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  • Cell & Developmental Biology  (25,032)
  • Biochemistry and Biotechnology  (13,095)
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  • 101
    ISSN: 0006-3592
    Keywords: chimeric antibody ; CHO cells ; dihydrofolate reductase ; flow cytometry ; gene copy number ; stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 μM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 μM, and thereafter, it gradually increased to 20 μg/106 cells/day at 4 μM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 μM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 μM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 μM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:73-84, 1998.
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  • 102
    ISSN: 0006-3592
    Keywords: furin ; proprotein convertases ; transforming growth factor beta ; baculovirus ; precursor proteins ; overexpression systems ; growth factors ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFβ1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFβ1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFβ1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFβ1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFβ1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFβ1 by coexpressing prohTGFβ1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFβ1 increased in a dose-dependent fashion reaching up to 7.8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFβ1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:85-91, 1998.
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  • 103
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 117-117 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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  • 104
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 101-116 
    ISSN: 0006-3592
    Keywords: biofilm ; structure ; shape ; surface ; cellular automata ; discrete ; modeling ; roughness ; fractal ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A hybrid differential-discrete mathematical model has been used to simulate biofilm structures (surface shape, roughness, porosity) as a result of microbial growth in different environmental conditions. In this study, quantitative two- and three-dimensional models were evaluated by introducing statistical measures to characterize the complete biofilm structure, both the surface structure and volume structure. The surface enlargement, coefficient of roughness, fractal dimension of surface, biofilm compactness, and solids hold-up were found to be good measures of biofilm structure complexity. Among many possible factors affecting the biofilm structure, the influence of biomass growth in relation to the diffusive substrate transport was investigated. Porous biofilms, with many channels and voids between the “finger-like” or “mushroom” outgrowth, were obtained in a substrate-transport-limited regime. Conversely, compact and dense biofilms occurred in systems limited by the biomass growth rate and not by the substrate transfer rate. The surface complexity measures (enlargement, roughness, fractal dimension) all increased with increased transport limitation, whereas the volume measures (compactness, solid hold-up) decreased, showing the change from a compact and dense to a highly porous and open biofilm. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:101-116, 1998.
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  • 105
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 409-419 
    ISSN: 0006-3592
    Keywords: fungal morphology ; dissolved oxygen tension ; pellets ; agitation intensity ; stirred vessels ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 409-419, 1998.
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  • 106
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    Biotechnology and Bioengineering 57 (1998), S. 420-429 
    ISSN: 0006-3592
    Keywords: pentachlorophenol ; dechlorinating bacteria ; methanogenic culture ; anaerobic mixed culture ; first-order kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method was developed to evaluate growth of a reductively dechlorinating bacterial population within a pentachlorophenol (PCP)- and acetate-fed, mixed, methanogenic culture. In 6- to 12-day experiments, a computer-monitored/feedback-controlled bioreactor was used to maintain constant pH, temperature, and acetate concentration, while transformation of multiple PCP additions was monitored. The potential at a platinum electrode, EPt, was not controlled externally, but was maintained constant at -0.25 ± 0.002 V (vs. SHE) by iron sulfides in the medium and the activity of the culture. PCP was reductively dechlorinated at the ortho position, yielding 3,4,5-trichlorophenol (3,4,5-TCP) via 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP). Below an initial PCP concentration of 0.5 μM, PCP was transformed to 3,4,5-TCP within 3 to 6 h. Biomass concentration changes were small during this period, and PCP and 2,3,4,5-TeCP transformations were modeled as pseudo-first-order reactions. Increases in pseudo-first-order rate constants for PCP and 2,3,4,5-TeCP were directly related to the amount of PCP transformed to 3,4,5-TCP, suggesting enrichment of a PCP-catabolizing population. Moreover, rate constant increases were independent of the amount of acetate consumed, changes in the overall volatile suspended solids (VSS) concentration, and the experimental duration. When PCP was added to the reactor at increasingly shorter time intervals in an exponential pattern, pseudo-first-order rate constants increased exponentially. An average rate constant doubling time of 1.7 days (1.4 to 2.3 d) was estimated. While the VSS concentration of the culture increased 60% in an 8-day period, pseudo-first-order rate constants increased by a factor of approximately 6. This large increase in transformation rate constants suggests growth of a bacterial population capable of using PCP and 2,3,4,5-TeCP as terminal electron acceptors. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 420-429, 1998.
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  • 107
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    Biotechnology and Bioengineering 57 (1998), S. 280-286 
    ISSN: 0006-3592
    Keywords: biofilm ; plasmid transfer ; conjugation ; mathematical models ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A strain of Pseudomonas putida that harbors plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor. Transfer of the RK2 mobilizable pDLB101 plasmid to B. azotoformans was monitored over a 4-day period. Experimental results demonstrated that the broad host range, RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. In the companion article to this work, the rate of plasmid transfer was quantified as a function of the limiting nutrient, succinate, and as a function of the mechanism of transfer. A biofilm process simulation program (AQUASIM) was modified to analyze resultant experimental data. Although the AQUASIM package was not designed to simulate or predict genetic events in biofilms, modification of the rate process dynamics allowed successful modeling of plasmid transfer. For the narrow range of substrate concentrations used in these experiments, nutrient level had only a slight effect on the rate and extent of plasmid transfer in biofilms. However, further simulations using AQUASIM revealed that under nutrient poor conditions, the number of transconjugants appearing in the biofilm was limited. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 280-286, 1998.
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  • 108
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    Biotechnology and Bioengineering 57 (1998), S. 306-313 
    ISSN: 0006-3592
    Keywords: Halomonas elongata ; osmotic shock ; fed-batch ; compatible solutes ; ectoine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel biotechnological process called “bacterial milking” has been established for the production of compatible solutes using the Gram-negative bacterium Halomonas elongata. Following a high-cell-density fermentation which provided biomass up to 48 g cell dry weight per liter, we applied alternating osmotic shocks in combination with crossflow filtration techniques to harvest the compatible solutes ectoine and hydroxyectoine. H. elongata, like other halophilic or halotolerant microorganisms, produces compatible solutes in response to the salinity of the medium. When transferred to a low salinity medium (osmotic downshock), H. elongata cells rapidly released their solutes to achieve osmotic equilibrium. Subsequent reincubation in a medium of higher salt concentration resulted in resynthesis of these compatible solutes and - after a defined regeneration time - the procedure could be repeated. By repeatedly performing this “bacterial milking” process (at least nine times) we were able to produce large amounts of ectoines with a biomass productivity of 155 mg of ectoine per cycle per gram cell dry weight. Further purification of the products was achieved by a simple two-step procedure based on cation exchange chromatography and crystallization. The principles described in this article may also be useful for the production of other low-molecular-weight compounds. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 306-313, 1998.
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  • 109
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    Biotechnology and Bioengineering 57 (1998), S. 314-320 
    ISSN: 0006-3592
    Keywords: Phaffia rhodozyma ; chemostat ; continuous fermentation ; astaxanthin ; peat ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Response surface methodology was applied to optimize the growth of the yeast Phaffia rhodozyma in continuous fermentation using peat hydrolysates as substrate. A second-order, complete, factorial design of the experiments was used to develop empirical models providing a quantitative interpretation of the relationships between the two variables studied, dilution rate and pH. Maximum biomass concentration in the fermentor was obtained by employing the following predicted optimum fermentation conditions: a dilution rate of 0.017/h and a pH level of 7.19. A verification experiment, conducted at previously optimized conditions for maximum biomass volumetric productivity (a dilution rate of 0.022/h, and a pH level of 6.90), produced values for biomass concentration, residual substrate concentration, biomass yield, and biomass volumetric productivity that were very close to the predicted values, indicating the reliability of the empirical model. The concentration of the pigment astaxanthin produced by the yeast under the optimized growth conditions was found to be 544 mg astaxanthin/kg dry cell biomass. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 314-320, 1998.
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  • 110
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    Biotechnology and Bioengineering 57 (1998), S. 462-470 
    ISSN: 0006-3592
    Keywords: mercury detoxification ; Pseudomonas aeruginosa ; bioreactor design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mercury-hyperresistant strain of Pseudomonas aeruginosa PU21 harboring plasmid Rip64 was utilized to develop bioprocesses able to detoxify and recover soluble mercuric ions in aquatic systems. The kinetics of mercury detoxification was investigated to determine the parameters needed for the design of the bioprocesses. Batch, fed-batch, and continuous bioreactors were utilized to evaluate the efficiency and feasibility of each mode of operation. The results showed that the specific mercury detoxification rate was dependent on cell growth phases, as well as the initial mercury concentrations. Cells at the lag growth phase exhibited the best specific detoxification rate of approximately 1.1 × 10-6 μg Hg/cell/h, and the rate was optimal at an initial mercury concentration of 8 mg/L. In batch operations with initial mercuric ions ranging from 2 to 10 mg/L, the mercuric ions added were rapidly volatilized from the media in less than 2-3 h. With periodic feeding of 3 or 5 mg Hg/L at fixed time intervals, the fed-batch processes had mercury removal efficiencies of 2.9 and 3.3 mg Hg/h/L, respectively. For continuous operations, the effluent cell concentration (Xe) was essentially invariant at 527 and 523 mg/L with the dilution rates (D) of 0.18 and 0.325 h-1, respectively. The increase in mercury feeding concentrations (Hgf) from 1.0 to 6.15 mg Hg2+/L did not affect the steady-state cell concentration (Xe) but forced the effluent mercury concentration (Hge) to increase. The decrease in the dilution rate, however, resulted in lower Hge values. It was also found that sequential mercury vapor absorption columns recovered over 80% of the Hg° released from the bioreactor while the residual mercury vapor was subsequently immobilized by an activated carbon trap in the down stream of the absorption column. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 462-470, 1998.
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  • 111
    ISSN: 0006-3592
    Keywords: specific metabolic rate ; calorimetry ; capacitance ; metabolic activity ; on-line biosensor ; animal cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: One of the requirements for enhanced productivity by the animal culture systems used in biotechnology is the direct assessment of the metabolic rate by on-line biosensors. Based on the fact that cell growth is associated with an enthalpy change, it is shown that the specific heat flow rate is stoichiometrically related to the net specific rates of substrates, products, and indeed to specific growth rate, and therefore a direct reflection of metabolic rate. Heat flow rate measured by conduction calorimetry has a technical advantage over estimates for many material flows which require assays at a minimum of two discrete times to give the rate. In order to make heat flow rate specific to the amount of the living cellular system, it would be advantageous to divide it by viable biomass. This requirement has been fulfilled by combining a continuous flow microcalorimeter ex situ with a dielectric spectroscope in situ, the latter measuring the viable cell mass volume fraction. The quality of the resulting biosensor for specific heat flow rate was illustrated using batch cultures of Chinese hamster ovary cells (CHO 320) producing recombinant human interferon-γ (IFN-γ) during growth in a stirred tank bioreactor under fully aerobic conditions. The measuring scatter of the probe was decreased significantly by applying the moving average technique to the two participant signals.It was demonstrated that the total metabolic rate of the cells, as indicated by the specific heat flow rate sensor, decreased with increasing time in batch culture, coincident with the decline in the two major substrates, glucose and glutamine, and the accumulation of the by-products, ammonia and lactate. Furthermore, the specific heat flow rate was an earlier indicator of substrate depletion than the flow rate alone. The calorimetric-respirometric ratio showed the intensive participation of anaerobic processes during growth and the related IFN-γ production. Specific heat flow rate was monotonically related to specific cell growth rate and associated with specific IFN-γ production. Specific heat flow rate is potentially a valid control variable for the growth of genetically engineered cell lines producing target proteins. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 464-477, 1998.
