceptor availability on the production of chloroform (CF) during carbon tetrachloride (CT) destruction by a denitrifying bacterial consortium. In one series of tests, acetate (electron donor) was present in excess while nitrate and nitrite (electron acceptor) were limiting. In the other series of tests, acetate was the limiting nutrient, and nitrate and nitrite were in excess. Under nitrate limiting conditions, 50% (±17%) of the CT transformed by the microorganisms was converted to CF. However, under acetate limiting conditions, only 4% (±4%) of the CT that was degraded appeared as CF. Previous research had suggested that denitrifying bacteria can degrade CT via two competing pathways. One of these pathways produces CF as the predominant end product. The second pathway produces CO2 as the primary end product. The results shown here suggest that the first pathway is dominant when nitrate and nitrite are depleted while the second pathway, which produces little CF, dominates when nitrate or nitrite are available.
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    Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five™) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 909-920 
    Details
    ISSN: 0006-3592
    Keywords: baculovirus ; insect cells ; metabolism ; Sf-9; high five™ ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five™) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five™ cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five™ cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five™ cell cultures, but not in Sf-9 cell cultures. In addition, High Five™ cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five™ cell cultures. It was also found that the medium had a significant effect on High Five™ cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.
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    On-line green fluorescent protein sensor with LED excitation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 921-926 
    Details
    ISSN: 0006-3592
    Keywords: green fluorescent protein ; sensor ; on-line monitoring ; quantitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:921-926, 1997.
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    Kinetic studies of fusarium solani pisi cutinase used in a gas/solid system: Transesterification and hydrolysis reactions (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 1-8 
    Details
    ISSN: 0006-3592
    Keywords: transesterification ; hydrolysis ; water activity ; cutinase ; gas ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (aw = 0.2, aw = 0.4 and aw = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from aw = 0.2 to aw = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.
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