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  • Inorganic Chemistry  (1,360)
  • Biochemistry and Biotechnology  (879)
  • 1995-1999  (1,487)
  • 1990-1994
  • 1930-1934  (752)
  • 1997  (1,487)
  • 1933  (752)
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  • 1995-1999  (1,487)
  • 1990-1994
  • 1930-1934  (752)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 727-736 
    ISSN: 0006-3592
    Keywords: acetate ; anaerobic ; biodegradation ; formaldehyde ; methanogenic ; toxicity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Formaldehyde is present in several industrial wastewaters including petrochemical wastes. In this study, the toxicity and degradability of formaldehyde in anaerobic systems were investigated. Formaldehyde showed severe toxicity to an acetate enrichment methanogenic culture. As low as 10 mg/L (0.33 mM) of formaldehyde in the reactor completely inhibited acetate utilization. Formaldehyde, however, was degraded while acetate utilization was inhibited. Degradation of formaldehyde (Initial concentration ≤30 mg/L) followed Monod model with a rate constant, k, of 0.35-0.46 d-1. At higher initial concentrations (≥60 mg/L), formaldehyde degradation was inhibited and partial degradation was possible. The initial formaldehyde to biomass ratio, S0/X0, was useful to predict the degradation potential of high formaldehyde concentrations in batch systems. When S0/X0 ≤ 0.1, formaldehyde was completely degraded with initial concentration of up to 95 mg/L; when S0/X0 ≥ 0.29, formaldehyde at higher than 60 mg/L was only partially degraded. The inhibition of formaldehyde degradation in batch systems could be avoided by repeated additions of low concentrations of formaldehyde (up to 30 mg/L). Chemostats (14-day retention time) showed degradation of 74 mg/L-d (1110 mg/L) of influent formaldehyde with a removal capacity of 164 mg/g VSS-day. A spike of 30 mg/L (final concentration in the chemostat) formaldehyde to the chemostat caused only a small increase in effluent acetate concentration for 3 days. But a spike of 60 mg/L (final concentration in the chemostat) formaldehyde to the chemostat resulted in a dramatic increase in acetate concentration in the effluent. The results also showed that the acetate enrichment culture was not acclimated to formaldehyde even after 226 days. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 727-736, 1997.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 737-746 
    ISSN: 0006-3592
    Keywords: ultrafiltration ; scale-up ; scale-down ; linear scale ; proteins ; membrane fouling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Tangential flow filtration has traditionally been scaled up by maintaining constant the filtrate volume to membrane surface area ratio, membrane material and pore size, channel height, flow path geometry and retentate and filtrate pressures. Channel width and the number of channels have been increased to provide increased membrane area. Several other parameters, however, have not been maintained constant. A new comprehensive methodology for implementation of linear scale up and scale down of tangential flow filtration processes has been developed. Predictable scale up can only be achieved by maintaining fluid dynamic parameters which are independent of scale. Fluid dynamics are controlled by operating parameters (feed flow rate, retentate pressure, fed batch ratio and temperature), geometry (channel length, height, turbulence promoter and entrance/exit design), materials (membrane, turbulence promoter, and encapsulant compression), and system geometry (flow distribution). Cassette manufacturing procedures and tolerances also play a significant role in achieving scale independent performance. Extensive development work in the aforementioned areas has resulted in the successful implementation of linear scale up of ultrafiltration processes for recovery of human recombinant DNA derived pharmaceuticals. A 400-fold linear scale up has been achieved without intermediate pilot scale tests. Scale independent performance has a direct impact on process yield, protein quality and product economics and is therefore particularly important in the biotechnology industry. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 737-746, 1997.
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  • 3
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 340-344 
    ISSN: 0006-3592
    Keywords: subtilisin ; chymotrypsin ; substrate specificity ; organic solvents ; lyophilized enzymes ; stereoselectivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple methodology has been successfully employed to explain the solvent dependence of the substrate specificity of enzymes in organic media. This methodology, which does not require the knowledge of the enzyme structure and is thus applicable to lyophilized and other noncrystalline enzyme preparations, predicts that the kcat/KM ratio for two substrates should be proportional to their Raoult's law activity coefficients. This approach has been validated for two enzymes, subtilisin Carlsberg and α-chymotrypsin, catalyzing the propanolysis of unnatural (in addition to natural) ester substrates in a variety of anhydrous solvents. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 340-344, 1997.
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  • 4
    ISSN: 0006-3592
    Keywords: Primatone RL ; sialylation ; interferon-γ ; serum substitutes ; cell ; CHO cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Although serum-free media have been widely used in mammalian cell culture for therapeutic protein production, the effects of serum-substitutes on product quality have not been extensively examined. This study observed an adverse effect of Primatone RL, an animal tissue hydrolysate commonly used as a serum-substitute to promote cell growth, on sialylation of interferon-γ (IFN-γ) derived from Chinese hamster ovary (CHO) cell culture in both batch and fed-batch modes. In batch cultures, decreased sialylation was observed at each of the glycosylation sites (i.e., Asn25 and Asn97) of IFN-γ with the use of elevated concentrations of the peptone. Although poorest sialylation was obtained with the use of a growth-inhibiting concentration of Primatone RL, diminished sialylation was observed at the optimal peptone concentration for cell growth and product yield. Since incubation of the product in Primatone RL-supplemented acellular medium did not result in decreased sialylation, the negative effect of Primatone RL could not be attributed to extracellular desialylation of IFN-γ by components of the peptone. In the fed-batch mode, a culture utilizing a serum-free feeding medium supplemented with Primatone RL demonstrated poorer sialylation than a similar culture not fed the peptone. The results of both the batch and fed-batch experiments indicate that the adverse effect of the peptone was not due solely to ammonia accumulation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 353-360, 1997.
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  • 5
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 380-390 
    ISSN: 0006-3592
    Keywords: insect cells ; baculovirus ; bcl-2 ; recombinant proteins ; cell viability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of bcl-2 expression on cell viability and recombinant protein synthesis was investigated in the Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five™) insect cell lines. It was found that coinfection with a baculovirus expressing bcl-2 [Autographa californica nuclear polyhedrosis virus (AcNPV)-bcl2] extended the life span of High Five™ cells but not Sf-9 cells when compared to infection with recombinant baculoviruses expressing either human tissue plasminogen activator (AcNPV-tPA) or Escherichia coli β-galactosidase (AcNPV-βgal). Similar results were obtained in coinfection experiments; i.e., AcNPV-bcl2 coinfection increased the life span of High Five™ cells over that of cells infected with either AcNPV-tPA or AcNPV-βgal alone, but they did not affect the life span of coinfected Sf-9 cells. Coinfection of Sf-9 cells with AcNPV-bcl2 and AcNPV-βgal resulted in a decrease in the maximum β-gal expression levels of over 90% when compared to infection with AcNPV-βgal alone. A similar trend was found in the β-gal mRNA levels. Coinfection also resulted in a reduced β-gal expression level in High Five™ cells, but the reduction was consistent with what would be expected when two recombinant viruses compete for use of the cellular machinery. In contrast to the inhibitory effect of AcNPV-bcl2 coinfection on βgal expression, t-PA expression levels were either not affected (Sf-9 cells) or were increased 50% (High Five™ cells) over those obtained by infection with AcNPV-tPA alone. These results support the hypotheses that bcl-2 can inhibit transcription of genes under polyhedrin promoter control and that β-gal expression levels, but not t-PA expression levels, are controlled at the transcriptional level. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 380-390, 1997.
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  • 6
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    Biotechnology and Bioengineering 56 (1997), S. 433-440 
    ISSN: 0006-3592
    Keywords: simple dissolution-reaction model ; enzymatic conversion ; solid substrate suspension ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Although reactions in substrate suspension are employed in industry for several bioconversion processes, there appears to be no quantitative model available in the literature to rationalize the optimization of these processes. We present a simple model that incorporates the kinetics of substrate dissolution and a simultaneous enzymatic reaction. The model was tested in the α-chymotrypsin-catalyzed hydrolysis of an aqueous suspension of dimethyl benzylmethylmalonate to a homogeneous solution of enantiomerically pure monoester. This reaction occurs in the bulk phase, so catalysis by enzyme absorbed at the solid-liquid interface plays no role. The value of the parameters in the model (i.e., the mass transfer coefficient of substrate dissolution (kL), the substrate solubility, and the rate constant for the enzymatic reaction) were determined in separate experiments. Using these parameter values, the model gave a good quantitative prediction of the rate of the overall dissolution-reaction process. When the particle size distribution is known, kL may also be calculated instead. The model seems to be applicable also for other poorly soluble substrates, other enzymes, and other solvents. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 433-440, 1997.
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  • 7
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 456-463 
    ISSN: 0006-3592
    Keywords: enzymatic fragment condensation ; α-chymotrypsin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR1 (R1: Et, Al, Cam) and H-Asp-(OR2)-Phe-NH2 (R2: H, But) catalyzed by α-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at aw = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at aw = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH2 with β-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.
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  • 8
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 473-484 
    ISSN: 0006-3592
    Keywords: transgenic plants ; recombinant protein ; gene expression ; downstream processing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This review is based on our recent experience in producing the first commercial recombinant proteins in transgenic plants. We bring forward the issues that have to be considered in the process of selecting and developing a winning transgenic plant production system. From the production point of view, transcription, posttranscription, translation, and posttranslation are important events that can affect the quality and quantity of the final product. Understanding the rules of gene expression is required to develop sound strategies for optimization of recombinant protein production in plants. The level of recombinant protein accumulation is critical, but other factors such as crop selection, handling and processing of transgenic plant material, and downstream processing are equally important when considering commercial production. In some instances, the cost of downstream processing alone may determine the economic viability of a particular plant system. Some of the potential advantages of a plant production system such as the high levels of accumulation of recombinant proteins, glycosylation, compartmentalization within the cell, and natural storage stability in certain organs are incentives for aggressively pursuing recombinant protein production in plants. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 473-484, 1997.
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  • 9
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 530-537 
    ISSN: 0006-3592
    Keywords: ammonium ; cell culture ; cell cycle ; cell death ; cell growth ; Jurkat cells, GH4 cells ; LLC-PK1 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The toxic effect of ammonium upon a variety of cell lines of lymphoid (Jurkat), pituitary (GH4), and renal (LLC-PK1) origin was studied. Millimolar concentrations of the ion mildly affected the growth of GH4 cells and prevented the growth of LLC-PK1 cells. The ion did not lead to the death of LLC-PK1 cells but it produced morphologic changes in these cells. The effects of ammonium upon Jurkat cells were different because cells died after accumulating at S phase. Cell death was due to apoptosis and might be related to ammonium-induced calcium mobilization from intracellular stores. These results indicate that the toxic effects caused by ammonium accumulation are different depending upon the cell type. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 530-537, 1997.
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  • 10
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 538-545 
    ISSN: 0006-3592
    Keywords: affinity ; separation ; purification ; continuous ; trypsin ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 μg of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.
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  • 11
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    Biotechnology and Bioengineering 56 (1997), S. 605-609 
    ISSN: 0006-3592
    Keywords: affinity immobilization ; glycoenzymes ; thermal stability ; non-inhibitory antienzyme antibodies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyclonal antibodies directed against the yeast invertase glycosyls were raised by immunizing rabbits with neoglycoprotein-I and neoglycoprotein-II. The neoglycoproteins were prepared by separately coupling the N-linked large and small molecular weight yeast invertase oligosaccharides respectively to bovine serum albumin with the help of glutaraldehyde. Antibodies specifically recognizing the invertase oligosaccharides were purified from the sera of rabbits immunized with either neoglycoprotein using an affinity column of sepharose 4B-linked yeast invertase. Specific immunoaffinity supports for the immobilization of invertase were constructed by coupling the affinity-purified antineoglycoprotein-I or antineoglycoprotein-II antibodies to cyanogen bromide activated sepharose-4B. Both the affinity adsorbants were effective in binding and improving the thermal stability of invertase. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 605-609, 1997.
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  • 12
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    Biotechnology and Bioengineering 56 (1997), S. 618-625 
    ISSN: 0006-3592
    Keywords: albumin ; silicon ; hydrophobicity ; adsorption ; Tween 20 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The ability of Tween 20 to reduce the adsorption of albumin on silicon surfaces of different hydrophobicity was investigated by ellipsometry. As expected, protein adsorption was found to depend on the degree of hydrophobicity of the surfaces and on the concentration of the surfactant. A reduction of 90% in albumin adsorption on hydrophobic methylated surfaces by 0.05% Tween 20 was achieved, whereas a reduction of only 15% on hydrophilic surfaces was observed. Experiments of time-dependent protein adsorption in both pure protein and protein-surfactant mixtures were conducted to ascertain the stability of physically adsorbed Tween 20 films on intermediate silicon surfaces. It was found that the adsorbed Tween 20 film was robust and there was no evidence of exchange of the Tween molecules with albumin for up to 240 min exposure. Adsorption minima were confirmed to correlate with minima in contact angle and critical micelle concentration (CMC). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 618-625, 1997.