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  • 112
    ISSN: 0006-3592
    Keywords: lipase immobilization ; lipases in fine chemistry ; interfacial activation ; solid hydrophobic interfaces ; lipase stereospecificity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these “well-defined” hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their “open and hyperactivated structure”. This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 486-493, 1998.
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  • 113
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    Biotechnology and Bioengineering 58 (1998), S. 494-501 
    ISSN: 0006-3592
    Keywords: mixture optimization ; cellulase ; experimental design ; synergism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A factorial experimental design approach was used to optimize mixtures of six cellulases (five Thermomonospora fusca cellulases and plus/minus Trichoderma reesei CBHI along with β-glucosidase) so as to maximize the glucose produced from filter paper. Optimized mixture A and mixture B produced glucose at 25 and 8.3 μmol glucose/μmol enzyme/min, respectively, which are 8 and 1.5 times higher than the sum of the activity of the individual cellulases. In both mixtures, the glucose yield depended on the ratio and the cellulases used. Most enzymes showed synergistic interactions that increased the glucose yield. The yield of glucose with the optimum mixtures depended on the total enzyme concentration. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 494-501, 1998.
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  • 114
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    Biotechnology and Bioengineering 58 (1998), S. 502-509 
    ISSN: 0006-3592
    Keywords: genetically structured mathematical model ; trp operon ; cloned gene expression control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A genetically structured mathematical model of the trp attenuator in Escherichia coli based on known coupling mechanisms of the transcription of the trp leader region and translation of the trp leader peptide region is proposed. The model simulates, both qualitatively and quantitatively, the effects of tryptophan on the repression of cloned gene products. It shows that repression by attenuation mechanism alone operates over a narrow trp concentration range of 1 to 5 μM compared with 1 to 100 μM for trp repressor mechanism. This implies that attenuation by transcription termination is not relaxed until tryptophan starvation is severe. Simulation results show that the attenuator starts to derepress when the repressor is about 40% repressed, and becomes significantly derepressed only when the repressor repression decreased to about 20%. Unlike the case of repressor-operator interaction, the operating range of tryptophan concentration in the attenuator mechanism is not sensitive to plasmid copy number. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 502-509, 1998.
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  • 115
    ISSN: 0006-3592
    Keywords: nitrate removal ; Klebsiella oxytoca ; Arthrobacter globiformis ; dinitroethylene glycol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two strains, a gram-negative bacterium Klebsiella oxytoca CECT 4460 and a gram-positive, mycelium-forming bacterium Arthrobacter globiformis CECT 4500, tolerant to up to 1 M nitrate, were isolated from the grounds of a munitions factory. Under strict aerobic conditions and with appropriate C-sources, growth of these bacteria took place when the nitrate concentration in the medium was below 150 mM. Optimal growth conditions regarding the culture medium composition for the biological removal of nitrate were established in batch cultures. Then, the system was scaled up to a 40-L pilot plant and operated under continuous conditions in a factory with direct waste streams from dinitroethylene glycol production after appropriate dilution with nontreated groundwaters. The level of nitrate in the effluent was below 0.5% of the initial N-load. Nitrite and ammonium were undetectable and the level of the C-source in the effluent was below 50 mg per L. On the basis of these results, we conclude that the system worked on site satisfactorily. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 510-514, 1998.
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  • 116
    ISSN: 0006-3592
    Keywords: drug delivery ; tissue engineering ; surface modification ; polymer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We describe the development of a novel biodegradable polymer designed to present bioactive motifs at the surfaces of materials of any architecture. The polymer is a block copolymer of biotinylated poly(ethylene glycol) (PEG) with poly(lactic acid) (PLA); it utilizes the high-affinity coupling of the biotin-avidin system to undergo postfabrication surface engineering. We show, using surface plasmon resonance analysis (SPR) and confocal microscopy that surface engineering can be achieved under aqueous conditions in short time periods. These surfaces interact with cell surface molecules and generate beneficial responses as demonstrated by the model study of integrin-mediated spreading of endothelial cells on polymer surfaces presenting RGD peptide adhesion sequences. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 529-535, 1998.
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  • 117
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    Biotechnology and Bioengineering 58 (1998), S. 536-540 
    ISSN: 0006-3592
    Keywords: ultrasonication ; cell disruption ; E. coli ; β-galactosidase ; fusion proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The release kinetics of β-galactosidase protein have been determined during small-scale ultrasonication of E. coli cells. Among several studied parameters, ionic strength and cell concentration have the least influence on the rate of protein recovery, whereas sample volume and acoustic power dramatically affect the final yield of soluble protein in the cell-free fraction. The analysis of these critical parameters has prompted us to propose a simple model for E. coli disintegration that only involves acoustic power and sample volume, and which allows prediction of optimal sonication times to recover significant amounts of both natural and recombinant proteins in a given set of relevant conditions. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 536-540, 1998.
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  • 118
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    Biotechnology and Bioengineering 57 (1998), S. 518-528 
    ISSN: 0006-3592
    Keywords: ammonium ; UDP-GlcNAc ; N -glycosylation ; BHK-21 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of different ammonium concentrations and glucosamine on baby hamster kidney (BHK)-21 cell cultures grown in continuously perfused double membrane bioreactors was investigated with respect to the final carbohydrate structures of a secretory recombinant glycoprotein. The human interleukin-2 (IL-2) mutant glycoprotein variant IL-Mu6, which bears a novel N-glycosylation site (created by a single amino acid exchange of Gln100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH4Cl, or glucosamine. Recombinant glycoprotein products were purified and characterized by amino acid sequencing and carbohydrate structural analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry, high-pH anion-exchange chromatography with pulsed amperometric detection, and methylation analysis. In the absence of glutamine, cells secreted glycoprotein forms with preponderantly biantennary, proximal fucosylated carbohydrate chains (85%) with a higher NeuAc content (58%). Under standard conditions in the presence of 7.5 mM glutamine, complex-type N-glycans were found to be mainly biantennary (68%) and triantennary structures (33%) with about 50% containing proximal α1-6-linked fucose; 37% of the antenna were found to be substituted with terminal α2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH4Cl, a significant and reproducible increase in tri- and tetraantennary oligosaccharides (45% of total) was detected in the secretion product. In glutamin-free cultures supplemented with glucosamine, an intermediate amount of high antennary glycans was detected. The increase in complexity of N-linked oligosaccharides is considered to be brought about by the increased levels of intracellular uridine diphosphate-GlcNAc/GalNAc. These nucleotide sugar pools were found to be significantly elevated in the presence of high NH3/NH4+ and glucosamine concentrations. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 518-528, 1998.
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  • 119
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    Biotechnology and Bioengineering 57 (1998), S. 529-535 
    ISSN: 0006-3592
    Keywords: bacteriophage λ ; Q - mutant ; Escherichia coli ; recombinant protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We previously demonstrated that the λ system integrated into the host chromosome can overcome the instability encountered in continuous operations of unstable plasmid-based expression vectors. High stability of a cloned gene in a lysogenic state and a high copy number in a lytic state provide cloned-gene stability and overexpression in a two-stage continuous operation. But the expression by the commonly used S- mutant λ was only twice as high as that of the single copy. To increase the expression in the λ system, we constructed a Q- mutant λ vector that can be used in long-term operations such as a two-stage continuous operation. The Q- mutant phage λ is deficient in the synthesis of proteins involved in cell lysis and λ DNA packaging, while the S- mutant is deficient in the synthesis of one of two phage proteins required for lysis of the host cell and liberation of the progeny phage. Therefore, it is expected that the replicated Q- λ DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of the cloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products. The β-galactosidase expression per unit cell by the Q- mutant in a lytic state was about 30 times higher than that in a lysogenic state, while the expression by the commonly used S- mutant in a lytic state was twice as high as that in a lysogenic state. The optimal switching time of the Q- mutant from the lysogenic state to the lytic state for the maximum production of β-galactosidase was 5.3 h, which corresponds to an early log phase in the batch operation. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 529-535, 1998.
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  • 120
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    Biotechnology and Bioengineering 58 (1998), S. 451-451 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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  • 121
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    Biotechnology and Bioengineering 58 (1998), S. 453-463 
    ISSN: 0006-3592
    Keywords: Saccharopolyspora erythraea ; airlift reactor ; propeller ; heterogeneity ; cycling dissolved oxygen tension ; hydrodynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bioreactor heterogeneity has been studied in a multiconfigurable pilot-scale airlift reactor (0.25 m3) which created different degrees of heterogeneity. The impact of the two sparger configurations, i.e. in the draft tube or the annulus, in conjunction with a marine propeller fitted at the base of the downcomer, on the physiology of Saccharopolyspora erythraea was studied. Cellular growth, morphology, and productivity were compared between airlift and stirred tank reactors. Dissolved oxygen tension heterogeneity caused by differences in dissolved oxygen tension around the vessel did not affect growth, but the reduction of heterogeneity improved the specific erythromycin production rate and final specific production. Erythromycin production was shown to be proportional to the energy dissipation rate. The enhancement of bubble coalescence with increasing apparent viscosity led to the reduction of the sectional gas holdups and the improvement of liquid mixing. The extent of the changes with increasing apparent viscosity was dependent on the broth morphology, reactor configurations, and operating conditions. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 453-463, 1998.
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  • 122
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    Biotechnology and Bioengineering 58 (1998), S. 478-485 
    ISSN: 0006-3592
    Keywords: Aspergillus awamori ; intrinsic kinetic parameters ; oxygen concentration profiles ; oxygen microelectrode ; immobilisation ; pellet formation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fungi like Aspergillus awamori may spontaneously form pellets, which introduces an extra oxygen transfer resistance and influences the activity of the microorganism. Consequently, dramatic variations of apparent kinetics are reported in literature, due to variations in culture conditions, e.g., oxygen bulk concentration and pellet morphology. True intrinsic growth parameters like maximum growth rate and biomass yield, are important for process modelling and design. Values for these parameters may be obtained from observed kinetics by properly accounting for the anaerobic activity of the fungus. The true aerobic carbon yield for A. awamori of 0.6 mol Cx/mol Cs could be determined from the observed biomass yield after macroscopic monitoring of the anaerobic activity, and correction for the ethanol production by the fungal pellets. The true maximum growth rate was obtained from artificially immobilised A. awamori. In such well-defined system, transport is only diffusive and the morphology is not influenced by the stirring conditions. A maximum growth rate of 0.4 h-1 at pH 4.5 could be established in gel beads after microscopic monitoring of the oxygen penetration with microelectrodes. The developing biomass concentration profiles in these beads may be inferred from an adequate theoretical description of the oxygen profiles in course of time. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 478-485, 1998.
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  • 123
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    Biotechnology and Bioengineering 59 (1998), S. 2-9 
    ISSN: 0006-3592
    Keywords: polyethyleneglycol ; murine macrophages ; fibroblasts ; cell adhesion ; peptide immobilization ; multinucleated giant cell formation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyethyleneglycol-based networks were employed as substrates to graft bioactive peptides to study macrophage interactions with materials. Our overall objective was to utilize biologically active factors to stimulate certain macrophage function on materials suitable for implantation in connective tissues. In this study, we sought to explore the bioactivity of several peptides derived from extracellular matrix adhesion proteins and macrophage-active proteins that are normally soluble. The candidate peptides examined corresponded to residues 63 to 77 of complement component C3a (C3a(63-77)), residues 178 to 207 of interleukin-1 beta (IL1β(178-207)), residues 1615 to 1624 of fibronectin (FN(1615-1624)), endothelial-macrophage activating polypeptide II, complement component C5a inhibitory sequence, macrophage inhibitory peptide, and YRGDG; materials lacking peptides were used as negative controls. An established murine cell-line IC-21 was employed as a macrophage model, and human dermal fibroblasts were used for comparison. Our results showed that the substrates without grafted peptides were free from artifactual cell adhesion associated with the adsorption of serum or cellularly secreted proteins for long duration of culture. Of all grafted samples, IL1β(178-207)- and C3a(63-77)-grafted surfaces supported higher adherent macrophage densities. C3a(63-77)- and FN(1615-1624)-grafted surfaces supported higher adherent fibroblast densities. From competitive inhibition studies, cell adhesion was determined to occur in a receptor-peptide specific manner. The presence of grafted YRGDG in addition to IL1β(178-207), C3a(63-77), or FN(1615-1624) synergistically increased macrophage and fibroblast adhesion. Materials grafted with IL1β(178-207) or C3a(63-77) co-grafted with or without YRGDG did not support the formation of multinucleated giant cells from the fusion of adherent macrophages in vitro. © 1998 John Wiley & Sons, Inc., Biotechnol Bioeng 59:2-9, 1998.
