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Kinetics of product inhibition in alcohol fermentation. Reprinted from Biotechnology and Bioengineering, Vol. X, Issue 6, Pages 845-864 (1968).

The inhibitory effect of ethanol concentration p in a medium on the specific rates of growth mu and ethanol production nu of a specific strain of baker's yeast was studied in a chemostat, where except for ethanol as the product, only the concentration of glucose S was controlled to limit the metabolic activity of the yeast. This was designed to supplement the previous findings from the batch experiment, in which ethanol was added artificially and no substrate components were limiting the metabolism of the same yeast, that mu = mu(0)e(-k(1)p) and nu = nu(0)e(-k(1)p), where k(1) and k(2) are empirical constants and subscript the 0 denotes respective values at p = 0. The effects of p on the values of mu and nu were confirmed by the Line-weaver-Burk plot to belong to noncompetitive inhibition. The formulas here for mu and nu as affected by p, if extrapolated to the case of no limiting substrates, were in good agreement in respective forms with those derived previously from the batch experiment, though the values of corresponding coefficients in these formulas were different. The differential equations for mu and nu as functions of both p and S and, in addition for the rate of glucose consumption as correlated by the yield factors either with the cell growth rate or the rate of ethanol production, were solved properly with a digital computer. A kinetic pattern calculated so far was discussed with reference to the data obtained in the batch experiment and those relevant to actual "sake" brewing.

Culture Media↗

Measurement of heat evolution and correlation with oxygen consumption during microbial growth. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 3, Pages 269-281 (1968).

A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent on the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.

Aspergillus niger↗

Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 6, Pages 1101-1110 (1969).

Streptomyces peucetius var. caesius, obtained from S. peucetius, the daunomycin producing microorganism, by mutagenic treatment, differs from the parent culture by the color of the vegetative and aerial mycelia and by its antibiotic producing ability. S. peucetius var. caesius accumulates adriamycin in submerged and aerated culture on a medium containing glucose, brewer's yeast, and inorganic salts both in shake flasks and in stirred fermenters. Isolation of the product is performed by solvent extraction, chromatography on buffered cellulose columns, and crystallization as the hydrochloride. The new antitumor agent, adriamycin, is the 14-hydroxy derivative of daunomycin.

Antibiotics, Antineoplastic↗

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 3, Pages 387-397 (1977).

Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was demonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.

Alginates↗

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Reprinted from Biotechnology and Bioengineering, Vol. XXIII, No. 12, Pages 2789-2802 (1981).

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10(4) [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L-leucine dehydrogenase (LEUDH) was used as catalyst for the reductive amination of alpha-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L-leucine. Formate dehydrogenase (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L-leucine. The kinetic constants V(max) and k(m) of the enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L-leucine was produced continuously out of alpha-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).

Bioreactors↗

Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models. Reprinted from Biotechnology and Bioengineering, Vol. 26, Issue 1, Pages 66-73 (1984).

The influence of growth rate on Escherichia coli plasmid content and expression of a cloned-gene product has been described by a mathematical model based upon the molecular mechanism of lambdadv plasmid replication and known relationships between growth rate and transcription and translation activities of the host cell. The model simulates correctly decreases in plasmid content with increasing growth rate as observed experimentally for pBR322, NR1, R1, and Col E1 plasmids. A maximum with respect to growth rate in intracellular product accumulation is indicated by the model, as is a transient overshoot in product concentration following a shift from smaller to larger growth rate. Available data, although very limited, show the same trends. These results, obtained without parameter or kinetic form adjustments or manipulation, clearly illustrate the advantages of kinetic descriptions of recombinant systems based upon the pertinent molecular mechanisms.

Escherichia coli↗

Computer model for glucose-limited growth of a single cell of Escherichia coli B/r-A. Reprinted from Biotechnology and Bioengineering, Vol. 26, Issue 3, Pp 203-216 (1984).

A computer model is described which is capable of predicting changes in cell composition, cell size, cell shape, and the timing of chromosome synthesis in response to changes in external glucose limitation. The model is constructed primarily from information on unrestricted growth in glucose minimal medium. The ability of the model to make reasonable quantitative predictions under glucose-limitation is a test of the plausibility of the basic biochemical mechanisms included in the model. Such a model should be of use in differentiating among competing hypotheses for biological mechanisms and in suggesting as yet unobserved phenomena. The last two points are illustrated with the testing of a mechanism for the control of the initiation of DNA synthesis and predictions on cell-width variations during the division cycle.

Computer Simulation↗

Bioengineering of elastic cartilage with aggregated porcine and human auricular chondrocytes and hydrogels containing alginate, collagen, and kappa-elastin.