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  • 13
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    Biotechnology and Bioengineering 55 (1997), S. 807-814 
    ISSN: 0006-3592
    Keywords: sulphate reduction ; sulphite reduction ; biofilm ; immobilization ; gas-lift reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Feasibility of thermophilic (55°C) sulphate and sulphite reduction with H2 and CO2 gas-mixtures was studied in gas-lift reactors, which contained pumice particles as carrier material. Particular attention was paid to biomass retention and the competition between hydrogenotrophic sulphate-reducers and other hydrogenotrophic thermophiles. A model medium with defined mineral nutrients was used.The results of the experiments clearly demonstrate that sulphate conversion rates up to 7.5 g SO42-/L per day can be achieved. With sulphite, a reduction rate of 3.7 g S/L per day was obtained, which equals a sulphate conversion rate of 11.1 g SO42-/L per day. Under the applied conditions, a strong competition for hydrogen between hydrogenotrophic sulphate-reducers, tentatively designated as Desulfotomaculum sp., and hydrogenotrophic methanogens was observed. The outcome of the competition could not be predicted. Growth of the mixed culture was totally inhibited at an H2S concentration of 250 mg/L. Poor attachment of sulphate-reducing bacteria was observed in all experiments. The biomass concentration did not exceed 1.2 g/L, despite the presence of 50 g/L of pumice. The reason for this phenomenon remains to be understood. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 807-814, 1997.
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  • 14
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    Biotechnology and Bioengineering 55 (1997), S. 821-830 
    ISSN: 0006-3592
    Keywords: Citrobacter ; actinides ; nitrate ; biomineralization ; biocatalysis ; phosphatase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A Citrobacter sp. accumulates heavy metals as cell-bound metal phosphates, utilizing phosphate released by the enzymatic cleavage of a phosphomonoester substrate. The effect of increased substrate (glycerol 2-phosphate, G2P) concentration on phosphate release and heavy metal accumulation was evaluated using a stirred tank reactor (STR) and a plug flow reactor (PFR). A significant improvement in metal removal was achieved with increased substrate concentration using immobilized Citrobacter cells in the PFR, which was not observed using free cells in the STR. Nitrate is an inhibitor of the Citrobacter phosphatase. This inhibition was concentration dependent and reversible. The rate of product release was restored by increasing the concentration of substrate (G2P). The ratio of rates of phosphate release under two different conditions (different nitrate and G2P concentrations) can be described by a equation developed from Michaelis-Menten kinetics. The concentration of substrate required for restoration of maximum velocity, Vmax, in a batch and continuous-flow system can be predicted by substitution and calculation; this was confirmed by an experiment in model systems using cell suspensions and polyacrylamide gel immobilized cells in a flow-though column. For use in industrial situations it may be uneconomical or infeasible to supply additional substrate. Bioreactor activity was also restored by increasing the flow residence time, in accordance with a Michaelis-Menten-based model to describe removal of lanthanum from nitrate-supplemented flow in a PFR. © 1997 John Wiley & Sons, Inc. Biotechnol Biotechnol Bioeng 55:821-830, 1997.
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  • 15
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    Biotechnology and Bioengineering 55 (1997), S. 527-534 
    ISSN: 0006-3592
    Keywords: Catharanthus roseus ; hairy roots ; indole alkaloids ; organic acids ; nutrients ; growth association ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of growth, the uptake of macronutrients, and the accumulation of indole alkaloids were investigated in long-term, heterotrophically cultured transgenic (“hairy”) roots of Catharanthus roseus.Tabersonine, ajmalicine, and serpentine were monitored over a 70-day period. The doubling time [dry-weight (DW) basis] of C. roseus hairy roots in B5/2 nutrients supplemented with 3% sucrose was 3.6 days. NH4+, NO3,- and Pi were depleted sequentially from culture medium by hairy roots, while sugars remained undepleted. The growth-limiting nutrient was inorganic nitrogen, NH4+ and NO3-, with exponential-phase overall biomass yields of 34.1 and 5.0 g DW/g nutrient, respectively. Extracellular pH decreased to 4.8 in early exponential phase of culture growth from the initially adjusted value of 5.7, increased subsequently to a maximum of 7.7 in late exponential phase of growth coincident with the maximum of fresh weight (FW)/DW ratio, before decreasing to 5.5-5.0. The organic acids, pyruvate, formate, lactate, and succinate were excreted by hairy roots starting in late phase of exponential growth, possibly resulting in the late-culture pH decrease. Tabersonine accumulation was distinctly growth associated with maximum specific and total yields of 1.15 mg/g DW and 5.6 mg/L, respectively, in late-exponential phase of growth. Serpentine accumulation was non growth associated with increasing specific and total levels in stationary growth phase: 1.3 mg/g DW and 10.5 mg/L, respectively. The accumulation of ajmalicine also appeared growth associated. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 527-534, 1997.
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  • 16
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    Biotechnology and Bioengineering 55 (1997), S. 542-546 
    ISSN: 0006-3592
    Keywords: virus ; antibody ; imaging ; real-time ; phage T7 ; diffusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The design and implementation of controlled environments to continuously culture and evolve viruses provides a means to track how their populations respond to natural and designed anti-viral agents. We have previously demonstrated how the growth of viruses in spreading plaques enables detection and characterization of their evolutionary dynamics. Using plaques of phage T7 growing on E. coli as a model system, we observe here that velocities of propagation can be readily controlled by the level of anti-viral antiserum incorporated into the propagation medium. Further, we develop a simple analytic expression for the radial velocity of propagation in terms of the microscopic rates of viral amplification, Fickian diffusion of the virions and their neutralization by antiserum. Our analysis captures the essential dependence of propagation velocity on antiserum concentration. This study provides an ex vivo foundation for exploring how medically relevant viruses escape suppression by the immune system. © 1997 John Wiley & Son, Inc. Biotechnol Bioeng 55: 542-546, 1997.
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  • 17
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    Biotechnology and Bioengineering 55 (1997), S. 571-576 
    ISSN: 0006-3592
    Keywords: substrate-sufficient culture ; anabolism ; catabolism ; energy uncoupling ; growth yield ; residual substrate concentration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The growth yields (Yobs) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism, which leads to energy spilling. Although the uncoupling between anabolism and catabolism has already been recognized in the microbiology literature, how to quantitatively describe such uncoupling remains unclear. Based on a balance on substrate reaction, a growth yield model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. On the basis of that yield model, the concept of an uncoupling coefficient between anabolism and catabolism is defined in this work. A model describing the effect of the residual substrate concentration on the uncoupling coefficient of anabolism to catabolism is proposed. This model agrees very well with literature data. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 571-576, 1997.
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  • 18
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    Biotechnology and Bioengineering 55 (1997), S. 556-564 
    ISSN: 0006-3592
    Keywords: cumulative sedimentation analysis ; cell debris size ; Escherichia coli ; homogenization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new method to measure Escherichia coli cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 μm to 0.3 μm as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or 10 passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. © 1997 John Wiley & Sons, Inc. Biotechnol Bioang 55: 556-564, 1997.
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  • 19
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    Biotechnology and Bioengineering 55 (1997), S. 565-570 
    ISSN: 0006-3592
    Keywords: hybridoma ; hypoosmotic stress ; specific antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (qAb). However, the cells subjected to hypoosmotic stress did not display enhanced qAb. Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/γ2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to qAb was different from that to hyperosmotic stress. © 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.
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  • 20
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    Biotechnology and Bioengineering 55 (1997), S. 547-555 
    ISSN: 0006-3592
    Keywords: ethanol ; cellulose ; hemicellulose ; endoglucanase ; cellulase ; lignocellulose ; biomass ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This study demonstrates a new approach to reduce the amount of fungal cellulase required for the conversion of cellulose into ethanol. Escherichia coli KO11, a biocatalyst developed for the fermentation of hemicellulose syrups, was used to produce recombinant endoglucanase as a co-product with ethanol. Seven different bacterial genes were expressed from plasmids in KO11. All produced cell-associated endoglucanase activity. KO11(pLOI1620) containing Erwinia chrysanthemi celZ (EGZ) produced the highest activity, 3,200 IU endoglucanase/L fermentation broth (assayed at pH 5.2 and 35°C). Recombinant EGZ was solubilized from harvested cells by treatment with dilute sodium dodecyl sulfate (12.5 mg/ml, 10 min, 50°C) and tested in fermentation experiments with commercial fungal cellulase (5 filter paper units/g cellulose) and purified cellulose (100 g/L). Using Klebsiella oxytoca P2 as the biocatalyst, fermentations supplemented with EGZ as a detergent-lysate of KO11(pLOI1620) produced 14%-24% more ethanol than control fermentations supplemented with a detergent-lysate of KO11(pUC18). These results demonstrate that recombinant bacterial endoglucanase can function with fungal cellulase to increase ethanol yield during the simultaneous saccharification and fermentation of cellulose. © 1997 Wiley & Sons, Inc. Biotechnol Bioeng 55: 547-555, 1997.
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  • 21
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    Biotechnology and Bioengineering 55 (1997), S. 577-580 
    ISSN: 0006-3592
    Keywords: mRNA stability ; hairpins ; gene expression control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An expression system has been developed for the introduction of DNA cassettes into the region between the transcription and translation start sites of a gene of interest. This cassette system was used to engineer mRNA stability through the introduction of hairpins at the 5′ end. A synthetic DNA cassette was designed so that the resulting mRNA hairpin would be positioned one nucleotide from the 5′ mRNA end. The hairpin-containing mRNA exhibited a half-life 3 times that of the mRNA with no hairpin, resulting in increases in both mRNA and protein levels. These results indicate that it is possible to engineer mRNA stability as an additional means of controlling gene expression. © 1997 John Wiley & Sons Inc. Biotechnol Bioeng 55: 557-580, 1997
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  • 22
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    Biotechnology and Bioengineering 55 (1997), S. 581-591 
    ISSN: 0006-3592
    Keywords: adsorptive membranes ; oscillatory flow ; integrated processes ; in situ product recovery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Preferential transport in adsorptive membranes can be used to selectively remove biochemicals directly from fermentation broths. During preferential transport, an adsorbing solute is selectively transported across the membrane while nonadsorbing solutes and cells are retained by the membrane. This technique was used to separate lysozyme directly from a feed containing lysozyme, myoglobin, and yeast cells. We found that because the oscillatory flows used in preferential transport involve strokes that are close to symmetric, they are very efficient in alleviating cake formation due to cell deposition on the membrane surface. Theoretical results suggest that, by optimizing process variables, preferential transport can lead to a continuous concentrated stream of the adsorbing protein. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 581-591, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 592-608 
    ISSN: 0006-3592
    Keywords: Saccharomyces cerevisiae ; metabolic modeling ; sensitivity analysis ; glycolysis ; compartmentation ; transient response ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model of glycolysis in Saccharomyces cerevisiae is presented. The model is based on rate equations for the individual reactions and aims to predict changes in the levels of intra- and extracellular metabolites after a glucose pulse, as described in part I of this study. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. The model structure and experimental observations are related to the aerobic growth of the yeast. The model is based on material balance equations of the key metabolites in the extracellular environment, the cytoplasm and the mitochondria, and includes mechanistically based, experimentally matched rate equations for the individual enzymes. The model includes removal of metabolites from glycolysis and TCC for biosynthesis, and also compartmentation and translocation of adenine nucleotides. The model was verified by in vivo diagnosis of intracellular enzymes, which includes the decomposition of the network of reactions to reduce the number of parameters to be estimated simultaneously. Additionally, sensitivity analysis guarantees that only those parameters are estimated that contribute to systems trajectory with reasonable sensitivity. The model predictions and experimental observations agree reasonably well for most of the metabolites, except for pyruvate and adenine nucleotides. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 592-608, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 609-615 
    ISSN: 0006-3592
    Keywords: interacting populations ; membrane reactor ; induced metabolic changes ; elicitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The design of a reactor in which two interacting cell populations (microorganisms and plants) could grow under controlled conditions was considered. In this reactor, the cell populations are separated by a membrane which permits semi-in vivo study of induced interaction-specific changes in metabolism. In this paper, the interaction of suspension culture of Nicotiana tabacum (tobacco) and the Oomycete, Phytophthora nicotiana was simulated. The results of the computer simulation show the induced metabolic changes as a consequence of the biological interaction. The paper introduces a novel approach in the strategy for the study of interacting population in suspension cultures. This type of system has potential applications in studies of the regulation of secondary metabolism and for the production of high values pharmaceuticals. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 609-615, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 616-629 
    ISSN: 0006-3592
    Keywords: cell adhesion ; radial-flow chamber ; hydrodynamic shear ; detachment kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The strength of adhesion and dynamics of detachment of murine 3T3 fibroblasts from self-assembled monolayers were measured in a radial-flow chamber (RFC) by applying models for fluid mechanics, adhesion strength probability distributions, and detachment kinetics. Four models for predicting fluid mechanics in a RFC were compared to evaluate the accuracy of each model and the significance of inlet effects. Analysis of these models indicated an outer region at large radial positions consistent with creeping flow, an intermediate region influenced by inertial dampening, and an inner region dominated by entrance effects from the axially-oriented inlet. In accompanying experiments patterns of the fraction of cells resisting detachment were constructed for individual surfaces as a function of the applied shear stress and evaluated by comparison with integrals of both a normal and a log-normal distribution function. The two functions were equally appropriate, yielding similar estimates of the mean strength of adhesion. Further, varying the Reynolds number in the inlet, Red, between 630 and 1480 (corresponding to volumetric flow rates between 0.9 and 2.1 mL/s) did not affect the mean strength of adhesion. For these same experiments, analysis of the dynamics of detachment revealed three temporal phases: 1) rapid detachment of cells at the onset of flow, consistent with a first-order homogeneous kinetic model; 2) time-dependent rate of detachment during the first 30 sec. of exposure to hydrodynamic shear, consistent with the first-order heterogeneous kinetic model proposed by Dickinson and Cooper (1995); and 3) negligible detachment, indicative of pseudo-steady state after 60 sec. of flow. Our results provide rigorous guidelines for the measurement of adhesive interactions between mammalian cells and prospective biomaterial surfaces using a RFC. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 616-629, 1997.