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  • 124
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    Biotechnology and Bioengineering 59 (1998), S. 10-20 
    ISSN: 0006-3592
    Keywords: surface marker density ; receptor density ; immunomagnetic cell separation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A theoretical analysis was performed to determine the number of fractions a multidisperse, immunomagnetically labeled cell population can be separated into based on the surface marker (antigen) density. A number of assumptions were made in this analysis: that there is a proportionality between the number of surface markers on the cell surface and the number of immunomagnetic labels bound; that this surface marker density is independent of the cell diameter; and that there is only the presence of magnetic and drag forces acting on the cell. Due to the normal distribution of cell diameters, a “randomizing” effect enters into the analysis, and an analogy between the “theoretical plate” analysis of distillation, adsorption, and chromatography can be made. Using the experimentally determined, normal distribution of cell diameters for human lymphocytes and a breast cancer cell line, and fluorescent activated cell screening data of specific surface marker distributions, examples of theoretical plate calculations were made and discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:10-20, 1998.
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  • 125
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    Biotechnology and Bioengineering 59 (1998), S. 28-39 
    ISSN: 0006-3592
    Keywords: enzyme activities ; central metabolism ; mammalian cells ; chemostat culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:28-39, 1998.
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  • 126
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    Biotechnology and Bioengineering 59 (1998), S. 40-51 
    ISSN: 0006-3592
    Keywords: fixed-film bioreactor ; biofilter model ; trichloroethylene degradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Burkholderia cepacia PR123(TOM23C), expressing constitutively the TCE-degrading enzyme toluene ortho-monooxygenase (Tom), was immobilized on SIRAN™ glass beads in a biofilter for the degradation and mineralization of gas-phase trichloroethylene (TCE). To interpret the experimental results, a mathematical model has been developed which includes axial dispersion, convection, film mass-transfer, and biodegradation coupled with deactivation of the TCE-degrading enzyme. Parameters used for numerical simulation were determined from either independent experiments or values reported in the literature. The model was compared with the experimental data, and there was good agreement between the predicted and measured TCE breakthrough curves. The simulations indicated that TCE degradation in the biofilter was not limited by mass transfer of TCE or oxygen from the gas phase to the liquid/biofilm phase (biodegradation limits), and predicts that improving the specific TCE degradation rates of bacteria will not significantly enhance long-term biofilter performance. The most important factors for prolonging the performance of biofilter are increasing the amount of active biomass and the transformation capacity (enhancing resistance to TCE metabolism). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:40-51, 1998.
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  • 127
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    Biotechnology and Bioengineering 59 (1998), S. 52-61 
    ISSN: 0006-3592
    Keywords: denitritification ; denitratification ; anoxic filter ; kinetic model ; distributed fraction of reductase ; parametric sensitivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Denitritification and denitratification in anoxic filters were performed to generate experimental data. Also, a kinetic model of denitratification that accounts for intrinsic biokinetics and hydrodynamic behavior of the biofilter is proposed. In denitritification, the simulated results are in good agreement with the experimental data; and a higher nitrite influent concentration gives a higher nitrite reduction efficiency if the denitrifying loading is kept the same. In denitratification, the intermediate nitrite tends to accumulate, and a higher denitrifying loading results in a higher nitrite effluent concentration. By inserting biological and physical parameter values into the kinetic model, the variations in distributed fractions of nitrate-reductase (f) and nitrite-reductase (1-f) with different denitrifying loadings can be estimated by fitting in experimental data. The estimated f increased with an increase in denitrifying loading, implying that a higher denitrifying loading results in a higher nitrite effluent concentration. From parametric sensitivity analyses, the parameter f is more sensitive than other biological and physical parameters. Accordingly, the proposed kinetic model of denitratification can be used to predict the treatment performance of anoxic filters appropriately. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:52-61, 1998.
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  • 128
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    Biotechnology and Bioengineering 59 (1998), S. 68-72 
    ISSN: 0006-3592
    Keywords: enzymatic ; solid-to-solid conversion ; peptide synthesis ; proteases ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have studied a thermolysin-catalyzed solid-to-solid dipeptide synthesis using equimolar amounts of Z-Gln-OH and H-Leu-NH2 as model substrates. The high substrate concentrations make this an effective alternative to enzymatic peptide synthesis in organic solvents. Water content was varied in the range of 0 to 600 mL water per mol substrate and enzyme concentration in the range of 0.5 to 10 g/mol of substrates. High yields around 80% conversion and initial rates from 5 to 20 mmol s-1 kg-1 were achieved. The initial rate increases 10-fold on reducing the water content, to reach a pronounced optimum at 40 mL water per mol substrate. Below this, the rate falls to much lower values in a system with no added water, and to zero in a rigorously dried system. This behavior is discussed in terms of two factors: At higher water contents the system is mass transfer limited (as shown by varying enzyme content), and the diffusion distances required vary. At low water levels, effects reflect the stimulation of the enzymatic activity by water. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:68-72, 1998.
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  • 129
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    Keywords: 5′-nucleotide ; ester ; alcoholysis ; esterification ; nucleotide pyrophosphatase ; phosphodiesterase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nucleotide alkyl esters are pharmacologically important as potential (ant)agonists of purinoceptors and inhibitors of enzymes. Potato nucleotide pyrophosphatase (PNP) was compared with snake venom phosphodiesterase (SVP) as a catalyst to synthesize nucleotide alkyl esters. In methanol-water mixtures, the methanolysis/hydrolysis ratio of PNP, but not SVP, changed with pH and temperature, being optimal at high pH and low temperature. In a semi-preparative experiment, a crude PNP preparation produced 0.17 mM AMP-O-methyl ester (AMP-OMe) from 1 mM diadenosine 5′,5‴-P1,P2-diphosphate (AppA) and 5M methanol, at pH 9 and 0°C. Drawbacks to large-scale use are: low rates inherent to low temperatures, ATP unsuitability as a substrate for alcoholysis, and high cost of AppA. Advantages of PNP vs. SVP are cheapness, non-toxicity, and availability of the enzyme source. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:62-67, 1998.
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    Biotechnology and Bioengineering 59 (1998), S. 80-89 
    ISSN: 0006-3592
    Keywords: effective diffusion coefficient ; biofilm reactor ; biofilm thickness ; mass transfer ; silicone rubber membrane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel technique has been used to determine the effective diffusion coefficients for 1,1,2-trichloroethane (TCE), a nonreacting tracer, in biofilms growing on the external surface of a silicone rubber membrane tube during degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 and monochlorobenzene (MCB) by Pseudomonas JS150. Experiments were carried out in a single tube extractive membrane bioreactor (STEMB), whose configuration makes it possible to measure the transmembrane flux of substrates. A video imaging technique (VIT) was employed for in situ biofilm thickness measurement and recording. Diffusion coefficients of TCE in the biofilms and TCE mass transfer coefficients in the liquid films adjacent to the biofilms were determined simultaneously using a resistances-in-series diffusion model. It was found that the flux and overall mass transfer coefficient of TCE decrease with increasing biofilm thickness, showing the importance of biofilm diffusion on the mass transfer process. Similar fluxes were observed for the nonreacting tracer (TCE) and the reactive substrates (MCB or DCE), suggesting that membrane-attached biofilm systems can be rate controlled primarily by substrate diffusion. The TCE diffusion coefficient in the JS150 biofilm appeared to be dependent on biofilm thickness, decreasing markedly for biofilm thicknesses of 〉 1 mm. The values of the TCE diffusion coefficients in the JS150 biofilms 〈1-mm thick are approximately twice those in water and fall to around 30% of the water value for biofilms 〉 1-mm thick. The TCE diffusion coefficients in the GJ10 biofilms were apparently constant at about the water value. The change in the diffusion coefficient for the JS150 biofilms is attributed to the influence of eddy diffusion and convective flow on transport in the thinner (〈1-mm thick) biofilms. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:80-89, 1998.
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  • 131
    ISSN: 0006-3592
    Keywords: aqueous two-phase systems ; immobilized enzymes ; continuous extraction of product ; penicillin G acylase ; synthesis of antibiotics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend.Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:73-79, 1998.
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    Biotechnology and Bioengineering 59 (1998), S. 99-107 
    ISSN: 0006-3592
    Keywords: Monod kinetics ; mixed substrate growth ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the μmax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:99-107, 1998.
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  • 133
    ISSN: 0006-3592
    Keywords: apoptosis ; necrosis ; bcl-2 ; amino acids ; cell culture ; cell death ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90-98, 1998.
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    Biotechnology and Bioengineering 59 (1998), S. 128-128 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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    Biotechnology and Bioengineering 59 (1998), S. 116-121 
    ISSN: 0006-3592
    Keywords: mixed-substrate growth ; mathematical model ; competing species ; dynamical analogy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: There is a similarity between the metabolic dynamics of a microbial species growing on a mixture of two substrates and the dynamics of growth of two competing populations. Specifically, the enzymes catalyzing the uptake and catabolism of substrates exhibit phenomena analogous to extinction and coexistence.“Extinction” of the enzymes associated with one of the substrates results in sequential utilization of the substrates (diauxie) (Monod, 1942). “Coexistence” of the enzymes associated with the substrates results in simultaneous utilization of the substrates (Egli, 1995). Here, we formulate a simple model that shows the basis for this dynamical similarity: The equations describing the evolution of the enzyme levels are dynamical analogs of the Lotka-Volterra model for two competing species. The analogy suggests ways of capturing the experimentally observed preculture-dependent growth patterns, i.e., growth patterns that vary depending on the physiological state of the preculture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:116-121, 1998.
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  • 136
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    Biotechnology and Bioengineering 59 (1998), S. 122-127 
    ISSN: 0006-3592
    Keywords: lipase ; organic solvent ; flavour esters ; interfacial activation ; enzyme conformers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to improve the lipase-catalyzed synthesis of flavour esters, we have used the reported strategy of interfacial activation-based molecular (bio)imprinting [Mingarro et al. 1995. Proc. Natl. Acad. Sci. U.S.A. 92: 3308], later called trapping in the presence of amphiphile interfaces (TPI) [Mingarro et al. 1996. Biochemistry 35: 9935]. Five lipases of fungal and mammalian origin typically used for esterification process have been explored to improve production by TPI treatment. A marked enhancement of enzymatic activity has been observed in all TPI-treated lipases assayed and the activation factor obtained was up to 90-fold. The dependence on chain length of acyl donors in the esterification of geranyl alcohol has been investigated, showing clear differences between activated and nonactivated lipase. The results indicate that this rational approach leads to conversion yields that are remarkably higher, not only than its counterpart pH-optimized control lipase, but also the “protected” lipase by conventional methods (lyoprotectans or salts). We propose this strategy as a promising tool to be used in more industrial biotransformations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:122-127, 1998.