Transplantation of isolated chondrocytes has long been acknowledged as a potential method for rebuilding small defects in damaged or deformed cartilages. Recent advances in tissue engineering permit us to focus on production of larger amounts of cartilaginous tissue, such as might be needed for reconstructive surgery of the entire auricle. In this report we describe modification of the basic techniques that lead to production of a large amount of elastic cartilage originated from porcine and human isolated chondrocytes. Small fragments of auricular cartilage were harvested from children undergoing ear reconstruction for microtia or extirpation of preauricular tags and from ears of juvenile pigs. Enzymatically isolated elastic chondrocytes were then agitated in suspension to form the chondronlike aggregates, which were further embedded in molded hydrogel constructs made of alginate and type I collagen augmented with kappa-elastin. The constructs were then implanted in nude mice and harvested 4 and 12 weeks after heterotransplantation. The resulting neocartilage closely resembled native auricular cartilage at the gross, microscopic, and ultrastructural levels. Immunohistochemistry and electron microscopy additionally confirmed that the newly produced cartilage contained the major components of the elastic cartilage-specific matrix, including collagen type II, proteoglycans, and well-assembled elastic fibers.

Alginates↗

Bioengineering nitrogen acquisition in rice: can novel initiatives in rice genomics and physiology contribute to global food security?

Rice is the most important crop species on earth, providing staple food for 70% of the world's human population. Over the past four decades, successes in classical breeding, fertilization, pest control, irrigation and expansion of arable land have massively increased global rice production, enabling crop scientists and farmers to stave off anticipated famines. If current projections for human population growth are correct, however, present rice yields will be insufficient within a few years. Rice yields will have to increase by an estimated 60% in the next 30 years, or global food security will be in danger. The classical methods of previous green revolutions alone will probably not be able to meet this challenge, without being coupled to recombinant DNA technology. Here, we focus on the promise of these modern technologies in the area of nitrogen acquisition in rice, recognizing that nitrogen deficiency compromises the realization of rice yield potential in the field more than any other single factor. We summarize rice-specific advances in four key areas of research: (1). nitrogen fixation, (2). primary nitrogen acquisition, (3). manipulations of internal nitrogen metabolism, and (4). interactions between nitrogen and photosynthesis. We develop a model for future plant breeding possibilities, pointing out the importance of coming to terms with the complex interactions among the physiological components under manipulation, in the context of ensuring proper targeting of intellectual and financial resources in this crucial area of research.

Biotechnology↗

Bioengineering and characterization of DNA-protein assemblies floating on supported membranes.

A biodevice involving thiolated ssDNA and engineered cytochrome b5 linked through a cis-platine bridge is described. This original nanostructure is associated to a supported membrane through a floating anchor, thus constituting a dynamic bidimensionnal DNA capture device contrasting with the constrained geometry of currently available DNA chips. Characterization by optical spectroscopy, surface plasmon resonance and gel electrophoresis demonstrated that an unique molecular supra-assembly featuring specific DNA recognition capability has been obtained. This device is characterized by the reversibility of its assembly, self-organization and fluidity properties and is of interest as a prototype to design new generations of DNA chip biosensors.

Base Sequence↗

A biologically active VEGF construct in vitro: implications for bioengineering-improved prosthetic vascular grafts.

Prosthetic arterial grafts are unable to develop an intact endothelial lining after implantation, predisposing them to fail. Strategies have been sought to enhance endothelialization using growth factors and cytokines. This study assessed the biologic activity of vascular endothelial growth factor (VEGF) covalently linked to bovine serum albumin (BSA). Native and modified VEGF were assayed for endothelial cell migration and proliferation. Migration assays were performed comparing the effects of 2% fetal bovine serum (FBS), 50 ng/mL, 100 ng/mL, and 200 ng/mL of native VEGF and VEGF-BSA. Proliferation assays were performed by using Alamar Blue comparing cellular growth in 1% FBS, 10% FBS, 100 ng/mL unbound VEGF, and 100 ng/mL VEGF-BSA. VEGF is a potent chemotactic agent for endothelial cells in both unbound and bound states. Native VEGF solutions (50 ng/mL, 100 ng/mL, and 200 ng/mL) stimulated 23.9 cells/high power field (HPF), 35.3 cells/HPF, and 49.1 cells/HPF (p < 0.005). VEGF-BSA solutions stimulated 25.9 cells/HPF, 39.1 cells/HPF, and 69.0 cells/HPF (p < 0.001). VEGF-BSA and native VEGF supported similar increased cellular proliferation compared with 1% FBS media (p < 0.002). Modified VEGF retains its chemotactic and proliferative properties in vitro. These findings suggest that bare prosthetic surfaces lined with VEGF bound to a "basecoat" albumin may support endothelial cell proliferation and migration and thereby offer new strategies to improve graft patency.

Blood Vessel Prosthesis↗