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  • 26
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    Keywords: scale-down ; homogenisation ; modelling ; size-distribution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experiments were carried out aimed at establishing the effects of equipment scale down on the disruption of Baker's yeast cells in high pressure homogenisers. Data are reported on the cell debris particle size distribution (PSD) and on total protein release as a function of the applied pressure for two valve geometries and three scales of operation covering flow rates of 28, 60 and 280 L/h. A comparison of the results from the experiments indicates that over the range of parameters investigated both the total protein release and the cell debris PSDs are independent of valve geometry and flow rate through the homogeniser. These observations are discussed in the light of relevant previous publications. The cell debris PSDs have been simulated by using a recently published model and the total protein release data are described by the well-established Hetherington expression (Hetherington et al., 1971). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 642-649, 1997.
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  • 27
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    Biotechnology and Bioengineering 55 (1997), S. 630-641 
    ISSN: 0006-3592
    Keywords: nitrification ; immobilized cells ; Nitrosomonas europaea ; substrate limitation ; biomass death ; staining techniques ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The dynamics of growth and death of immobilized Nitrosomonas europaea were studied. For this, the death rate of suspended cells was determined in the absence of ammonium or oxygen by following the loss of respiration activity and by fluorescein-diacetate (FDA)/lissamine-green staining techniques. The death rates obtained (1.06 × 10-6 s-1 or 4.97 × 10-6 s-1 in the absence of oxygen or ammonium, respectively) were incorporated in a dynamic growth model and the effects on the performance of the immobilized-cell process illustrated by model simulations.These model simulations and experimental validation show that if decay of biomass occurs the biomass concentration in the center of the bead decreases. As a result, the systems react slower to changes in substrate concentrations than if all cells remain viable.To show that cells in the center of the bead died, the FDA and lissamine-green staining techniques were adapted for immobilized cells. It was shown that biomass decay occurred, especially in the center of the bead; the amount of cells decreased there, and the remaining cells were all stained with lissamine green indicating cell death. After the substrate availability was decreased, also cells near the surface of the bead lost their viability. The number of viable cells increased again after increasing the substrate concentration as the result of cell multiplication. At low substrate concentrations and low hydraulic retention times, as for example in the treatment of domestic wastewater, the death rate of cells is thus an important parameter for the performance of the immobilized-cell system. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 630-641, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 650-659 
    ISSN: 0006-3592
    Keywords: cometabolism ; methanotroph ; trichloroethylene ; reactor ; aerobic ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this research was to evaluate several factors affecting the performance of a two-stage treatment system employing methane-oxidizing bacteria for trichloroethylene (TCE) biodegradation. The system consists of a completely mixed growth reactor and a plug-flow transformation reactor in which the TCE is cometabolized. Laboratory studies were conducted with continuous growth reactors and batch experiments simulating transformation reactor conditions. Performance was characterized in terms of TCE transformation capacity (TC, g TCE/g cells), transformation yield (TY, g TCE/g CH4), and the rate coefficient ratio kTCE/KS,TCE (L/mg-d). The growth reactor variables studied were solids retention time (SRT) and nutrient nitrogen (N) concentration. Formate and methane were evaluated as potential transformation reactor amendments. Comparison of cultures from 2- and 8-day SRT (nitrogen-limited) growth reactors indicated that there was no significant effect of growth reactor SRT or nitrogen availability on TC or TY, but N-limited conditions yielded higher kTCE/KS,TCE. The TCE cometabolic activity of the 8-day SRT, N-limited growth reactor culture varied significantly during a 7-year period of operation. The TC and TY of the resting cells increased gradually to levels a factor of 2 higher than the initial values. The reasons for this increase are unknown. Formate addition to the transformation reactor gave higher TC and TY for 2-day SRT growth reactor conditions and significantly lower TC, TY, and kTCE/KS,TCE for 8-day SRT N-limited conditions. Methane addition to the transformation reactor inhibited TCE cometabolism at low TCE concentrations and enhanced TCE cometabolism at high TCE concentrations, indicating that the TCE cometabolism in the presence of methane does not follow simple competitive inhibition kinetics. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 650-659, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 674-685 
    ISSN: 0006-3592
    Keywords: fixed-film bioreactor ; biofilter ; trichloroethylene ; mineralization ; toluene ortho -monooxygenase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An aerobic, single-pass, fixed-film bioreactor was designed for the continuous degradation and mineralization of gas-phase trichloroethylene (TCE). A pure culture of Burkholderia cepacia PR123(TOM23C), a Tn5transposon mutant of B. cepacia G4 that constitutively expresses the TCE-degrading enzyme, toluene ortho-monooxygenase (TOM), was immobilized on sintered glass (SIRAN™ carriers) and activated carbon. The inert open-pore structures of the sintered glass and the strongly, TCE-absorbing activated carbon provide a large surface area for biofilm development (2-8 mg total cellular protein/mL carrier with glucose minimal medium that lacks chloride ions). At gas-phase TCE concentrations ranging from 0.04 to 2.42 mg/L of air and 0.1 L/min of air flow, initial maximum TCE degradation rates of 0.007-0.715 nmol/(min mg protein) (equivalent to 8.6-392.3 mg TCE/L of reactor/day) were obtained. Using chloride ion generation as the indicator of TCE mineralization, the bioreactor with activated carbon mineralized an average of 6.9-10.3 mg TCE/L of reactor/day at 0.242 mg/L TCE concentration with 0.1 L/min of air flow for 38-40 days. Although these rates of TCE degradation and mineralization are two- to 200-fold higher than reported values, TOM was inactivated in the sintered-glass bioreactor at a rate that increased with increasing TCE concentration (e.g., in ∼2 days at 0.242 mg/L and 〈1 day at 2.42 mg/L), although the biofilter could be operated for longer periods at lower TCE concentrations. Using an oxygen probe and phenol as the substrate, the activity of TOM in the effluent cells of the bioreactor was monitored; the loss of TOM activity of the effluent cells corroborated the decrease in the TCE degradation and mineralization rates in the bioreactor. Repeated starving of the cells was found to restore TOM activity in the bioreactor with activated carbon and extended TCE mineralization by ∼34%. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 674-685, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 686-692 
    ISSN: 0006-3592
    Keywords: tolouene degradation ; biomass formation ; bioscrubber ; trickle-bed ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of degradation of toluene from a model waste gas and of biomass formation were examined in a bioscrubber operated under different nutrient limitations with a mixed culture. The applicability of the kinetics of continuous cultivation of the mixed culture was examined for a special trickle-bed reactor with a periodically moved filter bed. The efficiency of toluene elimination of the bioscrubber was 50 to 57% and depended on the toluene mass transfer as evident from a constant productivity of 0.026 g dry cell weight/L · h over the dilution rate. Under potassium limitation the biomass productivity was reduced by 60% to 0.011 g dry cell weight/L · h at a dilution rate of 0.013/h. Conversely, at low dilution rates the specific toluene degradation rates increased. Excess biomass in a trickle-bed reactor causes reduction of interfacial area and mass transfer, and increase in pressure drop. To avoid these disadvantages, the trickle-bed was moved periodically and biomass was removed with outflowing medium. The concentration of steady state biomass fixed on polyamide beads decreased hyperbolically with the dilution rate. Also, the efficiency of toluene degradation decreased from 72 to 56% with increasing dilution rate while the productivity increased. Potassium limitation generally caused a reduction in biomass, productivity, and yield while the specific degradation increased with dilution rate. This allowed the application of the principles of the chemostat to the trickle-bed reactor described here, for toluene degradation from waste gases. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 686-692, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 610-617 
    ISSN: 0006-3592
    Keywords: chitin ; chitosan ; macroporous membranes ; affinity separation ; ovalbumin ; lysozyme ; egg white ; affinity membranes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (≥1.1 mL/min/cm2) corresponding to pressure drops ≥2 psi. Their adsorption capacity for lysozyme (∼50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (〉98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610-617, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 635-644 
    ISSN: 0006-3592
    Keywords: optimal growth ; flux towards growth ; E. coli K12 ; multiple substrate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A comprehensive model is developed based on an optimal strategy describing varied microbial growth phenomenon involving sequential and simultaneous utilization of substrate. The model mimics the complex regulatory process of a cell which results in diverse growth process with the help of simple multi-variable constrained optimization, which aims at maximizing the specific cell growth. The metabolic processes of a cell are represented by simple flux balance equations. The different growth phenomenon exhibited by a microorganism are attributed to different levels of control present inside the cell. Provision is made in the model for these controls, in the form of constraints in the optimization formulation. The model prediction matches well with the experimental data of simultaneous growth of E. coli K12 on a mixture of glucose and organic acids like lactate, pyruvate, and acetate. Moreover, the model predictions are well in agreement with earlier published experimental data for the growth of E. coli K12 on other organic acids like fumarate, α-ketoglutarate, and succinate. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 635-644, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 650-655 
    ISSN: 0006-3592
    Keywords: substrate reactivity ; lignocellulose ; cellulase ; pretreated wood ; property ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In an effort to better understand the role of the substrate in the rapid fall off in the rate of enzymatic hydrolysis of cellulose with conversion, substrate reactivity was measured as a function of conversion. These measurements were made by interrupting the hydrolysis of pretreated wood at various degrees of conversion; and, after boiling and washing, restarting the hydrolysis in fresh buffer with fresh enzyme. The comparison of the restart rate per enzyme adsorbed with the initial rate per enzyme adsorbed, both extrapolated back to zero conversion, provides a measurement of the substrate reactivity without the complications of product inhibition or cellulase inactivation. The results indicate that the substrate reactivity falls only modestly as conversion increases. However, the restart rate is still higher than the rate of the uninterrupted hydrolysis, particularly at high conversion. Hence we conclude that the loss of substrate reactivity is not the principal cause for the long residence time required for complete conversion. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 650-655, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 671-680 
    ISSN: 0006-3592
    Keywords: immobilized enzymes ; Candida rugosa lipase ; organic solvents ; lovastatin ; dielectric constant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lipase from Candida rugosa immobilized on a nylon support has been used to synthesize lovastatin, a drug which lowers serum cholesterol levels, by the regioselective acylation of a diol lactone precursor with 2-methylbutyric acid in mixtures of organic solvents. Analogs of lovastatin having a different side chain were also obtained through this method by reacting the diol substrate with different carboxylic acids. The selection of reaction conditions that maximize the initial reaction rate is investigated. Since the diol substrate has very low solubility in non-polar solvents, reaction solvents consisting of mixtures of hexane with a different, more polar cosolvent are considered. For each of the cosolvent mixtures studied, the reaction rate is maximum for an intermediate percentage of cosolvent in hexane. With total concentrations of the diol lactone in the range 6.25-12.5 mM, maximum initial rates correspond approximately to those cosolvent concentrations that permit a complete solubilization of the substrate. At higher cosolvent concentrations, lower rates are obtained. When considering the same dissolved substrate concentration, the reaction rate was found to increase with increasing values of logPmix and decreasing values of the dielectric constant, when varying the composition of a binary solvent mixture. However, when comparing different cosolvents, no general trend with respect to these properties was observed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56:671-680, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 715-726 
    ISSN: 0006-3592
    Keywords: fungal morphology ; pellets ; hyphae ; hair of pellets ; agitation intensity ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Both parallel fermentations with Aspergillus awamori (CBS 115.52) and a literature study on several fungi have been carried out to determine a relation between fungal morphology and agitation intensity. The studied parameters include hyphal length, pellet size, surface structure or so-called hairy length of pellets, and dry mass per-wet-pellet volume at different specific energy dissipation rates. The literature data from different strains, different fermenters, and different cultivation conditions can be summarized to say that the main mean hyphal length is proportional to the specific energy dissipation rate according to a power function with an exponent of -0.25 ± 0.08. Fermentations with identical inocula showed that pellet size was also a function of the specific energy dissipation rate and proportional to the specific energy dissipation rate to an exponent of -0.16 ± 0.03. Based on the experimental observations, we propose the following mechanism of pellet damage during submerged cultivation in stirred fermenters. Interaction between mechanical forces and pellets results in the hyphal chip-off from the pellet outer zone instead of the breakup of pellets. By this mechanism, the extension of the hyphae or hair from pellets is restricted so that the size of pellets is related to the specific energy dissipation rate. Hyphae chipped off from pellets contribute free filamentous mycelia and reseed their growth. So the fraction of filamentous mycelial mass in the total biomass is related to the specific energy dissipation rate as well.To describe the surface morphology of pellets, the hyphal length in the outer zone of pellets or the so-called hairy length was measured in this study. A theoretical relation of the hairy length with the specific energy dissipation rate was derived. This relation matched the measured data well. It was found that the porosity of pellets showed an inverse relationship with the specific energy dissipation rate and that the dry biomass per-wet-pellet volume increased with the specific energy dissipation rates. This means that the tensile strength of pellets increased with the increase of specific energy dissipation rate. The assumption of a constant tensile strength, which is often used in literature, is then not valid for the derivation of the relation between pellet size and specific energy dissipation rate. The fraction of free filamentous mycelia in the total biomass appeared to be a function of the specific energy dissipation in stirred bioreactors. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 715-726, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 626-634 
    ISSN: 0006-3592
    Keywords: anaerobic digestion ; on-line control ; hydrogen concentration ; digester overloading ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of dissolved hydrogen as an early warning signal of digester failure and a control parameter to operate anaerobic digesters was investigated. A sensitive, on-line method was developed for measuring trace levels of dissolved hydrogen in a semi-permeable membrane, situated within the biomass of a 1 L laboratory anaerobic digester, using trace reduction gas analysis. At normal operating conditions, the dissolved hydrogen partial pressure (2 to 8 Pa) was found to be linearly correlated with the loading rate of the digester, and was a sensitive indicator of the effect of shockloads as well as gradual overloading. An increase in hydrogen partial pressure above a critical concentration of 6.5-7 Pa indicated the initial stage of digester overloading (i.e., volatile fatty acids accumulation). A H2-based computer control system, using a critical hydrogen partial pressure of 6.5 Pa as the setpoint, was found to be effective for the safe operation of a laboratory digester close to its maximum sustainable loading rate. The existence of a relationship between hydrogen level and organic loading rate was also confirmed on a 600 m3 industrial digester, with digester overloading occurring at hydrogen concentrations above 7 Pa. The results suggest that the dissolved hydrogen concentration is capable of being a sensitive on-line parameter for the automated management of anaerobic digesters near their maximum sustainable loading capacity. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 626-634, 1997.