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    Biotechnology and Bioengineering 59 (1998), S. 108-115 
    ISSN: 0006-3592
    Keywords: thermophilic β-glycosidase ; catalytic membranes ; nonisothermal bioreactors ; thermodialysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Catalytic membranes, obtained by immobilizing thermophilic β-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic β-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, α and β, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1°C or a temperature gradient of 1°C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:108-115, 1998.
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    Biotechnology and Bioengineering 59 (1998), S. 310-317 
    ISSN: 0006-3592
    Keywords: acetic acid fermentation ; liquid-gas equilibrium ; evaporation losses ; mathematical model ; open fermentation system ; semiclosed fermentation system ; closed fermentation system ; laboratory scale ; pilot plant scale ; industrial plant scale ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An experimental study was conducted to propose an adequate mathematical model for liquid-gas equilibrium in acetic acid fermentations. Three operation scales (laboratory, pilot plant, and industrial plant) were employed to obtain the sets of experimental data. The proposed model, based in the UNIFAC method for the estimation of activity coefficients of a solution consisting of several components, takes into account the effect of temperature. However, in the set of equations, it has been necessary to put in the degree of equilibrium (∊). This coefficient adequately reflects the physical conditions of fermentation equipment. The experimental and numerical results help to define the fundamental mechanisms for liquid-gas equilibrium in these systems and demonstrate the model validity in the three tested scales. It was also found that in an industrial setting, closed systems are those with lowest evaporation losses. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:310-317, 1998.
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  • 139
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    Biotechnology and Bioengineering 59 (1998), S. 302-309 
    ISSN: 0006-3592
    Keywords: biofilms ; microelectrodes ; local mass transfer coefficient ; effective diffusivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Microelectrodes were used to measure oxygen profiles and local mass transfer coefficient profiles in biofilm clusters and interstitial voids. Both profiles were measured at the same location in the biofilm. From the oxygen profile, the effective diffusive boundary layer thickness (DBL) was determined. The local mass transfer coefficient profiles provided information about the nature of mass transport near and within the biofilm. All profiles were measured at three different average flow velocities, 0.62, 1.53, and 2.60 cm sec-1, to determine the influence of flow velocity on mass transport. Convective mass transport was active near the biofilm/liquid interface and in the upper layers of the biofilm, independent of biofilm thickness and flow velocity. The DBL varied strongly between locations for the same flow velocities. Oxygen and local mass transfer coefficient profiles collected through a 70 μm thick cluster revealed that a cluster of that thickness did not present any significant mass transport resistance. In a 350 μm thick biofilm cluster, however, the local mass transfer coefficient decreased gradually to very low values near the substratum. This was hypothetically attributed to the decreasing effective diffusivity in deeper layers of biofilms. Interstitial voids between clusters did not seem to influence the local mass transfer coefficients significantly for flow velocities of 1.53 and 2.60 cm sec-1. At a flow velocity of 0.62 cm sec-1, interstitial voids visibly decreased the local mass transfer coefficient near the bottom. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:302-309, 1998.
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  • 140
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    Biotechnology and Bioengineering 59 (1998), S. 318-327 
    ISSN: 0006-3592
    Keywords: plasmid ; retention ; TCE ; biofilm ; segregational stability ; activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The activity and stability of the TCE degradative plasmid TOM31c in the transconjugant host Burkholderia cepacia 17616 was studied in selective and non-selective biofilm cultures. The activity of plasmid TOM31c in biofilm cultures was measured by both TCE degradative studies and the expression of the Tom pathway. Plasmid loss was measured using continuous flow, rotating annular biofilm reactors, and various analytical and microbiological techniques. The probability of plasmid loss in the biofilm cultures was determined using a non-steady-state biofilm plasmid loss model that was derived from a simple mass balance, incorporating results from biofilm growth and plasmid loss studies. The plasmid loss model also utilized Andrew's inhibition growth kinetics and a biofilm detachment term.Results from these biofilm studies were compared to similar studies performed on suspended cultures of Burkholderia cepacia 17616-TOM31c to determine if biofilm growth has a significant effect on either plasmid retention or Tom pathway expression (i.e., TCE degradation rates). Results show that the activity and expression of the Tom pathway measured in biofilm cultures was significantly less than that found in suspended cultures at comparable growth rates. The data obtained from these studies fit the plasmid loss model well, providing plasmid loss probability factors for biofilm cultures that were equivalent to those previously found for suspended cultures. The probability of plasmid loss in the B. cepacia 17616-TOM31c biofilm cultures was equivalent to those found in the suspended cultures. The results indicate that biofilm growth neither helps nor hinders plasmid stability. In both the suspended and the biofilm cultures, plasmid retention and expression could be maintained using selective growth substrates and/or an appropriate plasmid-selective antibiotic. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:318-327, 1998.
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  • 141
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    Biotechnology and Bioengineering 59 (1998), S. 328-343 
    ISSN: 0006-3592
    Keywords: biotrickling filters ; biotrickling filter modeling ; mono-chlorobenzene ; biodegradation kinetics of mono-chlorobenzene ; chlorinated VOC emissions ; biofiltration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Removal of mono-chlorobenzene (m-CB) vapor from airstreams was studied in a biotrickling filter (BTF) operating under counter-current flow of the air and liquid streams. Experiments were performed under various values of inlet m-CB concentration, air and/or liquid volumetric flow rates, and pH of the recirculating liquid. Conversion of m-CB was never below 70% and at low concentrations exceeded 90%. A maximum removal rate of about 60 gm-3-reactor h-1 was observed. Conversion of m-CB was found to increase as the values of liquid and air flow rate increase and decrease, respectively. The effects of pH and frequency of medium replenishment on BTF performance were also investigated. The process was successfully described with a detailed mathematical model, which accounts for mass transfer and kinetic effects based on m-CB and oxygen availability. Solution of the model equations yielded m-CB and oxygen concentration profiles in all three phases (airstream, liquid, biofilm). It is predicted that oxygen has a controling effect on the process at high inlet m-CB concentrations. From independent, suspended culture, experiments it was found that m-CB biodegradation follows Andrews inhibitory kinetics. The kinetic constants were found to remain practically unchanged after the culture was used in BTF experiments for 8 months. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:328-343, 1998.
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  • 142
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    Biotechnology and Bioengineering 59 (1998), S. 344-350 
    ISSN: 0006-3592
    Keywords: electrodialysis ; citric acid ; pH ; temperature ; Faraday efficiency ; solute recovery efficiency ; specific energy consumption ; solute flux ; water flux ; feed solute concentration ; electric current density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of pH and temperature (θ) on the overall performance indicators (i.e., solute recovery, ρ, and Faraday, η, efficiencies; specific energy consumption, ε, solute, JS, and water, JW, fluxes) of batch electrodialytic recovery of citric acid from model solutions was assessed at different values of feed solute concentration (cSf) and electric current density (j). Regardless of the initial feed concentration used, ρ and JS were found to be independent of θ; η and JW exhibited a positive trend with respect to θ, while ε a negative one. At the maximum temperature tested (33°C), as the pH of the feed solution was varied from 3 to 7, ρ increased from 0.90 ± 0.08 to 0.97 ± 0.02, η grew from 0.09 ± 0.02 to 0.50 ± 0.01, JS practically doubled, ε reduced about 8 times, but JW increased from 3 to 4 times. So, the optimal conditions for this technique are to be determined by balancing the savings in the investment and maintenance costs against the energy costs. © John Wiley & Sons, Inc. Biotechnol Bioeng 59:344-350, 1998.
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  • 143
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    Biotechnology and Bioengineering 58 (1998), S. 250-253 
    ISSN: 0006-3592
    Keywords: in vivo 13C-NMR ; Rhizobium meliloti ; polymer biosynthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of in vivo 13C-NMR approach for the monitoring of the synthesis of various polymers within cells of Rhizobium meliloti (M5N1 strain) is reported. Significant differences in polymer biosynthesis have been shown as a function of the metabolic state of the cells and the labeled carbon source used. Consumption of carbon source and produced glycogen was complete with mid-exponential phase harvested cells. This was not the case with stationary phase harvested cells, for which polyhydroxybutyrate synthesis was higher and gluconate synthesis was lower than the former. [1-13C]fructose-grown cells produced more exopolysaccharide and polyhydroxybutyrate, but less β-(1,2) glucan and gluconate than [1-13C]glucose-grown cells. This approach offers a suitable tool to examine the kinetics of polymer biosynthesis by Rhizobia. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:250-253, 1998.
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  • 144
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    Keywords: chymotrypsin ; enzyme stability ; reversed micelles ; interface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stability of α-chymotrypsin and δ-chymotrypsin was studied in reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. α-Chymotrypsin is inactivated at the interface and at the water pool, while δ-chymotrypsin is inactivated only at the water pool. The mechanism of inactivation at the interface is related to the interaction of N-terminal group alanine 149 (absent in δ-chymotrypsin) with the negative interface. The dependence of enzyme activity on water content of these two enzymes in reversed micelles of AOT is also related with the interface interaction, since δ-chymotrypsin does not have a bell-shaped curve as observed for α-chymotrypsin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:360-363, 1998.
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  • 145
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    Biotechnology and Bioengineering 60 (1998), S. 333-340 
    ISSN: 0006-3592
    Keywords: fluoride-modified zirconia ; expanded bed ; packed bed ; protein adsorption ; adsorption-desorption kinetics ; intraparticle diffusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The expanded bed characteristics of 75-103μm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2× expanded bed, and a 3× expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 ± 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 μm) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times (∼0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion.© 1998 John Wiley & Sons, Inc. Bioeng 60: 333-340, 1998.
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  • 146
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    Biotechnology and Bioengineering 58 (1998), S. 263-266 
    ISSN: 0006-3592
    Keywords: Streptomyces lividans ; simple structured modeling ; cybernetic modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The growth of Streptomyces lividans in defined media was modeled using a simple structured growth model. Conventional unstructured models like Monod kinetics, substrate inhibition kinetics, and the logistic equation were also used in an attempt to fit the data, but the results were all unsatisfactory. The main reason for failure in applying simple unstructured models is that they cannot describe the long lag phases sometimes observed during growth of S. lividans. The simple structured growth model was derived along similar principles to cybernetic growth models. This model quite accurately describes the growth of S. lividans. It assumes that the rate of assimilation of a substrate depends on the concentration of a specific key enzyme. This key enzyme is only produced in the presence of the substrate, and it is broken down at a steady rate. An enzyme synthesis allocation variable, w, similar to the cybernetic variable, u, described in cybernetic growth models, is proposed to control enzyme synthesis. Until the key enzyme concentration approaches its maximum level, very little substrate is consumed. And consequently, the lag phase is sustained. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:263-266, 1998.
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  • 147
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    Biotechnology and Bioengineering 58 (1998), S. 267-271 
    ISSN: 0006-3592
    Keywords: metabolic engineering ; mathematical programming ; mixed integer nonlinear programming (MINLP) ; metabolic control analysis ; Dictyostelium discoideum ; tricarboxylic acid cycle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The design of new generation bioprocessing plants is increasingly dependent on the design of process-compatible microorganisms. The latter, whether through genetic or physiological manipulations, can be greatly assisted by metabolic engineering. An emerging powerful tool in metabolic engineering research is computer-assisted cell design using mathematical programming. In this work, the problem of optimizing cellular metabolic networks has been formulated as a Mixed Integer Nonlinear Programming (MINLP) model. The model can assist genetic engineers to identify which cellular enzymes should be modified, and the new levels of activity required to produce an optimal network. Results are presented from the tricarboxylic acid cycle in Dictyostelium discoideum. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:267-271, 1998.
