Analysis of bifurcations in continuous fermentation using recombinant microorganisms with delayed responses

When the feed rate to a fermenter is varied periodically in order to favor the growth of plasmid-containing cells, a transition may occur from the starting stationary state to another state. The resulting state may be constant or oscillatory. A generalised model based on the adaption times of plasmid-free and plasmid-harboring cells has been used. Analytical conditions have been derived for bifurcation from one nonoscillatory state to another or to an oscillatory state (Hopf bifurcation). The frequency of oscillation is shown to have an upper bound, which can be controlled by manipulating certain process parameters. The production of tryptophan synthetase by the plasmid pPLc23trpAl in E. coli is used as an example to determine the nature of the Hopf bifurcations.     

Dependence of process variables on fermentation parameters during start-up of a continuous flow reactor with recombinant microorganisms

A mathematical model is proposed to describe how the parametric dependences (sensitivities) of key process variables in a continuous flow fermenter using recombinant microorganisms vary with time. Solution of the model, with typical values for the parameters, indicates that most sensitivities increase several fold in about 20 hours. But the sensitivities of the product concentration either vanish or remain constant at low values. The presence of some plasmid-free cells when the fermentation begins does not seem to affect the sensitivities.     

Protectants used in the cryopreservation of microorganisms

The cryoprotective additives (CPAs) used in the frozen storage of microorganisms (viruses, bacteria, fungi, algae, and protozoa) include a variety of simple and more complex chemical compounds, but only a few of them have been used widely and with satisfactory results: these include dimethylsulfoxide (Me2SO), glycerol, blood serum or serum albumin, skimmed milk, peptone, yeast extract, saccharose, glucose, methanol, polyvinylpyrrolidone (PVP), sorbitol, and malt extract. Pairwise comparisons of the cryoprotective activity of the more common CPAs used in cryomicrobiology, based on published experimental reports, indicate that the most successful CPAs have been Me2SO, methanol, ethylene glycol, propylene glycol, and serum or serum albumin, while glycerol, polyethylene glycol, PVP, and sucrose are less successful, and other sugars, dextran, hydroxyethyl starch, sorbitol, and milk are the least effective. However, diols (as well as some other CPAs) are toxic for many microbes. Me2SO might be regarded as the most universally useful CPA, although certain other CPAs can sometimes yield better recoveries with particular organisms. The best CPA, or combination of CPAs, and the optimum concentration for a particular cryosensitive microorganism has to be determined empirically. This review aims to provide a summary of the main experimental findings with a wide range of additives and organisms. A brief discussion of mechanisms of CPA action is also included.     

Production of resveratrol in recombinant microorganisms

Resveratrol production in Saccharomyces cerevisiae was compared to that in Escherichia coli. In both systems, 4-coumarate:coenzyme A ligase from tobacco and stilbene synthase from grapes were expressed. When p-coumaric acid was used as the precursor, resveratrol accumulations in the culture medium were observed to be comparable in E. coli (16 mg/liter) and yeast (6 mg/liter).     

In situ fermentation monitoring with recombinant firefly luciferase

A novel method is described for the on-line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 10(6) cells/mL, or an OD(600) of 0.004, are easily detectable and concentrations greater than 10(10) cells/mL are well within range. The data are collected by a computer, so adaptation to on-line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density. (c) 1993 John Wiley & Sons, Inc.     

Biotests for mineral waters with natural and recombinant luminescent microorganisms

We have developed methods of biotesting mineral waters involving use of natural or recombinant luminescent strains with elimination of the effect of salt concentration and pH. To overcome the adverse effect of high salt concentrations, disguising the action of chemical pollutants, a special method of mineral water sample preparation is proposed. In this method, the marine luminescent bacterium Photobacterium phosphoreum (Microbiosensor B17 677f) is used as a test object. Samples to be analyzed are supplemented with NaCl depending on their natural salt concentration to adjust it to 3 g/l. Another approach, more universal and efficient, involves pH adjustment in the samples to 7.5. This value is suitable for application of both Microbiosensor B17 677f and the recombinant Escherichia coli strain harboring the cloned lux operon of P. leiognathi (Ecolum 9). It has been shown that this treatment, retaining the natural luminescence level of the bacterial biosensors, allows bioluminescent detection of exogenous pollutants added to the samples, including benzene and Cr(VI).     