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  • 37
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    Biotechnology and Bioengineering 56 (1997), S. 656-670 
    ISSN: 0006-3592
    Keywords: unsaturated biofilm ; diffusion ; substrate utilization kinetics ; matric water potential ; toluene ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biofilms are frequently studied in the context of submerged or aquatic systems. However, much less is known about biofilms in unsaturated systems, despite their importance to such processes as food spoilage, terrestrial nutrient cycling, and biodegradation of environmental pollutants in soils. Using modeling and experimentation, we have described the biodegradation of toluene in unsaturated media by bacterial biofilms as a function of matric water potential, a dominant variable in unsaturated systems. We experimentally determined diffusion and kinetic parameters for Pseudomonas putida biofilms, then predicted biodegradation rates over a range of matric water potentials. For validation, we measured the rate of toluene depletion by intact biofilms and found the results to reasonably follow the model predictions. The diffusion coefficient for toluene through unsaturated P. putida biofilm averaged 1.3 × 107 cm2/s, which is approximately two orders of magnitude lower than toluene diffusivity in water. Our studies show that, at the scale of the microbial biofilm, the diffusion of toluene to biodegrading bacteria can limit the overall rate of biological toluene depletion in unsaturated systems. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 656-670, 1997.
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  • 38
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    Biotechnology and Bioengineering 56 (1997), S. 697-705 
    ISSN: 0006-3592
    Keywords: poly-β-hydroxybutyric acid ; Alcaligenes eutrophus ; fed-batch culture ; high cell density culture ; optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The optimal feed rate profiles of glucose and ammonium hydroxide were calculated using a proposed model, and implemented for the production of poly-β-hydroxybutyric acid (PHB) by Alcaligenes eutrophus. By implementing these optimal feed rates with a high glucose feed concentration of 700 g/L and an ammonium hydroxide concentration of 7%(w/w), it was possible to achieve a high final cell concentration of 141 g/L and a high PHB concentration of 105 g/L in 40 h of fed-batch operation. The PHB productivity was as high as 2.63 g/(L hr). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 697-705, 1997.
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  • 39
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    Biotechnology and Bioengineering 53 (1997), S. 01-09 
    ISSN: 0006-3592
    Keywords: transferrin ; conalbumin ; metalloprotein ; affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recently these laboratories have demonstrated that it is possible to use proteins as efficient, selective agents for heavy metal removal and recovery. In this study, transferrin was chemically bound to an insoluble support. The ability of immobilized transferrin to produce clean water was demonstrated. Copper loading was independent of feed concentration. The loaded copper could be readily eluted and concentrated into the gram per liter range. The mechanism of copper release was studied. It was shown that release was dependent on pH and the chelating ability of the stripping agent. Metal release occurred slowly at pH 〈 7. However, at low pH in the presence of a chelator, metal removal occurred much more efficiently. The binding constant of copper to immobilized transferrin was determined as a function of pH. This information was used to model metal binding and release to the protein/support matrix. © 1997 John Wiley & Sons, Inc.
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  • 40
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    Biotechnology and Bioengineering 53 (1997), S. 10-16 
    ISSN: 0006-3592
    Keywords: microfiltration ; fouling ; yeast ; antifoam agents ; depressurization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The fouling effects of yeast fermentation broths of Candida utilis in the presence of various commercial antifoam agents (PPG2000, B5600, and G832) up to 4.0 mL/L were studied, using Millipore polyvinylidene fluoride 0.22-μm hydrophilic membranes (GVWP), in a stirred-cell system at 50 kPa and 700 rpm. PPG2000, which has a low value of work of adhesion (Wa of 0.81 mN/m), gave a steady flux of broth of 29 L/(h m2) and was found to have no significant fouling effect on the microfiltration of broth. G832, which has a high Wa, (26.0 mN/m) reduced the flux of the broth to 17 L/(h m2); i.e., by 42% when only 1.0 mL/L was used. However, B5600, which has a Wa of 14.3 mN/m, was found to enhance the flux of broth to 54 L/(h m2); i.e., by 86%, due to the preferential adsorption of the B5600 components onto the hydrophobic cell contents released. These results were reinforced by the depressurization experiments performed with both hydrophilic (GVWP) and hydrophobic (GVHP) membranes, using both young and aged broths. B5600 was found to be the optimum antifoam agent in this study in terms of membrane performance and defoaming efficiency. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 17-20 
    ISSN: 0006-3592
    Keywords: P. chrysogenum ; alginate oligosaccharides ; oligomannuronate ; oligoguluronate ; penicillin G ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 μg/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 26-31 
    ISSN: 0006-3592
    Keywords: protease ; transesterification ; enantioselectivity ; organic solvent ; solvent effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30°C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN′ always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 21-25 
    ISSN: 0006-3592
    Keywords: starch fermentation ; recombinant yeast ; ethanol production ; glucoamylase activity ; fed-batch culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 41-48 
    ISSN: 0006-3592
    Keywords: nicotinamide adenine dinucleotide ; isourea ; imidocarbonate ; agarose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stability of NAD(H) immobilized to a crosslinked agarose support (Sepharose®-4B) was examined in buffer solutions at a pH of 7.0 and 8.5. Specifically, this study investigated particle attrition and ligand leakage rates from a cyanogen bromide activated agarose support. Particle attrition did not occur under the experimental conditions. Ligand leakage rates were found to be first order in immobilized ligand concentration with two labile populations of ligand. The two-population model is consistent with the cyanogen bromide coupling chemistry, which results in both an isourea and imidocarbonate ligand linkage. The rate of ligand leakage was found to occur over a time scale of days, with first order rate constants ranging from 0.007 to 0.15 d-1, depending on solution pH. © 1997 John Wiley & Sons, Inc.
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  • 45
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    Biotechnology and Bioengineering 53 (1997), S. 49-57 
    ISSN: 0006-3592
    Keywords: chromatography ; enzyme purification ; peptide immobilization ; peptide ligand ; trypsin purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (〉95% pure), exhibiting an Arg residue as their C-terminal (site P1) for trypsin bio-recognition, a Pro or Ala in site P2, and a Thr or Val in site P3. Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 μmol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 μmol/g gel. The KD values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The KD values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (KD 8.7 μM), whereas the sequence H-TAR-OH displayed the lowest (KD 38 μM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 μmol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 32-40 
    ISSN: 0006-3592
    Keywords: expanded-bed reactor ; sulfur ; Thiobacilli ; immobilization ; biofilm ; sludge ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The performance of a new sulfide-oxidizing, expanded-bed bioreactor is described. To stimulate the formation of well-settleable sulfur sludge, which comprises active sulfide-oxidizing bacterial biomass and elemental sulfur, the aeration of the liquid phase and the oxidation of sulfide to elemental sulfur are spatially separated. The liquid phase is aerated in a vessel and subsequently recirculated to the sulfide-oxidizing bioreactor. In this manner, turbulencies due to aeration of the liquid phase in the bioreactor are avoided. It appeared that, under autotrophic conditions, almost all biomass present in the reactor will be immobilized within the sulfur sludge which consists mainly of elemental sulfur (92%) and biomass (2.5%). The particles formed have a diameter of up to 3 mm and can easily be grinded down. Within time, the sulfur sludge obtained excellent settling properties; e.g., after 50 days of operation, 90% of the sludge settles down at a velocity above 25 m h-1 while 10% of the sludge had a sedimentation velocity higher than 108 m h-1. Because the biomass is retained in the reactor, higher sulfide loading rates may be applied than to a conventional “free-cell” suspension. The maximum sulfide-loading rate reached was 14 g HS- L-1 d-1, whereas for a free-cell suspension a maximum loading rate of 6 g HS- L-1 d-1 was found. At higher loading rates, the upward velocities of the aerated suspension became too high so that sulfur sludge accumulated in the settling zone on top of the reactor. When the influent was supplemented with volatile fatty acids, heterotrophic sulfur and sulfate reducing bacteria, and possibly also (facultatively) heterotrophic Thiobacilli, accumulated within the sludge. This led to a serious deterioration of the system; i.e., the sulfur formed was increasingly reduced to sulfide, and also the formation rate of sulfur sludge declined. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 58-70 
    ISSN: 0006-3592
    Keywords: control ; monitoring ; fractional precipitation ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Downstream processing operations are often carried out blind in the process timescale since product monitoring on-line is not common. Knowledge of the location and concentration of the product and key contaminants is complementary to other process information for process development and, if available on-line in conjunction with a suitable model, control. This article sets out to demonstrate a model describing a two-cut fractional protein precipitation process and how this may be used for control of the process to maximize yield in the face of variable process stream conditions. Estimation of the model parameters is achieved by means of data-fitting by least squares and in comparison prediction by a Kalman filter algorithm. A description and error analysis of equipment for at-line monitoring of the soluble product in a pilot plant environment is presented which includes a micro-centrifuge necessary to clarify small volumes of sample prior to analysis. Finally, an account of the successful implementation of this equipment and the Kalman filter algorithm for control at bench scale is given where conditions in the process stream are deliberately disturbed to test the control operation. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 71-78 
    ISSN: 0006-3592
    Keywords: Zymomonas ; yeast ; acetaldehyde ; ethanol ; stress ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The lag phase of Saccharomyces cerevisiae subjected to a step increase in temperature or ethanol concentration was reduced by as much as 60% when acetaldehyde was added to the medium at concentrations less than 0.1 g/L. Maximum specific growth rates were also substantially increased. Even greater proportional reductions in lag time due to acetaldehyde addition were observed for ethanol-shocked cultures of Zymomonas mobilis. Acetaldehyde had no effect on S. cerevisiae cultures started from stationary phase inocula in the absence of environmental shock and its lag-reducing effects were greater in complex medium than in a defined synthetic medium. Acetaldehyde reacted strongly with the ingredients of complex culture media. It is proposed that the effect of added acetaldehyde may be to compensate for the inability of cells to maintain transmembrane acetaldehyde gradients following an environmental shock. © 1997 John Wiley & Sons, Inc.