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  • 148
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    Biotechnology and Bioengineering 59 (1998), S. 239-247 
    ISSN: 0006-3592
    Keywords: metabolic design analysis ; gene engineering ; biochemical reaction networks ; modular/top-down approach ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A biotechnological aim of genetic engineering is to increase the intracellular concentration or secretion of valuable compounds, while making the other concentrations and fluxes optimal for viability and productivity. Efforts to accomplish this based on over-expression of the enzyme, catalyzing the so-called “rate-limiting step,” have not been successful. Here we develop a method to determine the enzyme concentrations that are required to achieve such an aim. This method is called Metabolic Design Analysis and is based on the perturbation method and the modular (“top-down”) approach - formalisms that were first developed for the analysis of biochemical regulation such as, Metabolic Control Analysis. Contrary to earlier methods, the desired alterations of cellular metabolism need not be small or confined to a single metabolite or flux. The limits to the alterations of fluxes and metabolite concentrations are identified. To employ Metabolic Design Analysis, only limited kinetic information concerning the pathway enzymes is needed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 239-247, 1998.
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  • 149
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    Biotechnology and Bioengineering 59 (1998), S. 227-238 
    ISSN: 0006-3592
    Keywords: Bacillus subtilis ; folic acid ; metabolic engineering ; metabolic fluxes ; purine nucleosides ; riboflavin ; stoichiometric model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We developed a stoichiometric model of Bacillus subtilis metabolism for quantitative analysis of theoretical growth and biochemicals production capacity. This work concentrated on biochemicals that are derived from the purine biosynthesis pathway; inosine, guanosine, riboflavin, and folic acid. These are examples of commercially relevant biochemicals for which Bacillus species are commonly used production hosts. Two previously unrecognized, but highly desirable properties of good producers of purine pathway-related biochemicals have been identified for optimally engineered product biosynthesis; high capacity for reoxidation of NADPH and high bioenergetic efficiency. Reoxidation of NADPH, through the transhydrogenase or otherwise, appears to be particularly important for growth on glucose, as deduced from the corresponding optimal carbon flux distribution. The importance of cellular energetics on optimal performance was quantitatively assessed by including a bioenergetic efficiency parameter as an unrestricted, ATP dissipating flux in the simulations. An estimate for the bioenergetic efficiency was generated by fitting the model to experimentally determined growth yields. The results show that the maximum theoretical yields of all products studied are limited by pathway stoichiometry at high bioenergetic efficiencies. Simulations with the estimated bioenergetic efficiency of B. subtilis, growing under glucose-limiting conditions, indicate that the yield of these biochemicals is primarily limited by energy and thus is very sensitive to the process conditions. The maximum yields that can reasonably be expected with B. subtilis on glucose were estimated to be 0.343, 0.160, and 0.161 (mol product/mol glucose) for purine nucleosides, riboflavin, and folic acid, respectively. Potential strategies for improving these maximum yields are discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 227-238, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 296-298 
    ISSN: 0006-3592
    Keywords: lac promoter ; galactose ; galactokinase mutant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred.Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:296-298, 1998.
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  • 151
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    Biotechnology and Bioengineering 58 (1998), S. 299-302 
    ISSN: 0006-3592
    Keywords: phosphoglucomutase ; site-directed mutagenesis ; kinetic constants ; Pm promoter ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mutants of Escherichia coli deficient in phosphoglucomutase accumulate amylose when the cells are grown on maltose or galactose as carbon source. In the presence of physiological levels of phosphoglucomutase, most of the sugar is catabolized, leading to strongly reduced levels of amylose accumulation. By varying the expression level of heterologous phosphoglucomutase, we show that the minimum level needed to block amylose accumulation corresponds to a phosphoglucomutase activity of 150-600 nmole substrate transformed per min per mg of total soluble protein. Mutant phosphoglucomutases with strongly reduced Vmax values and increased Km values for the substrate glucose-1-phosphate or the co-substrate glucose-1,6-diphosphate, could also reduce amylose accumulation, but much higher enzyme expression levels were required. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:299-302, 1998.
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  • 152
    ISSN: 0006-3592
    Keywords: glycerol ; Enterobacter agglomerans ; 3-hydroxypropionaldehyde ; catabolic limitation ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Batch fermentation of glycerol to 1,3-propanediol (1,3PPD) by Enterobacter agglomerans CNCM 1210 showed the lethal accumulation of 3-hydroxypropionaldehyde (3-HPA) when performed under initial substrate content higher than 40 g/L. Assigned to the inhibition by the NAD/NADH ratio of the 3-HPA converting enzyme: 1,3PPD dehydrogenase, intracellular assays were conducted in an attempt to identify the metabolic mechanisms involved in the increase of that ratio. An overflow metabolism through the 1,3PPD formation pathway was established, while a catabolic limitation in the oxidative branch at the level of glyceraldehyde-3-phosphate dehydrogenase occurred. Uncoupled activities of synthesis and consumption of reducing equivalents are thus suspected to provoke the increase of the NAD/NADH ratio and the subsequent accumulation of 3-HPA. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:303-305, 1998.
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  • 153
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    Biotechnology and Bioengineering 59 (1998), S. 754-761 
    ISSN: 0006-3592
    Keywords: polyphosphate ; Escherichia coli ; phosphate starvation ; phosphate secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:754-761, 1998.
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  • 154
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    Biotechnology and Bioengineering 58 (1998), S. 321-324 
    ISSN: 0006-3592
    Keywords: yeast cell wall porosity and permeability ; β-1,3-glucanase ; selective protein release ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:321-324, 1998.
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  • 155
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    Biotechnology and Bioengineering 58 (1998), S. 316-320 
    ISSN: 0006-3592
    Keywords: ATP allocation ; celluloytic microorganisms ; consolidated bioprocessing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Under anaerobic, carbon limited conditions, celluloytic fermentative microorganisms face a metabolic choice with respect to the allocation of relatively scarce ATP: to invest it in cells or in hydrolytic enzymes. A model is proposed that defines an allocation parameter reflecting the fractional expenditure of ATP on cell synthesis relative to the total ATP available (gross ATP synthesized less maintenance). This parameter is then incorporated into an ATP-centered model of anaerobic cellulose fermentation based on the ethanol fermentation of yeast and the cellulase system of Trichoderma reesei. Results indicate that high processing rates are possible via a consolidated bioprocessing strategy, especially at high cellulase specific activities, and that cell/cellulase allocation represents an interesting system in which to study, and perhaps exploit, microbial evolution and metabolic control. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:316-320, 1998.
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  • 156
    ISSN: 0006-3592
    Keywords: l-ascorbic acid ; vitamin C ; 2-keto-l-gulonic acid ; l-sorbose dehydrogenase ; l-sorbosone dehydrogenase ; Gluconobacter ; chemical mutation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from d-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of l-sorbosone and 2-KLGA, l-sorbose dehydrogenase (SDH) and l-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine resulted in improvement of the production level. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:309-315, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 325-328 
    ISSN: 0006-3592
    Keywords: poly(3-hydroxybutyrate) ; Escherichia coli ; filamentation suppression ; defined medium ; high cell density culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E. coli that underwent filamentation. Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E. coli. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:325-328, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 329-332 
    ISSN: 0006-3592
    Keywords: tobacco cultured cells ; heat-shock promoter of Arabidopsis thaliana ; strong promoter from tobacco cell ; β-glucuronidase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the β-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor.Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-α, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5′-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells.Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:329-332, 1998.
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  • 159
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    Biotechnology and Bioengineering 60 (1998), S. 24-35 
    ISSN: 0006-3592
    Keywords: in situ microscopy ; image analysis ; cell concentration ; cell morphology ; biomass ; histogram ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A sensor is presented, which allows on-line microscopic observation of microorganisms during fermentations in bioreactors. This sensor, an In Situ Microscope (ISM) consists of a direct-light microscope with a measuring chamber, integrated in a 25 mm stainless steel tube, two CCD-cameras, and two frame-grabbers. The data obtained are processed by an automatic image analysis system.The ISM is connected with the bioreactor via a standard port, and it is immersed directly in the culture liquid - in our case Saccharomyces cerevisiae in a synthetic medium. The microscopic examination of the liquid is performed in the measuring chamber, which is situated near the front end of the sensor head. The measuring chamber is opened and closed periodically. In the open state, the liquid in the bioreactor flows unrestricted through the chamber. In closing, a defined volume of 2,2 · 10-8 mL of the liquid becomes enclosed. After a few seconds, when the movement of the cells in the enclosed culture has stopped, they are examined with the microscope. The microscopic images of the cells are registered with the CCD-cameras and are visualized on a monitor, allowing a direct view of the cell population. After detection, the measuring chamber reopens, and the enclosed liquid is released.The images obtained are evaluated as to cell concentration, cell size, cell volume, biomass, and other relevant parameters simultaneously by automatic image analysis. With a PC (486/33 MHz), image processing takes about 15 s per image. The detection range tested when measuring cells of S. cerevisiae is about 106 to 109 cells/mL (equivalent to a biomass of 0.01 g/L to 12 g/L). The calculated biomass values correlate very well with those obtained using dry weight analysis. Furthermore, histograms can be calculated, which are comparable to those obtained by flow cytometry. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 24-35, 1998.
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  • 160
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    Biotechnology and Bioengineering 60 (1998), S. 580-588 
    ISSN: 0006-3592
    Keywords: soybean oil ; ammonium sulfate ; secondary metabolite production ; streptomyces ; lipase ; homologs ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A valine-overproducing mutant (MA7040, Streptomyces hygroscopicus) was found to produce 1.5 to 2.0 g/L of the immunoregulant, L-683,590, at the 0.6 m3 fermentation scale in a simple batch process using soybean oil and ammonium sulfate-based GYG5 medium. Levels of both lower (L-683,795) and higher (HH1 and HH2) undesirable homolog levels were controlled adequately. This batch process was utilized to produce broth economically at the 19 m3 fermentation scale. Material of acceptable purity was obtained without the multiple pure crystallizations previously required for an earlier culture, MA6678, requiring valine supplementation for impurity control.Investigations at the 0.6 m3 fermentation scale were conducted, varying agitation, pH, initial soybean oil/ammonium sulfate charges, and initial aeration rate to further improve growth and productivity. Mid-cycle ammonia levels and lipase activity appeared to have an important role. Using mid-cycle soybean oil additions, a titer of 2.3 g/L of L-683,590 was obtained, while titers reached 2.7 g/L using mid-cycle soybean oil and ammonium sulfate additions. Both higher and lower homolog levels remained acceptable during this fed-batch process. Optimal timing of mid-cycle oil and ammonium sulfate additions was considered a critical factor to further titer improvements. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 580-588, 1998.
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  • 161
    ISSN: 0006-3592
    Keywords: CH0 cells ; sialidase activity ; recombinant DNase ; sialic acid ; antisense DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Under some cell culture conditions, recombinant glycoprotein therapeutics expressed in Chinese hamster ovary (CHO) cells lose sialic acid during the course of the culture (Sliwkowski et al., 1992; Munzert et al., 1996). A soluble sialidase of CHO cell origin degrades the expressed recombinant protein and has been shown to be released into the culture fluid as the viability of the cells decreases. To reduce the levels of the sialidase and to prevent desialylation of recombinant protein, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA. Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5′ 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values. To test if this level of sialidase would lead to increased sialic acid content of an expressed recombinant protein, the 474 antisense clone was employed as a host for expression of human DNase as a model glycoprotein. The sialic acid content of the DNase produced in the antisense cultures was compared with material made in the wild-type parental cell line. About 20-37% increase in sialic acid content, or 0.6-1.1 mole of additional sialic acid out of a total of 3.0 mole on the product, was found on the DNase made in the antisense cell lines. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 589-595, 1998.
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  • 162
    ISSN: 0006-3592
    Keywords: micellar electrokinetic capillary chromatography ; capillary isoelectric focusing ; Chinese hamster ovary ; interferon-gamma ; perfusion culture ; glycosylation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chinese hamster ovary cells producing recombinant human interferon-γ were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 × 107 cells (mL microcarrier)-1 at a specific growth rate (μ) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-γ was monitored by capillary electrophoresis of intact IFN-γ proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-γ by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-γ (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-γ by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-γ proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-γ during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-γ produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 596-607, 1998.