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  • 49
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    Biotechnology and Bioengineering 53 (1997), S. 79-87 
    ISSN: 0006-3592
    Keywords: polyacrylamide ; magnetic ; stabilized ; fluidized bed ; chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Spherical polyacrylamide/magnetite (PAM) composite beads, suitable for use in a magnetically stabilized fluidized bed (MSFB), were manufactured by a suspension polymerization method. Yield of beads depended on the type and concentration of buffer used during polymerization as well as the pH. More stabilizer was needed to prevent bead agglomeration as magnetite concentration increased. Bead diameter ranged from less than 60 to 600 μm, depending on reaction conditions, and the bead mean diameter and size distribution decreased with increasing impeller speed. The density and roundness factor of the beads were 1.19 ± 0.02 g cm-3 and 1.08 ± 0.03, respectively. The beads had high magnetization at a low applied magnetic field strength (60 mT at 75 kA m-1) and retained little residual magnetization (〈2 mT) after the field was removed. Incorporation of magnetite did not significantly affect the physical strength of the beads: the beads' average elastic modulus was 14 ± 4 kPa, similar to reported values for polyacrylamide gels (15.8 kPa). The beads were stable in a range of buffers from pH 1 to 10 and were resistant to microbial degradation. The fluidization and stabilization behavior of the beads was examined in a bench-scale MSFB. The minimum fluidization velocity (Umf) of the beads (0.035 mm s-1) allowed the MSFB to be operated at superficial velocities close to those used in HPLC systems. Against expectations, at high superficial velocities, the stabilized bed of the MSFB had a greater expansion than the unstabilized bed. The PAM beads could be derivatized and activated for soybean trypsin inhibitor immobilization by a standard carbodiimide method, and the affinity separation of trypsin from chymotrypsin was demonstrated. The PAM beads show excellent potential for use in MSFB chromatography. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 88-99 
    ISSN: 0006-3592
    Keywords: biofilm structure ; detachment ; abrasion ; collisions ; airlift-reactor ; hydrodynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The detachment of biomass from suspended biofilm pellets in three-phase internal loop airlift reactors was investigated under nongrowth conditions and in the presence of bare carrier particles. In different sets of experiments, the concentrations of biofilm pellets and bare carrier particles were varied independently. Gas hold-up, bubble size, and general flow pattern were strongly influenced by changes in volume fractions of biofilm pellets and bare carrier particles. In spite of this, the rate of biomass detachment was found to be linear with both the concentration of biofilm pellets and the bare carrier concentration up to a solids hold-up of 30%. This implies that the detachment rate was dominated by collisions between biofilm pellets and bare carrier particles. These collisions caused an on-going abrasion of the biofilm pellets, leading to a reduction in pellet volume. Breakage of the biofilm pellets was negligible. The biofilm pellets were essentially ellipsoidal, which made three-dimensional size determination necessary. Calculating particle volumes from two-dimensional image analysis measurements and assuming a spherical shape led to serious errors. The abrasion rate was not equal on all sides of the biofilm pellets, resulting in an increasing flattening of the pellets. This flattening was oriented with the basalt carrier inside the biofilm and independent of the absolute abrasion rate. These observations suggest that the collisions causing abrasion are somehow oriented. The internal structure of the biofilms showed two layers, a cell-dense outer layer and an interior with a low biomass density. Taking this density gradient into account, the washout of detached biomass matched observed changes in volume of the biofilm pellets. No gradient in biofilm strength with biofilm depth was indicated. © 1997 John Wiley & Sons, Inc.
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  • 51
    ISSN: 0006-3592
    Keywords: citrobacter sp. ; immobilized cells ; low pH ; phosphatase ; uranium ; wastewater ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A biotechnological process for the removal of heavy metals from aqueous solution utilizes enzymatically liberated phosphate ligand which precipitates with heavy metals (M) as cell-bound MHPO4. The enzyme, a phosphatase, obeys Michaelis-Menten kinetics in resting and immobilized cells; an integrated form of the Michaelis-Menten equation was used to calculate the apparent Km (Km app.) as operating in immobilized cells in flow-through columns by a ratio method based on the use of two enzyme loadings (Eo1, Eo2) or two input substrate concentrations (So1, So2). The calculated Km app. (4.08 mM) was substituted into an equation to describe the removal of metals by immobilized cells. In operation the activity of the bioreactor was in accordance with that predicted mathematically, within 10%. The initial tests were done at neutral pH, whereas the pH of industrial wastewaters is often low; an increase in the Km app. at low pH was found in previous studies. Immobilized cells were challenged with acidic mine drainage wastewaters, where the limiting factors were chemical and not biochemical. Bioreactors initially lost activity in this water, but recovered to remove uranyl ion with more than 70% efficiency under steady-state conditions in the presence of competing cations and anions. Possible reasons for the bioreactor recovery are chemical crystallization factors. © 1997 John Wiley & Sons, Inc.
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  • 52
    ISSN: 0006-3592
    Keywords: isoelectric membranes ; immobilized reactors ; urease ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel type of immobilized enzyme reactor operating under an electric field is here reported: a multicompartment immobilized enzyme reactor (MIER). In this experimental set-up, the enzyme and zwitterionic buffering ions are trapped in between two isoelectric membranes, having isoelectric point (pl) values so far apart as to trap the enzyme by an isoelectric mechanism, while allowing operation at pH optima, even when the latter pH value is quite removed from the enzyme pl. As an example, urease (pl 4.9) is trapped between a pl 4.0 and a pl 8.0 membranes, thus permitting operation (via suitable amphoteric ions buffering at pH 7.5) at the pH of optimum of activity (pH 7.5). The charged product (ammonium ions) quickly leaves the enzyme chamber under the influence of the electric field, thus allowing sustained activity for much longer time periods than in conventional reactors. As an example, while in a batch reactor 90% of original enzyme activity is lost in 200 min, only 2% activity is lost in the same period in the MIER reactor. As an additional bonus, the MIER reactor allows conversion rates of ∼95% in a wide range of substrate concentrations, whereas batch-type reactors rarely achieve better than 50% conversion under comparable experimental conditions. © 1997 John Wiley & Sons, Inc.
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  • 53
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    Biotechnology and Bioengineering 53 (1997), S. 121-131 
    ISSN: 0006-3592
    Keywords: biocalalysis ; microemulsion ; gelatin ; organogel ; lipase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chromobacterium viscosum (CV) lipase was immobilized in gelatin-containing Aerosol-OT (AOT) microemulsion-based organogels (MBGs). The behavior of this novel, predominantly hydrophobic matrix as an esterification catalyst has been examined. The biocatalyst was most effective when the MBG was granulated to yield gel particles of ∼500 μm diameter, providing a total surface area of ca. 106 mm2 per 10 cm3 of gel. The gel was generally contacted with a solution of the substrate(s) in a hydrocarbon oil. Under most conditions reaction was not diffusion limited. Apparent lipase activity was influenced by certain compositional changes in the MBG, but most significantly when the R value, the mole ratio of water to surfactant, was altered. Higher activities were observed at lower R values. Although gels of lowest R value expressed the highest condensation activity, such formulations were physically unsuitable as immobilization matrices due to their proximity to the gel-solution phase boundary. MBGs of intermediate R values (between 60 and 80) were considered most suitable because they offer relatively high condensation activity and good physical stability. The gelatin concentration also exerted a small but measurable influence on the observed condensation rates. Apparent lipase activity was also influenced to some extent by the nature of the parent hydrocarbon used to prepare the MBG. Higher activities were obtained using formulations derived from isooctane and cyclohexane rather than the n-alkanes. Condensation activities expressed by CV lipase in the MBGs were broadly comparable to those expressed in the analogous parent water-in-oil (w/o) microemulsions. The MBGs functioned effectively in neat substrate solutions, but the condensation activity expressed by the MBGs in a series of successive batch syntheses was adversely affected by the formation and retention of the water coproduct. Selective removal of the water was achieved using a concentrated solution of dry reverse micelles, which resulted in recovery of lost activity. Pretreatment of lipase-containing MBGs resulted in the formation of MBGs with enhanced catalytic properties and modified composing the conventional procedure. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 139-150 
    ISSN: 0006-3592
    Keywords: filamentous fungus ; tridimensional structure ; fractal dimension ; spore aggregates ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The tridimensional growth of a filamentous fungus was simulated, based on a model for the evolution of the microscopic morphology of Trichoderma reesei. When supplemented with a spatial representation of growth, the model correctly simulates the evolution from a single spore to a pellet. Diffusion of oxygen is included in the model. The simulated tridimensional structures have a fractal nature; and the fractal dimension, determined by a box-counting method, increases during growth. The fractal dimension only depends on the mass of the pellet and is not affected by model parameters such as tip extension rate and branching frequency. Realistic pictures are obtained and the radius of the pellet increases at a constant rate. The influence of model parameters (tip extension rate, branching frequency, minimum porosity) on dissolved oxygen concentration profiles, biomass concentration profiles, rate at which the pellet diameter increases, and the evolution of the fractal dimension was determined. The dissolved oxygen profiles were found to be very different from the profiles, obtained by assuming a homogenous biomass distribution within the pellet. Finally, the formation of pellets from spore aggregates is calculated and the size of the spore aggregate is found to only influence the time needed before the appearance of a pellet and not its morphology. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 132-138 
    ISSN: 0006-3592
    Keywords: metabolic engineering ; metabolic control analysis ; transaldolase ; aromatics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Aromatic metabolites in Escherichia coli and other microorganisms are derived from two common precursors: phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). During growth on glucose, the levels of both E4P and PEP are insufficient for high throughput of aromatics because of the low carbon flux through the pentose pathway and the use of PEP in the phosphotransferase system. It has been shown that transketolase and PEP synthase are effective in relieving this limitation and promoting high throughput of aromatics. To determine whether transaldolase, another E4P-producing enzyme, is also a limiting factor in directing carbon flux to the aromatic pathway, E. coli transaldolase gene (tal) was cloned and overexpressed in an aroB strain which excretes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), the first intermediate in the aromatic pathway. We found that overexpression of transaldolase did significantly increase the production of DAHP from glucose. This result further supports the contention that the supply of E4P is limiting when glucose is the carbon source. However, overexpression of transaldolase in strains which already overexpress transketolase did not show a further increase in production of aromatics. This result was attributed to the saturation of E4P supply when TktA was overexpressed. The flux control of DAHP production was discussed on the basis of Metabolic Control Analysis. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 151-158 
    ISSN: 0006-3592
    Keywords: biofilms ; biofilm structure ; diffusivity ; mass transport in biofilms ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new technique for the determination of local diffusion coefficients in biofilms is described. It is based on the microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using confocal laser microscopy. The diffusion coefficients of fluorescein (MW 332), TRITC-IgG (MW 150000) and phycoerythrin (MW 240000) were measured in the cell clusters and interstitial voids of a heterogeneous biofilm. The diffusivities measured in the voids were close to the theoretical values in water. Fluorescein had the same diffusivity in cell clusters, voids, and sterile medium. TRITC-IgG did not diffuse in cell clusters, presumably due to binding to the cell cluster matrix. After treatment of the biofilm with bovine serum albumin, binding capacity decreased and the diffusion coefficient could be measured. The diffusivity of phycoerythrin in cell clusters was impeded by 41%, compared to interstitial voids. From the diffusion data of phycoerythrin it was further calculated that the cell cluster matrix had the characteristics of a gel with 0.6 nm thick fibers and pore diameters of 80 nm. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 159-167 
    ISSN: 0006-3592
    Keywords: amino acid ; flor yeast ; L-proline ; urea ; wine aging ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Urea, ammonium, and free amino acid contents were quantified in biological aging of a young wine under two flor film forming yeast strains, Saccharomyces cerevisiae race capensis and S. cerevisiae race bayanus, and compared. Cell viability in the film was different for the two yeast strains. Thus, capensis maintained a much greater number of viable cells per surface area than bayanus and hence used greater amount of nitrogen compounds. The main source of nitrogen for the yeasts during the biological aging process was L-proline. The two yeast strains also differed in the amounts of assimilable nitrogen they utilized, in their preferences for amino acid consumption, and kinetics. To accelerate the aging process, the effect of controlled monthly aeration of the wine aged with capensis strain was investigated. The results revealed that short aeration did not appreciably increase the overall consumption of assimilable nitrogen, but consumption of some nitrogen compounds was accelerated (particularly L-proline, L-tryptophan, L-glutamic acid, ammonium ion, L-lysine, and L-arginine); the use of L-ornithine was inhibited; and GABA, L-methionine, and urea were depletes. Probably the aeration increases the aroma compounds, thereby producing wines with improved sensory properties. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 168-178 
    ISSN: 0006-3592
    Keywords: airlift reactor ; BAS reactor ; biofilm ; nitrification ; nitrite ; oxygen transfer ; residence time ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biofilm airlift suspension (BAS) reactor can treat wastewater at a high volumetric loading rate combined with a low sludge loading. Two BAS reactors were operated, with an ammonium load of 5 kg N/(m3 d), in order to study the influence of biomass and oxygen concentration on the nitrification process. After start-up the nitrifying biomass in the reactors gradually increased up to 30 g VSS/L. Due to this increased biomass concentration the gas-liquid mass transfer coefficient was negatively influenced. The resulting gradual decrease in dissolved oxygen concentration (over a 2-month period) was associated with a concomitantly nitrite build-up. Short term experiments showed a similar relation between dissolved oxygen concentration (DO) and nitrite accumulation. It was possible to obtain full ammonium conversion with approximately 50% nitrate and 50% nitrite in the effluent. The facts that (i) nitrite build up occurred only when DO dropped, (ii) the nitrite formation was stable over long periods, and (iii) fully depending on DO levels in short term experiments, led to the conclusion that it was not affected by microbial adaptations but associated with intrinsic characteristics of the microbial growth system. A simple biofilm model based on the often reported difference of oxygen affinity between ammonium and nitrite oxydizers was capable of adequately describing the phenomena.Measurements of biomass density and concentration are critical for the interpretation of the results, but highly sensitive to sampling procedures. Therefore we have developed an independent method, based on the residence time of Dextran Blue, to check the experimental methods. There was a good agreement between procedures.The relation between biomass concentration, oxygen mass transfer rate and nitrification in a BAS reactor is discussed. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 179-184 
    ISSN: 0006-3592
    Keywords: flocculation ; on-line measurement ; floc formation rate ; floc dispersion rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The ability of yeast to flocculate is important in different separation processes, especially in the beer industry. Because of the regulation purposes, there is a need for online monitoring. With the presented measuring set-up, consisting of a peristaltic pump, a photometer, and a computer, it is possible to determine the onset of flocculation as well as to follow flocculation intensity and the concentration of nonflocculated cells. It was found that for the yeast strain Saccharomyces cerevisiae ZIM 198 the decrease of nonflocculated cells (after flocculation has occurred) during the exponential growth can be described by an exponential equation for the first-order process, whereas the increase of free cells due to dispersion of the flocs during the stationary phase follows the form of the growth curve. It was also demonstrated that the absorbency profiles of yeast sedimentation can be described by the second-order equation suggested by Stradford and Keenan for the decrease of cell concentration during sedimentation. © 1997 John Wiley & Sons, Inc.