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  • 163
    ISSN: 0006-3592
    Keywords: site-directed mutagenesis ; oriented immobilization ; subtilisin ; membrane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN′ on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 608-616, 1998.
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  • 164
    ISSN: 0006-3592
    Keywords: Klebsiella pneumoniae ; glycerol ; pyruvate kinase ; pyruvate:formate-lyase ; pyruvate dehydrogenase ; in vitro and in vivo activities ; dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The activities of pyruvate kinase (PK), pyruvate: formate-lyase (PFL), pyruvate dehydrogenase (PDH), and citrate synthase (CS) involved in the anaerobic glycerol conversion by Klebsiella pneumoniae were studied in continuous culture under conditions of steady states and sustained oscillations. Both the in vitro and in vivo activities of PK, PFL, and PDH are strongly affected by the substrate concentration and its uptake rate, as is the in vitro activity of CS. The flux from phosphoenolpyruvate to pyruvate is found to be mainly regulated on a genetic level by the synthesis rate of PK, particularly at low substrate concentration and low growth rate. In contrast, the conversion of pyruvate to acetyl-CoA is mainly regulated on a metabolic level by the in vivo activities of PFL and PDH. The ratio of in vitro to in vivo activities is in the range of 1 to 1.5 for PK, 5 to 17 for PFL and 5 to 80 for PDH under the experimental conditions. The regulation of in vivo activity and synthesis of these enzymes is sensitive to fluctuations of culture conditions, leading to oscillations of both the in vitro and in vivo activities. In particular, PFL is strongly affected during oscillations; its average in vitro activity is only about half of its corresponding steady-state value under similar environmental conditions. The average in vitro activities of PDH and PK under oscillations are close to their corresponding steady-state values. In contrast to all other enzymes measured for the glycerol metabolism by K. pneumoniae PFL and PDH are more effectively in vivo utilized under oscillations than under steady state, underlining the peculiar role of pyruvate metabolism in the dynamic responses of the culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 617-626, 1998.
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  • 165
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    Biotechnology and Bioengineering 57 (1998), S. 1-10 
    ISSN: 0006-3592
    Keywords: pTRIDENT ; tricistronic expression vectors ; gene expression ; mammalian cells ; tetracycline-responsive promoter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We constructed tricistronic expression vectors for the simultaneous and coordinated expression of three independent genes in mammalian cells. One single promoter allows high level and, in some vectors, adjustable transcription of all three cistrons. Whereas the first cistron is translated in a cap-dependent manner, the subsequent ones utilize intercistronic regions of viral origin such as the internal ribosomal entry site of poliovirus or the cap-independent translation enhancer of encephalomyocarditis virus for enhanced translation. Three multiple cloning sites with a total of up to 18 unique restriction sites allow sequential cloning of the genes of interest. The modular structure of this pBluescript®-based high copy number vector system allows straightforward movement of individual cistrons among members of the pTRIDENT family, and facilitates their combination with existing expression vectors. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 1-10, 1998.
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  • 166
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    Biotechnology and Bioengineering 57 (1998), S. 11-21 
    ISSN: 0006-3592
    Keywords: proteins ; salts ; intermolecular interactions ; potentials of mean force ; precipitation ; crystallization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 11-21, 1998.
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  • 167
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    Biotechnology and Bioengineering 57 (1998), S. 55-61 
    ISSN: 0006-3592
    Keywords: synthetic antimicrobial peptide ; prochymosin ; recombinant ; expression ; purification ; fusion protein ; inclusion bodies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 55-61, 1998.
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  • 168
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    Biotechnology and Bioengineering 57 (1998), S. 228-237 
    ISSN: 0006-3592
    Keywords: AlkB ; Pseudomonas oleovorans ; alkane hydroxylase ; iron ; Escherichia coli ; alk + recombinants ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The alk genes enable Pseudomonas oleovorans to utilize alkanes as sole carbon and energy source. Expression of the alk genes in P. oleovorans and in two Escherichia coli recombinants induced iron limitation in minimal medium cultures. Further investigation showed that the expression of the alkB gene, encoding the integral cytoplasmic membrane protein AlkB, was responsible for the increase of the iron requirement of E. coli W3110 (pGEc47).AlkB is the non-heme iron monooxygenase component of the alkane hydroxylase system, and can be synthesized to levels up to 10% (w/w) of total cell protein in E. coli W3110 (pGEc47). Its synthesis is, however, strictly dependent on the presence of sufficient iron in the medium. Our results show that a glucose-grown E. coli alk+ strain can reach alkane hydroxylase activities of about 25 U/g cdw, and are consistent with the recent finding that catalytically active AlkB contains two, rather than one iron atom per polypeptide chain. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 228-237, 1998.
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  • 169
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    Biotechnology and Bioengineering 57 (1998), S. 238-244 
    ISSN: 0006-3592
    Keywords: expression ; membrane protein ; glycophorin ; neomycin resistance ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gene for the integral membrane protein glycophorin A (GPA) was cloned in frame to the 5′ end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT). Protein expression was achieved in Escherichia coli as well as in mammalian cells. In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks after transfection; the amount of GPA-NPT fusion protein produced was constant from experiment to experiment. Neomycin resistance was directly correlated with GPA expression, thus allowing the direct selection for a stable GPA-expressing cell population without the need of a cloning step. The amount of GPA-NPT produced was further increased by weakening the specific NPT enzymatic activity via site-directed mutagenesis. Detection was simplified by the fact that all different fusion proteins could be detected by the same anti-NPT antibody. This approach may be also applicable to other membrane proteins. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 238-244, 1998.
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  • 170
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    Biotechnology and Bioengineering 57 (1998), S. 272-279 
    ISSN: 0006-3592
    Keywords: biofilm ; plasmid transfer ; conjugation ; retrotransfer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 272-279, 1998.
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  • 171
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    Biotechnology and Bioengineering 57 (1998), S. 251-261 
    ISSN: 0006-3592
    Keywords: continuous culture ; metabolic overflow ; multiplicity ; stability analysis ; dynamics ; growth inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 251-261, 1998.
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  • 172
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    Biotechnology and Bioengineering 57 (1998), S. 287-296 
    ISSN: 0006-3592
    Keywords: expression ; plasmid ; stability ; TCE ; continuous culture ; activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min · mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 287-296, 1998.
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  • 173
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    Biotechnology and Bioengineering 57 (1998), S. 297-305 
    ISSN: 0006-3592
    Keywords: Penicillium chrysogenum, autolysis ; image analysis ; penicillin V degradation ; enzymology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and β-1,3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in β-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 297-305, 1998.
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  • 174
    ISSN: 0006-3592
    Keywords: structured model ; morphology ; DiOC6 ; image analysis ; Aspergillus oryzae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A morphologically structured model is well suited for obtaining a good description of growth and product formation of filamentous fungi for use in a process model, for example. This article describes a new morphologically structured model and its application to an α-amylase producing strain of Aspergillus oryzae. The model is based on a division of the fungal hyphae into three different regions: an extension zone, representing the tips of the hyphae; an active region, which is responsible for growth and product formation; and an inactive hyphal region. Two metamorphosis reactions describing branching and inactivation are included in the model, and the kinetics of branching and tip extension are based on known experimentally verified models of fungal microscopic morphology. To verify the structure of the model a double-staining method, based on a combination of fluorescence microscopy and automated image analysis, has been developed for measuring the fraction of active cells. The method employs the fluorescent dye 3,3′-dihexyloxocarbocyanin to stain organelles inside the hyphae and Calcoflour White to stain the cell wall. The ratio between the projected areas of the organelles and of the entire hyphal element is then taken to be proportional to the fraction of active cells. When applied to chemostat and fed-batch experiments, the double-staining method seemed to confirm the basic morphological structure of the model. The model is able to produce accurate simulations of steady-state and transient conditions in chemostats, of batch cultivations, and even the formation of a single hyphal element from a spore, all with the same values of the model parameters. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 321-329, 1998.
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  • 175
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    Biotechnology and Bioengineering 57 (1998), S. 342-355 
    ISSN: 0006-3592
    Keywords: methanogenic population dynamics ; anaerobic digesters ; solid waste ; biosolids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37°C) and thermophilic (55°C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 342-355 1998.
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  • 176
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    Biotechnology and Bioengineering 57 (1998), S. 356-366 
    ISSN: 0006-3592
    Keywords: biodegradation kinetics ; naphthalene ; nonaqueous phase liquid ; mass transfer ; naphthoquinone ; coaltar ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model is formulated to describe dissolution of naphthalene from an insoluble nonaqueous phase liquid (NAPL) and its subsequent biodegradation in the aqueous phase in completely mixed batch reactors. The physicochemical processes of equilibrium partitioning and mass transfer of naphthalene between the NAPL and aqueous phases were incorporated into the model. Biodegradation kinetics were described by Monod's microbial growth kinetic model, modified to account for the inhibitory effects of 1,2-naphthoquinone formed during naphthalene degradation under certain conditions. System parameters and biokinetic coefficients pertinent to the NAPL-water systems were determined either by direct measurement or from nonlinear regression of the naphthalene mineralization profiles obtained from batch reactor tests with two-component NAPLs comprised of naphthalene and heptamethylnonane. The NAPLs contained substantial mass of naphthalene, and naphthalene biodegradation kinetics were evaluated over the time required for near complete depletion of naphthalene from the NAPL. Model predictions of naphthalene mineralization time profiles compared favorably to the general trends observed in the data obtained from laboratory experiments with the two-component NAPL, as well as with two coal tars obtained from the subsurface at contaminated sites and composed of many different PAHs (polycyclic aromatic hydrocarbon compounds). The effects of varying the NAPL mass and the naphthalene mole fractions in the NAPL are discussed. It was observed that the time to achieve a given percent removal of naphthalene does not change significantly with the initial mass of naphthalene in a fixed volume of the NAPL. Significant changes in the mineralization profiles are observed when the volume (and mass) of NAPL in the system is changed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 356-366, 1998.
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  • 177
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    Biotechnology and Bioengineering 57 (1998), S. 367-379 
    ISSN: 0006-3592
    Keywords: fluidized-bed ; consecutive reaction kinetics ; distributed fraction of methanogens ; rate-limiting ; parametric sensitivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A kinetic model involving the distributed fractions of acidogens and methanogens is proposed. To determine the fluxes and biochemical reaction rates of the substrate sucrose and its intermediates, volatile fatty acids (VFAs) in bulk liquid and within the biofilm, a kinetic model was developed by combining the solid-phase model with the liquid-phase model. The predicted substrate removal efficiencies of the conventional and tapered fluidized-bed bioreactors (CFB, TFBs) are in good agreement with the experimental results. The biofilm thickness in TFBs are thicker than that in CFB, resulting in performance enhancement with TFBs. The simulated results obtained from the kinetic model show that methanogenesis is the rate-limiting step of degradation of the simple organic compound (sucrose), and the chemical oxygen demand (COD) concentration in the effluent is mainly contributed by the intermediates VFAs. The distributed fractions of acidogens and methanogens determined experimentally are 0.4 and 0.6, respectively. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 367-379, 1998.