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  • 60
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    Keywords: antioxidant vitamins ; vitamin C ; vitamin E ; β-carotene ; Euglena gracilis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Euglena gracilis Z is one of the few microorganisms which simultaneously produces antioxidant vitamins such as β-carotene and vitamins C and E. Photoheterotrophically cultured E. gracilis Z produced larger levels of biomass but with a lower content of antioxidant vitamins than photoautotrophically grown cultures. For efficient production of these vitamins, a two-step culture was performed. Cells were grown photoheterotrophically and then transferred to photoautotrophic conditions. When E. gracilis Z cells were grown in fed-batch culture under photoheterotrophic conditions, their density reached 19 g/L after 145 h. Subsequent transfer of these cells to photoautotrophic conditions increased vitamin content, enhancing the total vitamin yields, which were 71.0 mg/L of β-carotene, 30.1 mg/L of vitamin E, and 86.5 mg/L of vitamin C. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 191-201 
    ISSN: 0006-3592
    Keywords: pellet morphology on-line image analysis submerged growth Streptomyces tendae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The morphology of filamentous microorganisms in submerged culture is of great interest. On the one hand, morphology influences rheology and mass transfer in the fermentation broth. On the other hand, morphology could be a visible expression of physiology and metabolism of the microorganisms. An algorithm for the morphological characterization and the estimation of biomass of filamentous microorganisms by means of digital image analysis has been developed. After measurement of eight features the objects in the broth are classified into different morphological classes, i.e., pellet aggregates, rough pellets, smooth pellets, mycelial flocks, and medium components. The classification is based on the measured object parameters and a knowledge base, which was generated in a preceding training phase. The method was tested on Streptomyces tendae Tü 901/8c. A typical batch fermentation in a defined medium is presented. It could be shown that both morphology and physiology have been changed in the course of the fermentation, especially during the transition from trophophase to idiophase. In order to supervise the fermentation processes continuously, an on-line image analysis system has been developed. Sampling, dilution, and image acquisition of the culture were performed under the control of a personal computer. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 202-206 
    ISSN: 0006-3592
    Keywords: salting-out precipitation ; initial protein concentration ; protein solubility ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of initial protein concentration on the performance of salting-out precipitation is examined. In the precipitation of bovine albumin by ammonium sulfate, a peak occurs in the plot of protein solubility versus initial protein concentration; that is, the solubility first increases and then falls with increasing initial protein concentration. In addition, the dependence of solubility on the initial protein concentration is less significant if using higher salt concentrations. The solubility behavior of bovine albumin may be representative, because it covers all possible alternatives; namely, the solubility is independent of, increases with, decreases with, or first increases and then decreases with the initial protein concentration. The appearance of a solubility peak can be explained based on the occurrence of a primary particle during the precipitation process. However, inclusion of the influence of initial protein concentration into the Cohn equation is not feasible with the use of a logarithmic scale, which does not sensitively reflect the change in protein solubility. Increasing initial protein concentration favors protein recovery because it reduces the resultant volume of the supernatant phase. © l997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 207-213 
    ISSN: 0006-3592
    Keywords: mineral support ; cephamycin C ; soybean oil ; Streptomyces ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mineral support was used for cephamycin C production in a culture using soybean oil as the sole carbon source. When the support was added into an oil-water system, the soybean oil was emulsified as fine oil droplets, which was observed by a photomicroscope. Mycelia were also twined around the support, which was observed by a scanning electron microscope. That caused the formation of an oil-mycelia complex on or around the support, which provided a larger specific surface area of oil. When 15 g/L of the support was used in a batch culture of Streptomyces sp. P6621 with 50 g/L of soybean oil, the maximum cephamycin C concentration was 2.8 g/L, which was 2.2 times higher than that without the support. This indicates that the mineral support is useful for the culture system using vegetable oil as a carbon source. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 226-231 
    ISSN: 0006-3592
    Keywords: plant cell culture ; bacitracin ; protease ; peptide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Production of useful peptides using recombinant plant cells is often limited by the stability of the newly translated peptides in the plant expression system. As an initial step toward producing peptides in transformed plant cell cultures, we examined the stability of exogenously added arginine vasopressin (AVP) peptide in a suspension culture of Nicotiana plumbaginifolia cells. The peptide was lost rapidly from the plant cell culture, but the rate of loss was slowed significantly by the addition of the peptide, bacitracin, at a concentration of 150 μg/mL. At higher concentrations, bacitracin substantially inhibited the growth of the plant cells in culture. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 214-219 
    ISSN: 0006-3592
    Keywords: Taxol ; plant cell culture ; continuous production ; immobilization ; Taxus cuspidata ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production characteristics for Taxol (paclitaxel) using free and immobilized cells of Taxus cuspidata were investigated in a perfusion culture bioreactor. Although the cell growth was inhibited by higher dilution rates, the specific production rate of Taxol was increased by perfusion compared with that using batch operation. Perfusion cultures using a nylon-mesh cell separator for free suspension cells showed similar production profiles to those obtained using immobilized cells. Continuous Taxol production was successfully obtained at an approximate specific production rate of 0.3 mg/g DCW (dry cell weight) per day for up to 40 days. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 238-241 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; bacterial motility ; geometrical restriction ; capillary tube ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Motility of Escherichia coli K-12 cells was studied in a capillary tube with an inside diameter of 3 μm, which is comparable to the size of the cells' body. The extreme restriction, imposed by the capillary on the bacterial motility, caused a series of phenomena such as cell aggregation, swimming as a cluster, and break-up of aggregates, which were observable for the first time, and which are reported here. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 232-237 
    ISSN: 0006-3592
    Keywords: supercritical fluid ; rapid expansion ; antisolvent ; biocompatible polymer ; drug delivery system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Three micronization techniques, based on the use of supercritical carbon dioxide, were investigated to produce microspheres of a natural biocompatible polysaccharide. Particles smaller than 20 μm were obtained by means of the rapid expansion of a supercritical solution method (RESS), both with and without cosolvents. The mean diameter of the particles was reduced to about 0.5 μm when a solution of the polymer in an organic solvent was expanded by using carbon dioxide as a supercritical antisolvent (SAS). The SAS process was operated both in a continuous and in a batch mode. The former leads to aggregated structures and fibers, and the latter to the formation of micronic spherical particles. It was found that the experimental temperature did not substantially affect the shape and dimension of the particles. A stronger dependence is shown with respect to the solute concentration in the starting solution. The proposed method is attractive as the basis of a new process for the preparation of drug delivery systems. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 220-225 
    ISSN: 0006-3592
    Keywords: anaerobic processes ; immobilized anaerobic sludge ; intrinsic kinetic parameters ; fixed-bed reactors ; horizontal-flow anaerobic immobilized sludge reactor ; polyurethane foam ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (robs) under a diversity of external mass transfer resistance conditions. The robs curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters rmax, the maximum specific substrate utilization rate, and Ks, the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30°C resulted in intrinsic rmax and Ks, respectively, of 0.330 mg chemical oxygen demand (COD) · mg-1 volatile suspended solids (VSS) · h-1 and 72 mg COD · L-1. In comparison with the values found in the literature, intrinsic rmax is significantly high and intrinsic Ks is relatively low. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 243-252 
    ISSN: 0006-3592
    Keywords: carbon dioxide evolution rate ; mass transfer ; modeling ; biodegradation ; pH ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Respirometry is a precious tool for determining the activity of microbial populations. The measurement of oxygen uptake rate is commonly used but cannot be applied in anoxic or anaerobic conditions or for insoluble substrate. Carbon dioxide production can be measured accurately by gas balance techniques, especially with an on-line infrared analyzer. Unfortunately, in dynamic systems, and hence in the case of short-term batch experiments, chemical and physical transfer limitations for carbon dioxide can be sufficient to make the observed carbon dioxide evolution rate (OCER) deduced from direct gas analysis very different from the biological carbon dioxide evolution rate (CER).To take these transfer phenomena into account and calculate the real CER, a mathematical model based on mass balance equations is proposed. In this work, the chemical equilibrium involving carbon dioxide and the measured pH evolution of the liquid medium are considered. The mass transfer from the liquid to the gas phase is described, and the response time of the analysis system is evaluated.Global mass transfer coefficients (KLa) for carbon dioxide and oxygen are determined and compared to one another, improving the choice of hydrodynamic hypotheses. The equations presented are found to give good predictions of the disturbance of gaseous responses during pH changes.Finally, the mathematical model developed associated with a laboratory-scale reactor, is used successfully to determine the CER in nonstationary conditions, during batch experiments performed with microorganisms coming from an activated sludge system. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 243-252, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 253-258 
    ISSN: 0006-3592
    Keywords: biofilm ; deep biofilm reactor (DBFR) ; kinetics ; linearity ; operational control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Various reported field studies on the performance of biofilm reactors suggest that the linear control of the system is effective for maintaining the consistent treatment efficiency under changing environmental conditions. However, no theoretical basis is available in the literature to substantiate such a claim. In this article, inherent linearity of the biofilm process has been identified along with the conditions under which this linearity exists. Exploiting the linear state of the system, operational criteria for regulating the performance of the biofilm reactors are obtained. The utility and applicability of the developed criteria are numerically demonstrated. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 253-258, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 259-266 
    ISSN: 0006-3592
    Keywords: waste gas ; styrene ; fungi ; biofilter performance ; biofilm ; Exophiala jeanselmei ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A general mathematical model developed for a description of pollutant degradation in a biofilm was used to evaluate the performance of a biofilter for the purification of styrene-containing gas. The biofilter contained perlite as an inert support on which a biofilm was present composed of a mixed microbial population containing the fungus Exophiala jeanselmei as a major styrene-degrading microorganism. Although styrene is a moderately hydrophobic compound, the biofilter was reaction limited at a styrene gas phase concentration of 0.1-2.4 g/m3. Limitation of biofilter performance by the mass transfer of styrene was only observed at styrene concentrations lower than 0.06 g/m3. A maximal styrene degradation rate of 62 g/(m3 · h) was maintaind for over 1 year. At a high styrene concentration, the maximal styrene degradation rate could be increased to 91 g/(m3 · h) by increasing the oxygen concentration in the gas from 20 to 40%. After 300 days of operation, the dry-weight biomass concentration of the filter bed was 41% (w/w), and an average biofilm thickness of 240-280 μm, but maximal up to 600 μm, was observed. Experimental results and model calculations indicated an effective biofilm thickness of about 80 μm. It is postulated that the thickness of the effective biofilm is determined by the oxygen availability in the biofilm. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 259-266, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 267-273 
    ISSN: 0006-3592
    Keywords: liquid emulsion membrane ; reversed micelles ; protein extraction ; α-chymotrypsin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new type of liquid emulsion membrane containing reversed micelles for protein extraction is introduced. A three-step extraction mechanism is proposed including solubilization, transportation, and release of the protein. The surfactants Span80 and sodium di(2-ethylhexyl)sulfosuccinate (AOT) are used to stabilize the membrane phase and to build up the reversed micelles, respectively. α-Chymotrypsin was used as the model protein. The condition in the internal phase inhibits the solubilization process of the already extracted protein back into reversed micelles. Concerning the solubilization, we studied the influence of the AOT concentration in the membrane phase and the ionic strength in the external phase. The extraction rate increases with higher AOT concentration and decreases with higher ionic strength. Using NaCl in the external phase led to better extraction results than using KCl. Maximum extraction results of 98% into the membrane phase and 65% into the internal phase were obtained. This condition retained 60% of the enzyme's activity. The concentration of KCl in the internal phase does not affect the solubilization rate but the release into the internal phase. By this way the ionic strength in the internal phase is used as the driving force for the protein release. The solubilization process is much faster than the diffusion and the releasing process, as found by variation of the extraction time. The influence of the operating conditions on the membrane swelling is also discussed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 267-273, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 290-295 
    ISSN: 0006-3592
    Keywords: yeast lysis ; Monte Carlo simulation ; cell rupture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The overall reaction in the enzymatic lysis of yeast takes place in three major steps: (i) the two-layer wall is digested, (ii) the cell bursts under the osmotic pressure difference to release its intracellular material, and (iii) the intracellular material is digested by the enzymes still present in the solution. The first and third steps are continuous processes, adequately described by Michaelis-Menten kinetic models. The second step is a discrete event, statistical in nature. A model of engineering value should effectively bridge the gap between the two continuous processes (first and third steps). In this work, Monte Carlo simulations are used to identify a suitable function that captures the statistical nature of cell rupture and represents the rate of release of intracellular material. It is shown that the two-parameter beta distribution function serves this purpose most effectively. Comparisons with experimental results indicate that the cell rupture ratio is a widely distributed statistical function. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 290-295, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 296-303 
    ISSN: 0006-3592