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  • 178
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    Biotechnology and Bioengineering 57 (1998), S. 718-731 
    ISSN: 0006-3592
    Keywords: biofilm ; modeling ; reaction-diffusion-growth ; cellular automata ; immobilized cells ; structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The theoretical basis and quantitative evaluation of a new approach for modeling biofilm growth are presented here. Soluble components (e.g., substrates) are represented in a continuous field, whereas discrete mapping is used for solid components (e.g., biomass). The spatial distribution of substrate is calculated by applying relaxation methods to the reaction-diffusion mass balance. A biomass density map is determined from direct integration in each grid cell of a substrate-limited growth equation. Spreading and distribution of biomass is modeled by a discrete cellular automaton algorithm. The ability of this model to represent diffusion-reaction-microbial growth systems was tested for a well-characterized system: immobilized cells growing in spherical gel beads. Good quantitative agreement with data for global oxygen consumption rate was found. The calculated concentration profiles of substrate and biomass in gel beads corresponded to those measured. Moreover, it was possible, using the discrete spreading algorithm, to predict the spatial two- and three-dimensional distribution of microorganisms in relation to, for example, substrate flux and inoculation density. The new technique looks promising for modeling diffusion-reaction-microbial growth processes in heterogeneous systems as they occur in biofilms. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 718-731, 1998
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  • 179
    ISSN: 0006-3592
    Keywords: PAH degradation ; white rot fungus ; Bjerkandera sp. ; surfactant ; toxicity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of nonionic surfactants on the polycyclic aromatic hydrocarbon (PAH) oxidation rates by the extracellular ligninolytic enzyme system of the white-rot fungus Bjerkandera sp. strain BOS55 was investigated. Various surfactants increased the rate of anthracene, pyrene, and benzo[a]pyrene oxidation by two to fivefold. The stimulating effect of surfactants was found to be solely due to the increased bioavailability of PAH, indicating that the oxidation of PAH by the extracellular ligninolytic enzymes is limited by low compound bioavailability. The surfactants were shown to improve PAH dissolution rates by increasing their aqueous solubility and by decreasing the PAH precipitate particle size. The surfactant Tween 80 was mineralized by Bjerkandera sp. strain BOS55; as a result both the PAH solubilizing activity of Tween 80 and its stimulatory effect on anthracene and pyrene oxidation rates were lost within 24 h after addition to 6-day-old cultures. It was observed that the surfactant dispersed anthracene precipitates recrystallized into larger particles after Tween 80 was metabolized. However, benzo[a]pyrene precipitates remained dispersed, accounting for a prolonged enhancement of the benzo[a]pyrene oxidation rates. Because the endogenous production of H2O2 is also known to be rate limiting for PAH oxidation, the combined effect of adding surfactants and glucose oxidase was studied. The combined treatment resulted in anthracene and benzo[a]pyrene oxidation rates as high as 1450 and 450 mg L-1 d-1, respectively, by the extracellular fluid of 6-day-old fungal cultures. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 220-227, 1998.
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  • 180
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    Biotechnology and Bioengineering 57 (1998), S. 245-249 
    ISSN: 0006-3592
    Keywords: human parathyroid hormone ; proteolysis ; L-arginine ; secretory production ; KEX2 endoproteinase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor α pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (≥0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases. © 1998 John Wiley & Sons, Inc. Biotechnol. Bioeng. 57: 245-249, 1998.
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  • 181
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    Biotechnology and Bioengineering 57 (1998), S. 262-271 
    ISSN: 0006-3592
    Keywords: Herpes Simplex Virus type 2 ; DISC HSV-2 ; heparin ; dextran sulphate ; cell rupture ; virus release ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production and extracellular release of a recombinant Herpes Simplex Virus (type 2) from monolayers of infected complementing Vero cells (CR2) are addressed. Growth and virus production conditions are identified that provide adequate virus titers with cell seeding densities and viral multiplicities of infection that could be reasonably handled in manufacturing. Harvesting by sonication of cell monolayers is shown to give the highest recovery of infectious virus (to 2.5 × 106 pfu/mL) but leads to process stream contamination by cellular proteins through the rupturing of cells (to 28 pg protein/pfu). By comparison, freeze-thaw cycles and osmotic rupture by hypotonic saline or glycerol shock procedures yield only low virus recovery (typically 〈10% of that by sonication), and are accompanied by yet higher levels of protein contamination (up to 30-fold higher pg protein/pfu). Addition of the polyanionic polymers, heparin or dextran sulphate to a harvest using either hypotonic saline, glycerol shock or isotonic phosphate buffered saline increased the yield of infectious virus in the supernatant. By contrast, addition of polycationic poly-l-lysine resulted in negligible increase in the supernatant virus titer. The highest virus titers (4.7 × 107 pfu/mL) were achieved following treatment of roller bottle cultured cells displaying a high cytopathic effect with heparin at 50 μg/mL for at least 3 h post harvest. This procedure also gave the lowest levels of protein contamination (〈2 pg protein/pfu). The fivefold lower yield of infectious virus from cultures displaying a low cytopathic effect (〈70% CPE) indicates the importance of cell physiological state at harvest. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 262-271, 1998.
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  • 182
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    Biotechnology and Bioengineering 57 (1998), S. 438-446 
    ISSN: 0006-3592
    Keywords: DNA ; alginate ; encapsulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 438-446, 1998.
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  • 183
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    Biotechnology and Bioengineering 57 (1998), S. 430-437 
    ISSN: 0006-3592
    Keywords: glucose ; glycerol ; γ-poly(glutamic acid) ; Bacillus licheniformis ATCC 9945a ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bacillus licheniformis ATCC 9945a is one of the bacterial strains that produce γ-poly(glutamic acid) (γ-PGA). The use of carbohydrate medium components for γ-PGA production was explored. Cells were grown in shake flasks or in controlled pH fermentors using medium formulations that contain different carbon sources. During the cultivations, aliquots were removed to monitor cell growth, carbon utilization, polymer production, and polymer molecular weight. Glucose was a better carbon source than glycerol for cell growth. Furthermore, glucose was utilized at a faster rate than glycerol, citrate, or glutamate. However, by using mixtures of glucose and glycerol in medium formulations, the efficiency of γ-PGA production increased. For example, by increasing the glycerol in medium formulations from 0 to 40 g/L, the γ-PGA broth concentration after 96 h increased from 5.7 to 20.5 g/L. Considering that glycerol utilization was low for the glucose/glycerol mixtures studied, it was unclear as to the mechanism by which glycerol leads to enhanced product formation. Cell growth and concomitant γ-PGA production (12 g/L) at pH 6.5 was possible using glucose as a carbon source if trace amounts (0.5 g/L each) of citrate and glutamate were present in the medium. We suggested that citrate and glutamate were useful in preventing salt precipitation from the medium. In addition, glutamate may be preferred relative to ammonium chloride as a nitrogen source. The conversion of glucose to γ-PGA by the strain ATCC 9945a was believed to occur by glycolysis of glucose to acetyl-CoA and tricarboxylic acid (TCA) cycle intermediates that were then metabolized via the TCA cycle to form α-ketoglutarate, which is a direct glutamate precursor. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 430-437, 1998.
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    Biotechnology and Bioengineering 57 (1998), S. 454-461 
    ISSN: 0006-3592
    Keywords: propionic acid ; extractive fermentation ; solvent ; partition ; acid recovery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Solvent selection for extractive fermentation for propionic acid was conducted with three systems: Alamine 304-1 (trilaurylamine) in 2-octanol, 1-dodecanol, and Witcohol 85 NF (oleyl alcohol). Among them, the solvent containing 2-octanol exhibited the highest partition coefficient in acid extraction, but it was also toxic to propionibacteria. The most solvent-resistant strain among five strains of the microorganism was selected. Solvent toxicity was eliminated via two strategies: entrapment of dissolved toxic solvent in the culture growth medium with vegetable oils such as corn, olive, or soybean oils; or replacement of the toxic 2-octanol with nontoxic Witcohol 85 NF. The complete recovery of acids from the Alamine 304-1/Witcohol 85 NF was also realized with vacuum distillation. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 454-461, 1998.
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  • 185
    ISSN: 0006-3592
    Keywords: crossflow filtration ; tubular mineral membrane ; slug flow ; flux enhancement ; yeast suspension ; alcoholic fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This study deals with the use of an upward gas/liquid slug flow to reduce tubular mineral membrane fouling. The injection of air into the feedstream is designed to create hydrodynamic conditions that destabilize the cake layer over the membrane surface inside the filtration module complex. Experimental study was carried out by filtering a biological suspension (yeast) through different tubular mineral membranes. The effects of operating parameters, including the nature of the membrane, liquid and gas flowrates, and transmembrane pressure, were examined. When external fouling was the main limiting phenomenon, flux enhancements of a factor of three could be achieved with gas sparging compared with single liquid phase crossflow filtration. The economic benefits of this unsteady technique have also been examined. To investigate the possibility of long-term operation of the two-phase flow principle, dense cell perfusion cultures of Saccharomyces cerevisiae were carried out in a fermentor coupled with an ultrafiltration module. The air injection allowed a high and stable flux to be maintained over 100 h of fermentation, with a final cell concentration of 150 g dry weight/L. At equal biomass level, a twofold gain in flux could be attained compared with classical steady crossflow filtration at half the cost. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:47-57, 1998.