    Keywords: biofilters ; microbial ecocystems ; compost ; granular activated carbon ; phospholipid fatty acid analysis ; POTW ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Compost and granular activated carbon biofilters operated at a wastewater treatment plant simultaneously removed low concentrations of hydrogen sulfide and volatile organic compounds. Through the use of phospholipid fatty acid analyses, the effects of declining pH caused by sulfide oxidation were established for microbial growth, microorganism stress, and microbial community structure. Microorganisms on both media demonstrated increases in microbial densities, varying degrees of environmental stress, and domination by gram-negative bacteria. However, the declining pH had little effect on compound removal, which was greater than 99% for the hydrogen sulfide and greater than 70% for the oxygenated and aromatic hydrocarbons. The microbial communities adjusted to difficult environmental conditions through acclimation of the species present or by growth of low-pH-tolerant species. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 296-303, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 283-289 
    ISSN: 0006-3592
    Keywords: regulated secretion ; insulin processing ; insulin secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Certain classes of prohormones and other neuroendocrine or endocrine-derived secretory proteins are post-translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine-derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine-derived cell line βTC-3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T-flask experiments with confluent cultures of βTC-3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversed-phase high-performance liquid chromatography (HPLC). Under conditions of steady-state secretion in glucose-rich growth medium the cells turned over their (pro)insulin inventory (90 ± 5% mature insulin) at 2-3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 μM) and isobutylmethylxanthine (1 mM), ∼80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 ± 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady-state rate with a treatment which attenuated calcium influx (20 μM verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only ∼50% converted to mature insulin, but 85 ± 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long-term and predominantly mature stores. Thus, a cyclic controlled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady-state secretion. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 283-289, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 274-282 
    ISSN: 0006-3592
    Keywords: insulinoma cells ; regulated secretion ; insulin secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Controlled secretion of proteins from endocrine-derived cell lines has been proposed as a means to produce some classes of post-translationally modified proteins in bioreactors. Under the right biological and environmental conditions it may be possible to improve the product purity or quality relative to that obtained through steady (constitutive) secretion. The pancreatic-islet-derived cell line, βTC-3, was selected as a model system to explore the secretory dynamics of insulin under various combinations of stimulatory or inhibitory environmental conditions. The βTC-3 cells exhibited a glucose-mediated stimulus-response pattern which was saturated above 1 mM glucose and with an apparent “Kg” of 0.1 mM glucose. However, the kinetics of insulin synthesis were closely coupled to those of secretion such that βTC-3 cells cycled between saturating and basal levels of glucose were never perturbed far from an intracellular synthesis-secretion equilibrium. When more powerful and selective agents were used to control secretion, the system performance improved markedly. A combination of 1 mM isobytylmethylxanthine (IBMX) and 1 μM carbachol (with saturating levels of glucose) could discharge 75% of stored insulin in 2 h. When this treatment was followed by incubation in media adjusted to attenuate the influx of calcium into the cells, intracellular pools were efficiently replenished within 24 h. Calcium attenuating treatments included hyperpolarization with reduced potassium (1 mM), calcium channel blockade with the dihydropyridine verapamil (1 μM), and the direct mass-action effect of reduced environmental calcium (0.5 mM versus 1.8 mM). Other inhibitory treatments were explored, but these tended to reduce both insulin synthesis and secretion. The best recharging treatment found was a combination of verapamil (1 μM) with reduced calcium level (0.5 mM).To demonstrate the feasibility of a controlled secretion process, βTC-3 T-flask cultures were grown to confluence, then cycled through two periods of discharging (2 h) and recharging (20 h) with the best combinations of secretagogues and calcium attenuators. The overall process was quite efficient: Only 15% of the overall insulin secretion took place during the recharging episodes, and this residual secretion represented only 10% of the net insulin synthesis during these episodes. Discharging was very effective in the first episode (80% recovery of stored insulin), but slightly less efficient in subsequent discharging episodes, possibly due to a desensitization effect of the calcium attenuating media. Nevertheless, the regulated secretory pathway of βTC-3 cells could be successfully harnessed to a controlled secretion process for the selective recovery of stored insulin. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 274-282, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 310-319 
    ISSN: 0006-3592
    Keywords: T. ferrooxidans ; iron oxidation ; energetics ; model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A structured model for Thiobacillus ferrooxidans growth dependence on ferrous and ferric iron, arsenic, oxygen, carbon dioxide, pH, and temperature is presented. A new kinetic mechanism for ferrous oxidation by T. ferrooxidans is introduced. Data from several earlier experimental studies of T. ferroaxidans growth are used for model development. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 310-319, 1997.
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  • 78
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    Biotechnology and Bioengineering 53 (1997), S. 332-338 
    ISSN: 0006-3592
    Keywords: α-hydroxynitrile lyase ; cassava ; cyanogenesis ; cyanohydrin ; Escherichia coli expression vector ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The coding sequence of the cyanogenic α-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid contained the same sequence as the cDNA clone pHNL10. Peptide sequencing of the recombinant protein showed that the N-terminus was heterogeneous, with either four or six additional amino acid residues compared with the native protein. Circular dichroism spectra indicated similar secondary structure contents for both proteins. Enzyme assays showed that specific activity of native and recombinant proteins were 0.24 and 0.26 mmol CN-/mg/min, respectively; that both proteins had optimal activity at 40°C and pH 5.5; and that both proteins were inhibited by the serine protease inhibitor phenyl-methane sulfonyl flouride (PMSF). Isoelectric focusing of native and recombinant protein revealed multiple isoforms for both proteins; the recombinant protein had a more basic mean isoelectric point (pl) (5.1) than the native protein (4.5). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 332-338, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 320-331 
    ISSN: 0006-3592
    Keywords: numerical modeling ; uncertainty ; statistics ; cometabolism ; trichloroethylene ; methanotroph ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The rates of methane utilization and trichloroethylene (TCE) cometabolism by a methanotrophic mixed culture were characterized in batch and pseudo-steady-state studies. Procedures for determination of the rate coefficients and their uncertainties by fitting a numerical model to experimental data are described. The model consisted of a system of differential equations for the rates of Monod kinetics, cell growth on methane and inactivation due to TCE transformation product toxicity, gas/liquid mass transfer of methane and TCE, and the rate of passive losses of TCE. The maximum specific rate of methane utilization (kCH4) was determined by fitting the numerical model to batch experimental data, with the initial concentration of active methane-oxidizing cells (X0a) also used as a model fitting parameter. The best estimate of kCH4 was 2.2 g CH4/g cells-d with excess copper available, with a single-parameter 95% confidence interval of 2.0-2.4 mg/mg-d. The joint 95% confidence region for kCH4 and X0a is presented graphically. The half-velocity coefficient (KS,CH4) was 0.07 mg CH4/L with excess copper available and 0.47 mg CH4/L under copper limitation, with 95% confidence intervals of 0.02-0.11 and 0.35-0.59 mg/L, respectively. Unique values of the TCE rate coefficients kTCE and KS,TCE could not be determined because they were found to be highly correlated in the model fitting analysis. However, the ratio kTCE/KS,TCE and the TCE transformation capacity (TC) were well defined, with values of 0.35 L/mg-day and 0.21 g TCE/g active cells, respectively, for cells transforming TCE in the absence of methane or supplemental formate. The single-parameter 95% confidence intervals for kTCE/KS,TCE and TC were 0.27-0.43 L/mg-d and 0.18-0.24 g TCE/g active cells, respectively. The joint 95% confidence regions for kTCE/KS,TCE and TC are presented graphically. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 320-331, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 339-344 
    ISSN: 0006-3592
    Keywords: cell culture ; tissue engineering ; thermoresponsive polymer ; cell adhesion ; insulin conjugate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 304-309 
    ISSN: 0006-3592
    Keywords: biological phosphorus removal ; activated sludge ; phosphate uptake kinetics ; overplus phenomenon ; luxury uptake ; Acinetobacter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Acinetobacter isolates from activated sludge treatment plants of forest industry were used as model organisms for polyphosphate accumulating bacteria to study excess phosphate uptake by the overplus phenomenon as well as luxury uptake of phosphate during growth. The initial, rapid phosphate uptake by the phosphorus-starved Acinetobacter isolates (the overplus phenomenon) followed the Michaelis-Menten model (maximum initial phosphate uptake rate 29 mg P g-1 dry mass (DM) h-1, half-saturation constant for excess phosphate uptake 17 mg P L-1). During the rapid uptake no growth was observed, but most cells contained polyphosphate granules. Also growth and luxury uptake of phosphate could be modeled with the Michaelis-Menten equation (maximum phosphate uptake rate 3.7-12 mg P g-1 DM h-1, half-saturation constant for growth 0.47-6.0 mg P L-1, maximum specific growth rate 0.15-0.55 h-1). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 304-309, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 345-348 
    ISSN: 0006-3592
    Keywords: fourier transform infrared (FTIR) spectroscopy ; lyophilization ; pKa ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lyophilized proteins exhibit “pH memory,” i.e., their behavior in the solid form corresponds to the pH of the aqueous solution from which they were freeze dried. Herein, we directly tested whether the ionization state is “remembered” by model organic compounds containing various protein functional groups (amino, carboxyl, and phenolic). The fraction of ionized species was quantitated from the infrared spectra of both the aqueous and lyophilized states. The pKa values in the aqueous and lyophilized forms for each compound were found to be quite similar, within 0.3 units from each other. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 345-348, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 363-371 
    ISSN: 0006-3592
    Keywords: biofilm ; autotrophic bacteria ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An autotrophic biofilm has been investigated for over 10 months in a biofilm tube reactor. The objective of this investigation was the verification and improvement of a biofilm model. The use of a Clark-type oxygen microelectrode in situ allowed the determination of the substrate flux in the biofilm. Also, the population dynamics of the autotrophic bacteria could be evaluated by varying the substrate conditions. Simulation of the experimental results showed that the liquid phase of the biofilm decreased with biofilm depth. This could be described by a logistic function. The density of the inert volume fraction was found to be higher than that of the viable bacteria. This was verified in a nonsubstrate phase of 5 weeks. Growth and decay of the autotrophic bacteria could be described by the growth, endogenous respiration, and death processes. Mass transfer coefficients at the bulk/biofilm interface were evaluated. They were found to be one order of magnitude higher than those known from hydrodynamics in tubes without a biofilm. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 363-371, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 351-362 
    ISSN: 0006-3592
    Keywords: Fourier-transform infrared (FTIR) ; excipients ; lyophilization ; organic solvents ; subtilisin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We developed an FTIR (Fourier transform infrared) methodology for quantitatively assessing the secondary structure of proteins suspended in nonaqueous media. This methodology was used to measure the percentages of α-helices and β-sheets of subtilisin Carlsberg, prepared under different conditions, placed in various organic solvents. The title question was addressed with respect to some instances of markedly influencing the subtilisin activity in organic solvents reported in the literature. It is concluded that the mechanism of subtilisin activation by KCl and N-Ac-L-Phe-NH2 present in the aqueous solution of the enzyme prior to lyophilization may be due to their preservation of the secondary structure, otherwise altered by the dehydration. Likewise, subtilisin inactivation in the protein-dissolving solvent DMSO (dimethyl sulfoxide) is likely caused by enzyme denaturation (the loss of both α-helices and β-sheets). On the other hand, some other ligands, as well as protein nondissolving organic solvents, while greatly affecting the subtilisin activity, have little effect on its secondary structure, thus ruling out the causal relationship between the two. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 351-362, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 391-396 
    ISSN: 0006-3592
    Keywords: assay ; cholesterol ; cholesterol oxidase ; free and total cholesterol ; cholesterol esterase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A dynamic method for free and total cholesterol assay based on the oxidation of cholesterol by cholesterol oxidase, and conversion of cholesteryl oleate to cholesterol by cholesterol esterase is discussed in this article. The reaction conditions for total cholesterol assay were a temperature of 310 K and pH of 7.4. For conversion of cholesteryl oleate to cholesterol, the samples were incubated with 0.6 unit/mL of cholesterol esterase and 0.02 g/mL of taurocholate. The determination of initial reaction rates in the oxidation of free cholesterol, which is directly related to the cholesterol concentration, was found to be rapid, reliable, and inexpensive. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 391-396, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 372-378 
    ISSN: 0006-3592
    Keywords: glucose ; lactate ; on-line monitoring ; mammalian cell culture ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: On-line monitoring and control of cell culture fermentation is important for optimal and consistent production of biologicals. In this work, glucose and lactate concentrations are monitored on-line using a commercially available analyzer (Model 2700, Yellow Springs Instruments, Yellow Springs, OH) during batch and perfusion hybridoma cell fermentation. Cell free samples from the reactor are obtained using a 0.45 μm hollow fiber filtering system placed in a circulation loop. The samples were analyzed at specified times and the data are collected on a computer. A process control strategy was developed to control the concentrations of glucose and lactate in a perfusion reactor where the feed rate is adjusted to maintain their concentrations at desired set points. Hybridoma cells (A10G10) were cultivated in a high density perfusion culture where cell density increased from 2 to 14 million cells/mL. During this period the control algorithm successfully adjusted the perfusion rate while maintaining constant glucose and lactate concentrations. Glucose consumption and lactate accumulation rates as well as net lactate yield on glucose were monitored continuously during perfusion culture. These metabolic rates were observed to be independent of cell concentration and were used for the estimation of viable cell density in the reactor. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 372-378, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 387-390 
    ISSN: 0006-3592
    Keywords: enzymatic synthesis ; peptide synthesis ; Vibrio ; protease ; thermolysin ; organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Condensation of Cbz-Asp and PheOMe catalyzed by a neutral protease from Vibrio sp. T1800 (Vimelysin: VLN) was studied. VLN showed a relatively higher catalytic activity of condensation and an apparently larger yield after 3 h or 24 h, in comparison with thermolysin (TLN), especially at lower pH and temperatures.VLN showed higher solvent-tolerance than TLN. TLhe apparent highest yield (25%) was obtained in 30% DMSO by using VLN; under similar conditions, TLN gave only about a half of this value. The rate of the condensation reaction per mole of enzyme (v/[E]o) in DMSO 50% at 37°C and pH 6.5 was 0.16 s-1 for VLN and 0.047 s-1 for TLN. In 30% ethanol VLN showed more than three-fold peptide yield than TLN after 5 h reaction. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 387-390, 1997.