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  • 186
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    Biotechnology and Bioengineering 58 (1998), S. 380-386 
    ISSN: 0006-3592
    Keywords: reverse micelles ; cutinase ; deactivation ; conformational changes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380-386, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 408-415 
    ISSN: 0006-3592
    Keywords: nitric oxide ; NOx ; flue gas ; denitrification ; aerobic ; biofilter ; aerosol ; biomass control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The presence of significant denitrification activity in an aerobic toluene-treating biofilter was demonstrated under batch and flow-through conditions. N2O concentrations of 9.2 ppmv were produced by denitrifying bacteria in the presence of 15% acetylene, in a flow-through system with a bulk gas phase O2 concentration of 〉17%. The carbon source for denitrification was not toluene but a byproduct or metabolite of toluene catabolism. Denitrification conditions were successfully used for the reduction of 60 ppmv nitric oxide to 15 ppmv at a flow rate of 3 L min-1 (EBRT of 3 min) in a fully aerated, 17% v/v O2 (superficially aerobic) biofilter. Higher NO removal efficiency (97%) was obtained by increasing the toluene supply to the biofilter. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:408-415, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 400-407 
    ISSN: 0006-3592
    Keywords: abrasion ; airlift reactor ; biofilm ; structure ; density ; surface shape ; thickness ; shear ; carrier concentration ; substrate loading ; detachment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of process conditions (substrate loading rate and detachment force) on the structure of biofilms grown on basalt particles in a Biofilm Airlift Suspension (BAS) reactor was studied. The structure of the biofilms (density, surface shape, and thickness) and microbial characteristics (biomass yield) were investigated at substrate loading rates of 5, 10, 15, and 20 kg COD/m3 · day with basalt concentrations of 60 g/L, 150 g/L, and 250 g/L. The basalt concentration determines the number of biofilm particles in steady state, which is the main determining factor for the biofilm detachment in these systems. In total, 12 experimental runs were performed. A high biofilm density (up to 67 g/L) and a high biomass concentration was observed at high detachment forces. The higher biomass content is associated with a lower biomass substrate loading rate and therefore with a lower biomass yield (from 0.4 down to 0.12 gbiomass/gacetate). Contrary to general beliefs, the observed biomass detachment decreased with increasing detachment force. In addition, smoother (fewer protuberances), denser and thinner compact biofilms were obtained when the biomass surface production rate decreased and/or the detachment force increased. These observations confirmed a hypothesis, postulated earlier by Van Loosdrecht et al. (1995b), that the balance between biofilm substrate surface loading (proportional to biomass surface production rate, when biomass yield is constant) and detachment force determines the biofilm structure. When detachment forces are relatively high only a patchy biofilm will develop, whereas at low detachment forces, the biofilm becomes highly heterogeneous with many pores and protuberances. With the right balance, smooth, dense and stable biofilms can be obtained. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:400-407, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 440-444 
    ISSN: 0006-3592
    Keywords: xylitol production ; redox potential ; dissolved oxygen ; Candida parapsilosis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of redox potential on xylitol production by Candida parapsilosis was investigated. The redox potential was found to be useful for monitoring the dissolved oxygen (DO) level in culture media, especially when the DO level was low. An increase in the agitation speed in a 5 L fermentor resulted in an increased culture redox potential as well as enhanced cell growth. Production of xylitol was maximized at a redox potential of 100 mV. As the initial cell concentration increased from 8 g/L to 30 g/L, the volumetric productivity of xylitol increased from 1.38 g/L · h to 4.62 g/L · h. A two-stage xylitol production strategy was devised, with stage 1 involving rapid production of cells under well-aerated conditions, and stage 2 involving cultivation with reduced aeration such that the culture redox potential was 100 mV. Using this technique, a final xylitol concentration of 180 g/L was obtained from a culture medium totally containing 254.5 g/L xylose in a 3,000 L pilot scale fermentor after 77 h fermentation. The volumetric productivity of xylitol during the fermentation was 2.34 g/L · h. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:440-444, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 428-439 
    ISSN: 0006-3592
    Keywords: water vaporization ; ethanol stripping ; condensation ; absorption ; elemental recoveries ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Systematic errors due to the neglect of water and/or ethanol partition between liquid and gaseous phases are discussed for bioreactors equipped with or without a condenser. Both water vapor and ethanol vapor are present in the off-gas leaving the condenser. Presence of residual water vapor largely influences the gas measurements by dilution. As a consequence, the oxygen consumption rate can be overestimated by a factor of 3 if calculations are not corrected for water vapor content or if no additional device is implemented after the condenser to completely dry the off-gases. The mass balance and partition equations predict that the condenser has only a small effect on reduction of the ethanol vapor content of the off-gas. The reason is the high ethanol concentration of the condensate droplets on the condenser wall in contact with the off-gases. Model predictions as well as experimental results show that ethanol evaporation represents a large fraction of the ethanol production rate and influences greatly the elemental recoveries. For a reactor working at 30°C without condensation of the vapors and for a volumetric aeration rate of 0.63vvm, stripping of ethanol resulted in a gaseous dilution rate of 0.016 h-1 for ethanol. The dilution rate by stripping was reduced to 0.014 h-1 when a condenser at 12°C was implemented. The fraction of ethanol that is stripped is mainly dependent on the ratio D/vvm (liquid to gaseous flow rates), and the effect is only slightly influenced by low condenser temperature. The evaporation of ethanol may account for more than 20% of the ethanol formation rate. Therefore, the condenser does not succeed to reflux all ethanol to the reactor broth. In terms of a unit operation, ethanol vapor can be efficiently reduced by absorption instead of condensation. To demonstrate the feasibility, a simple modification of the reactor was tested for continuous cultures: the feed port was changed from the top-plate to the top of the condenser, which was used as an absorption column. Ethanol stripping was reduced by a factor of 4 as compared to the condensation setup (at 12°C): it accounted for 2% of the ethanol production rate as compared to 8.2% at D = 0.19 h-1 and 0.63vvm. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:428-439, 1998.
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    Biotechnology and Bioengineering 58 (1998), S. 416-427 
    ISSN: 0006-3592
    Keywords: protein separation ; isolation and purification ; aqueous two-phase extraction ; hydrogels ; gels ; polymer solution thermodynamics ; partition coefficients ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex®) are common chromatographic media.The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex® G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10,000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics.Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:416-427, 1998.
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  • 192
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    Biotechnology and Bioengineering 58 (1998), S. 445-450 
    ISSN: 0006-3592
    Keywords: on-line control ; pH control ; growth monitoring ; proton titration ; yeast ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The amount of acid or base consumed in yeast cultures has been recently assigned to the pathway of nitrogen assimilation under respiratory conditions with no contribution by carbon metabolism (Castrillo et al., 1995). In this investigation, experiments under respirofermentative conditions have shown that production or consumption of ethanol does not contribute significantly to the specific rate of proton production (qH+), thus extending the previously obtained relationships for all aerobic conditions in which other major acid/base contributions are not involved. Tests in batch and chemostat culture confirm the validity of qH+ as a formal control parameter in aerobic fermentations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:445-450, 1998.
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  • 193
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    Biotechnology and Bioengineering 57 (1998), S. 536-544 
    ISSN: 0006-3592
    Keywords: biofilm ; streamers ; biofouling ; drag ; fast Fourier transform analysis ; hydrodynamics ; oscillations ; pressure drop ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mixed population biofilms consisting of Pseudomonas aeruginosa, P. fluorescens, and Klebsiella pneumoniae were grown in a flow cell under turbulent conditions with a water flow velocity of 18 cm/s (Reynolds number, Re, =1192). After 7 days the biofilms were patchy and consisted of cell clusters and streamers (filamentous structures attached to the downstream edge of the clusters) separated by interstitial channels. The cell clusters ranged in size from 25 to 750 μm in diameter. The largest clusters were approximately 85 μm thick. The streamers, which were up to 3 mm long, oscillated laterally in the flow. The motion of the streamers was recorded at various flow velocities up to 50.5 cm/s (Re 3351) using confocal scanning laser microscopy. The resulting time traces were evaluated by image analysis and fast Fourier transform analysis (FFT). The amplitude of the motion increased with flow velocity in a sigmoidal shaped curve, reaching a plateau at an average fluid flow velocity of approximately 25 cm/s (Re 1656). The motion of the streamers was possibly limited by the flexibility of the biofilm material. FFT indicated that the frequency of oscillation was directly proportional to the average flow velocity (u(ave)) below 9.5 cm/s (Re 629). At u(ave) greater than 9.5 cm/s, oscillation frequencies were above our measurable frequency range (0.12-6.7 Hz). The oscillation frequency was related to the flow velocity by the Strouhal relationship, suggesting that the oscillations were possibly caused by vortex shedding from the upstream biofilm clusters. A loss coefficient (k) was used to assess the influence of biofilm accumulation on pressure drop. The k across the flow cell colonized with biofilm was 2.2 times greater than the k across a clean flow cell. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 536-544, 1998.
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  • 194
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    Biotechnology and Bioengineering 57 (1998), S. 545-551 
    ISSN: 0006-3592
    Keywords: enzyme array ; pulsed amperometric detection ; carbohydrate analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The introduction of an enzyme array-electrochemical detection method for carbohydrate analysis is demonstrated by using two complex and one high mannose N-linked oligosaccharides. Instead of measuring the remaining uncleaved oligosaccharides in enzymatic digestion, released monosaccharides are directly quantified by pulsed amperometric detection at a gold electrode. The measured monosaccharide concentrations in combination with the enzyme array analysis provide structural characterization of oligosaccharides. The enzyme array-electrochemical detection method does not require any separation procedure or any prior labeling of oligosaccharides. However, this method is limited by the use of purified oligosaccharide samples and the nature of the enzyme array. The development of more sophisticated enzyme arrays relies upon the introduction of a bank of highly specific (bond, arm, aglycon) exoglycosidases. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 545-551, 1998.
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  • 195
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    Biotechnology and Bioengineering 57 (1998), S. 552-556 
    ISSN: 0006-3592
    Keywords: baroenzymology ; reversed micelles ; α-chymotrypsin ; catalytic activity and stability ; effect of pressure, temperature, and glycerol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Thermostability of α-chymotrypsin at normal pressure in reversed micelles depends on both an effective surfactant solvation degree and glycerol content in the system. The difference in α-chymotrypsin stability in reversed micelles at various glycerol concentrations [up to 60% (v/v)] was more pronounced at high surfactant degrees of solvation, R ≥ 16. After a 1-h incubation at 40°C in “aqueous” reversed micelles (in the absence of glycerol), α-chymotrypsin retained only 1% of initial catalytic activity and 10, 22, 59, and 48% residual activity in glycerol-solvated micelles with 20, 30, 50, and 60% (v/v) glycerol, respectively. The explanation of the observed effects is given in the frames of micellar matrix structural order increasing in the presence of glycerol as a water-miscible cosolvent that leads to the decreasing mobility of the α-chymotrypsin molecule and, thus the increase of its stability. It was found that glycerol or hydrostatic pressure could be used to stabilize α-chymotrypsin in reversed micelles; a lower pressure is necessary to reach a given level of enzyme stability in the presence of glycerol. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 552-556, 1998.
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  • 196
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    Biotechnology and Bioengineering 57 (1998), S. 557-570 
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; polyhydroxyalkanoates ; metabolic engineering ; mathematical modeling ; enzyme kinetics ; regulation of metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 557-570, 1998.
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  • 197
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    Biotechnology and Bioengineering 59 (1998), S. 428-437 
    ISSN: 0006-3592
    Keywords: enzymes ; polyesters ; bulk polymerization ; calorimetry ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biocatalytic polytransesterification at high concentrations of monomers proceeds rapidly and is accompanied by an increase in the temperature of the reaction mixture due to liberation of heat of reaction during the initial phase. We have used principles of reaction calorimetry to monitor the kinetics of polymerization during this initial phase, thus relating the temperature to the extent of polymerization. Rate of polymerization increases with the concentration of monomers. This is also reflected by the increase in the temperature of the reaction mixture. Using time-temperature-conversion contours, a differential method of kinetic analysis was used to calculate the energy of activation (∼15.1 Kcal/mol). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:428-437, 1998.
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  • 198
    ISSN: 0006-3592
    Keywords: Aspergillus niger ; chemostat culture ; glucoamylase (GAM) ; protein secretion ; recombinant protein ; strain stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch culture and 373 ± 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:407-418, 1998.
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  • 199
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    Biotechnology and Bioengineering 59 (1998), S. 445-450 
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosylation engineering ; antisense ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:445-450, 1998.
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  • 200
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    Biotechnology and Bioengineering 59 (1998), S. 438-444 
    ISSN: 0006-3592
    Keywords: bioremediation ; plasma discharge ; dichlorophenol degradation ; perchloroethylene degradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pulsed electric discharge (PED) and bioremediation were combined to create a novel two-stage system which dechlorinates the halogenated pollutants, 2,4-dichlorophenol and perchloroethylene, with repetitive (0.1-1 kHz), short pulse (∼100 ns), low voltage (40-80 kV) discharges and then mineralizes the less chlorinated products with aerobic bacteria. A 6.1 mM aqueous dichlorophenol sample was cycled through the PED reactor (60 kV of applied pulsed voltage and 300 Hz) 6 times, resulting in the release of 55% of the initial dichlorophenol chloride ions (1 mM Cl- removed each cycle). The respective average specific efficiency is 0.4-0.6 keV/(Cl- molecule). Pseudomonas mendocina KR1, which grows in minimal medium supplemented with phenol but not with dichlorophenol, increased in cell density in all cultures supplemented with the PED-treated DCP samples and yielded a maximum of two-fold additional Cl- released compared to the PED-related alone. The number of PED-treatment cycles, voltage, and frequency were also varied, showing that both cell densities and overall dichlorophenol dechlorination were highly dependent upon the number of PED-treatment cycles, rather than the tested voltages and frequencies. Using this two-stage treatment system, PED released 31% of the initial chloride ions from dichlorophenol (after three cycles at 40-45 kV and 1.2 kHz) while P. mendocina KR1 in the second stage increased dechlorination to 90%. These results were corroborated by the 35% additional chloride release found with activated sludge cultures. Perchloroethylene (0.6 mM) was similarly treated in a first-stage PED reactor (80% chloride removal after four cycles) followed by biodegradation of the dechlorinated products with a recombinant toluene o-monooxygenase-expressing Pseudomonas fluorescens strain. Gas chromatographic analysis showed that the PED reactor created less-chlorinated byproducts (i.e., trichloroethylene) that were removed (74%) upon exposure to the recombinant bacterium. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:438-444, 1998.
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