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  • 88
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    Biotechnology and Bioengineering 53 (1997), S. 379-386 
    ISSN: 0006-3592
    Keywords: ppGpp ; recombinant protein synthesis ; translational machinery ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Maintaining a metabolically productive state for recombinant Escherichia coli remains a central problem for a wide variety of growth-dependent biosynthesis. This problem becomes particularly acute under conditions of minimal cell growth such as fed-batch fermentations. In this, we investigated the possibility of manipulating the protein synthesis machinery of E. coli whereby synthesis of foreign proteins might be decoupled from cell growth. In particular, the effects of eliminating intracellular ppGpp on the synthesis of foreign proteins were studied in both batch and fed-batch operations. A significant increase in CAT production was observed from the ppGpp-deficient strain during both exponential and fed-batch phases. The increase in CAT production during exponential growth was accompanied by a simultaneous increase in CAT mRNA levels. Interestingly, CAT production was increased five-fold, while the level of CAT-specific mRNA increased only three-fold. Thus, eliminating intracellular ppGpp appears to have increase the production of recombinant protein by increasing not only the pool sizes of CAT mRNA but also possible alternations in the post-transcriptional processes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 379-386, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 415-426 
    ISSN: 0006-3592
    Keywords: hepatocytes ; cell adhesion ; spheroids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary - spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 409-414 
    ISSN: 0006-3592
    Keywords: acylation ; pea isolate ; plant protein ; torus bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Acetylation, which acts on the amino groups of proteins, allows to increase the solubility and the emulsifying properties of pea isolate. Acetylation by acetic anhydride was carried out in a torus microreactor in semibatch and continuous conditions. The mixing characteristics, obtained by a residence time distribution (RTD) method, are the same in batch and continuous processes. The maximum acetylation degree reached by the torus reactor is higher than with the stirred reactor. Torus reactors are more efficient than stirred ones as shown by a conversion efficiency, defined by the quantity of modified lysine groups by consumed acetic anhydride. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 409-414, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 406-408 
    ISSN: 0006-3592
    Keywords: bioaffinity separation ; reverse micelles ; trypsin-trypsin inhibitor ; nonionic surfactant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Trypsin inhibitor was converted to hydrophobic states by covalently combining cholesteryl groups using an acylation reaction, and was immobilized in reverse micelles composed of a nonionic surfactant. Using this reverse micellar phase containing trypsin inhibitor as an affinity ligand, trypsin was selectively separated with high recoveries from a mixture of several kinds of contaminating proteins by forward and backward extraction. No loss of activity of the recovered trypsin was observed through these operations. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 406-408, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 397-405 
    ISSN: 0006-3592
    Keywords: airlift reactor ; biofilm ; biofilm detachment ; control biofilm formation ; heterotrophic layer ; hydraulic retention time ; nitrification ; oxygen diffusion limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A Biofilm Airlift Suspension (BAS) reactor was operated with nitrifying biofilm growth and heterotrophic suspended growth, simultaneously converting ammonium and acetate. Growth of heterotrophs in suspension decreases the diffusion limitation for the nitrifiers, and enlarges the nitrifying capacity of a biofilm reactor. Neither nitrifiers nor heterotrophs suffer from additional oxygen diffusion limitation when the heterotrophs grow in suspension. Control of the location of heterotrophic growth, either in suspension or in biofilms over the nitrifying biofilms, was possible by manipulation of the hydraulic retention time. A time delay for formation and disappearance of the heterotrophic biofilms of 10 to 15 days was observed. Surprisingly, it was found that in the presence of the heterotrophic layers the maximum specific activity on ammonia of the nitrifying biofilms increased. The reason for the increase in activity is unknown. The effect of heterotrophic biofilm formation on oxygen diffusion limitation for the nitrifiers is discussed. Some phenomena compensating the increased mass transfer resistance due to the growth of a heterotrophic layer are also presented. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 397-405, 1997.
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  • 93
    ISSN: 0006-3592
    Keywords: malate dehydrogenase ; protein chromatography ; Saccharomyces cerevisiae ; direct extraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel technique for protein chromatography has been developed, which can be used to extract proteins from particulate-containing solutions (such as fermentation broths or preparations of disrupted cells) on a continuous basis, and delivers clarified streams of purified product. Adsorbents deployed in this type of contactor are based on PVA-coated perfluorocarbons derivitized with affinity ligands such as triazine dyes. In this article, we describe the application of this equipment for the continuous purification of malate dehydrogenase from an unclarified homogenate of Saccharomyces cerevisiae, using a Procion Red HE-7B-derivitized adsorbent. Although operating conditions were not optimized to produce a product of maximized purification factor, concentration, and yield, we have shown that MDH can be purified continuously in 78% yield at a rate of 70 U/min, with a purification factor of approximately 10. This corresponds to specific productivity of approximately 0.35 U/min per milliliter of settled adsorbent, a higher specific productivity than was feasible with the same adsorbent using expanded bed adsorption (EBA). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 427-441, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 470-477 
    ISSN: 0006-3592
    Keywords: fluidized bed bioreactor ; recombinant ; yeast ; kinetics ; modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) by Saccharomyces cerevisiae strain XV2181 (a/a, Trp 1) containing plasmid pαADH2 and immobilized on porous glass beads in a fluidized bed bioreactor was studied. Kinetic models for plasmid stability, cell growth, and protein production in the three-phase fluidized bed bioreactor were developed and used to study the effects of solid loading or cell immobilization on plasmid stability and recombinant protein production. With increasing cell immobilization or solid loading in the bioreactor, plasmid stability and protein production improved significantly. The improvements could be attributed to the decreased θ value, which is the plasmid loss probability during cell division and is an indication of segregational instability of the recombinant cell, and the increased α value, which is the ratio of the specific growth rate of a plasmid-carrying cell to that of a plasmid-free cell and is indicative of competitive stability of the recombinant cell culture. θ decreased from 0.552 to 0.042 and α increased from 0.351 to 0.991 when solid loading in the bioreactor was increased from 5% (v/v) to 33%. The model simulation also showed that the specific growth rate of cells in the bioreactor was lower at higher solid loading. This indicated that there was significant mass transfer limitation, particularly for oxygen transfer, when the total cell density in the bioreactor was high at high solid loading. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 470-477, 1997.
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  • 95
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    Biotechnology and Bioengineering 53 (1997), S. 453-458 
    ISSN: 0006-3592
    Keywords: chemical permeabilization ; cell disruption ; urea ; EDTA ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetet-raacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 453-458, 1997.
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  • 96
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    Biotechnology and Bioengineering 53 (1997), S. 443-452 
    ISSN: 0006-3592
    Keywords: fermentation ; database mining ; pattern recognition ; dbminer© ; decision trees ; wavelets ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A large volume of data is routinely collected during the course of typical fermentation and other processes. Such data provide the required basis for process documentation and occasionally are also used for process analysis and improvement. The information density of these data is often low, and automatic condensing, analysis, and interpretation (“database mining”) are highly desirable. In this article we present a methodology whereby process variables are processed to create a database of derivative process quantities representative of the global patterns, intermediate trends, and local characteristics of the process. A powerful search algorithm subsequently attempts to extract the specific process variables and their particular attributes that uniquely characterize a class of process outcomes such as high- or low-yield fermentations.The basic components of our pattern recognition methodology are described along with applications to the analysis of two sets of data from industrial fermentations. Results indicate that truly discriminating variables do exist in typical fermentation data and they can be useful in identifying the causes or symptoms of different process outcomes. The methodology has been implemented in a user-friendly software, named db-miner, which facilitates the application of the methodology for efficient and speedy analysis of fermentation process data. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 443-452, 1997.
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  • 97
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    Biotechnology and Bioengineering 53 (1997), S. 478-486 
    ISSN: 0006-3592
    Keywords: Thiobacillus ferrooxidans ; kinetic model ; biological oxidation ; ferrous iron ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of bacterial oxidation of ferrous iron in the presence of Thiobacillus ferrooxidans cells were studied using an initial-rate method. Measurements of the redox potential of the solution during the oxidation of ferrous iron were used to assess the initial rate of the reaction. Effects on the rate of reaction were determined for ferrous iron concentration in the range 0.25 to 30 kg m-3, bacterial concentration in the range 3.25 × 107 to 4.47 × 108 cells mL-1, and temperature in the range 20 to 35°C. Using these experimental results and an approach based on Michaelis-Menten kinetics, a model for biological oxidation of ferrous iron was developed. The model, which incorporates terms for the effect of temperature and substrate and cell inhibition, was successfully used to simulate the full range of experimental data obtained. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 478-486, 1997.
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  • 98
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    Biotechnology and Bioengineering 53 (1997), S. 459-469 
    ISSN: 0006-3592
    Keywords: population dynamics ; biofilms ; fluorescent microbeads ; confocal scanning laser microscope (CSLM) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inert fluorescent microparticles were used as tracers to investigate the dynamics of spatial distribution of particulate components in mixed population biofilms. The tracer bead spatial distributions in the biofilm were experimentally measured by sectioning the biofilms with a microslicer. The experimental results were compared with model simulations using the biofilm model (BIOSIM) to evaluate the assumption that advective transport (displacement) of particulates balances with cell growth in the model. The tracer beads could traverse throughout a biofilm 360 μm thick within less than 23 minutes, which cannot be explained solely by their attachment to the surface followed by molecular diffusion. Advective transport of the tracer beads via “voids and pores” could be responsible for such rapid bead penetration. Observation by confocal scanning laser microscopy (CSLM) clearly showed that the biofilm consisted of a thick loose surface layer, varying in thickness, and a semicontiguous base layer separated by water channels. About 80% of attached tracer beads remained in the biofilm for over 20 days. The trapped tracer beads were gradually transferred from the depth of the biofilm to the surface. The observed bead release rate was much slower than the model predictions. This is probably because the cell density increased predominantly near the substratum, resulting in an unbalance of advective transport of the tracer beads and cell growth. The pores, voids, and cell-free spaces in the biofilm were first filled with growing biomass, thereafter, displacement of the beads took place once the cell density reached certain levels. The model assumptions of the temporal and spatial constant cell density and the continuum concept (flat biomass) are clearly oversimplified and should be revised. It was concluded that the dynamics of the inert microbeads in the biofilm was strongly influenced by not only microbial growth, but also by the biofilm structure and growth pattern. Therefore, one dimensional modeling is not adequate for the accurate description of the transport of particulates in a biofilm. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 459-469, 1997.
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  • 99
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    Biotechnology and Bioengineering 53 (1997), S. 497-506 
    ISSN: 0006-3592
    Keywords: immobilized enzymes ; polyacrylamide gels ; experimental design ; response surface methodology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed a methodology based on experimental design, to optimize a polyacrylamide gel as the support for enzyme immobilization, taking advantage of all the properties which this type of gel has. Monomer and crosslinking agent proportions are responsible for both the porous structure and pore size of the gel. A correct selection of those variables and suitable synthesis conditions leads to an increase in the activity retained by the gel. The path of steepest ascent method was used to obtain the relative maximum activity. The maximum retained activity was chosen with a central composite design in terms of the gel composition. The retained activity in the network, loss activity in the wash water, and loss activity due to steric impediment or blockage was modeled in terms of the variables responsible for the gel structure. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 497-506, 1997.
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  • 100
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 487-496 
    ISSN: 0006-3592
    Keywords: bacterial chemotaxis ; Escherichia coli ; random motility ; mathematical model ; sand core ; porous media ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The migration of chemotactic bacteria in liquid media has previously been characterized in terms of two fundamental transport coefficients - the random motility coefficient and the chemotactic sensitivity coefficient. For modeling migration in porous media, we have shown that these coefficients which appear in macroscopic balance equations can be replaced by effective values that reflect the impact of the porous media on the swimming behavior of individual bacteria. Explicit relationships between values of the coefficients in porous and liquid media were derived. This type of quantitative analysis of bacterial migration is necessary for predicting bacterial population distributions in subsurface environments for applications such as in situ bioremediation in which bacteria respond chemotactically to the pollutants that they degrade.We analyzed bacterial penetration times through sand columns from two different experimental studies reported in the literature within the context of our mathematical model to evaluate the effective transport coefficients. Our results indicated that the presence of the porous medium reduced the random motility of the bacterial population by a factor comparable to the theoretical prediction. We were unable to determine the effect of the porous medium on the chemotactic sensitivity coefficient because no chemotactic response was observed in the experimental studies. However, the mathematical model was instrumental in developing a plausible explanation for why no chemotactic response was observed. The chemical gradients may have been too shallow over most of the sand core to elicit a measurable response. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 487-496, 1997.
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