Antioxidant stabilization of the antifoaming agents used in the fermentation process

Oils used as antifoaming agents in fermentation are subjected to oxidation to form various products, such as aldehydes, carboxylic acids, peroxides, etc. The negative effect of such products on biosynthesis of chlortetracycline by strains VUA 84 and Iz 22/VUPL of Str. aureofaciens was recorded. The level of the oil oxidation was characterized by the contents of peroxides and epihydrinaldehydes. The Kreiss qualitative test was modified for quantitative estimation of epihydrinalydehydes. The oil antifoaming agents characterized by the routine parameters used in determination of oil quality, such as acid number, saponification number and iodine number. In the control experiments lard and soybeen oil were oxidized by air at a temperature of 100 degrees C without an antioxidant (2,6-ditetrabutyl-4-methylphenol). The oil antifoaming agents such as soybeen oil and lard were tested in biosynthesis of chlortetracycline carried out in flasks and pilot plant 750 1 apparatus. Antioxidant 4K, an alkylphenol antioxidant, in a 2% concentration proved to be effective for prevention of oxidation and nontoxic for the culture.     

Factors influencing productivity of fermentations employing recombinant microorganisms

Advances in molecular biology and the exploitation of recombinant DNA procedures for the expression of heterologous genes in microbial hosts offer an opportunity to develop fermentation processes more scientifically and less empirically than has been feasible previously. Nevertheless, the highly interdisciplinary nature of the task makes this a complex undertaking. The relationships between microbial physiology and plasmid copy number, plasmid stability, and gene expression are not well documented for host/vector systems and appear to be highly system specific. In an effort to account for these complex biological interactions which can exceed our intuitive capabilities, mathematical models have been designed to aid in process analysis and development activities. Fermentation strategies based on certain physiological assumptions are beginning to be reported for recombinant systems. Representative efforts in these areas indicate the broad scope of considerations involved in developing fermentation processes for recombinant microorganisms and the opportunities for further research in this field.     

Metabolic approaches for the optimisation of recombinant fermentation processes

The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number. Received: 30 April 1999 / Received revision: 26 July 1999 / Accepted: 1 August 1999     

Physiological bases of oligotrophy of microorganisms and the concept of microbial community

Three groups of physiological processes in microorganisms are considered the physiological basis of oligotrophy: the greater substrate affinity of the oligotrophs' transport systems, efficient or “economical” metabolism, and existence of a “master reaction” or “rate-determining steps” controlling the rate of metabolism. Heterotrophic microorganisms are divided into three unequal groups according to “reaction norma.” Two groups representing the extremes are small groups with the “narrow” reaction norma, regarding the concentrations and structure of the assimilated organic compounds and variability limits of the physiological characteristics mentioned above. The third, intermediate group includes the majority of microorganisms with the “wide” reaction norma.     

Dispersion-induced behavior in sub-critical operation of a recombinant fed-batch fermentation with run-away plasmids

Fermentations with recombinant bacteria containing run-away plasmids are typically operated alternately above and below a critical temperature. To minimize the risks of run away reactions, it is preferable to keep the high temperature periods as short as possible. In this study the possibility of sustained low temperature (sub-critical) operation in a suitably non-homogeneous broth is analyzed. Fluid dispersion is used as a measure of non-homogeneity. The fed-batch production of β-galactosidase by Escherichia coli containing the plasmid pOU140 and operated below 37 ^°C is analysed as a model system. To characterize non-homogeneity, an earlier model visualizing the broth as a set of two reactors with internal recycle has been modified for fed-batch fermentation. Three dilution rates, two internal and one external, quantify fluid dispersion. While plasmid replication and fermentation become quenched in sub-critical operation in a well-mixed reactor, with finite dispersion there may be an increase in the concentration of plasmid-containing cells and the recombinant protein. The concentration profiles many also have one or more peaks in the time domain. Thus, sustained fermentation with run-away plasmids appears feasible in a bioreactor with controlled non-homogeneity.     

Production of recombinant protein in Pichia pastoris by fermentation

This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.     

Benzoate fermentation by the anaerobic bacterium Syntrophus aciditrophicus in the absence of hydrogen-using microorganisms.

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (DeltaG') of -9.2 kJ/mol was reached (-4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO(2), and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than -20 kJ/mol, the postulated minimum free energy value for substrate metabolism.     

Toxicity of organic solvents used in situ in microbial fermentation

Summary Toxicity of organic solvents for in situ solvent extraction of carboxylic acids from aqueous solutions was studied on bacteria Bacillus subtillis and Acetobacter spp., yeasts Kluyveromyces marxianus and Pichia fermentans and fungi Aspergillus terreus and Rhizopus arrhizus. It has been found that trialkylamine and tributylphosphate are much more toxic than other carriers as trialkylphosphineoxide, triisobutylphosphinesulfide and trihexylphosphate. For R. arrhizus trihexylphosphate was found to be the less toxic solvent.     

Neural simulation of an unsteady state continuous recombinant fermentation with imperfect mixing

Summary A continuous fermentation based on a recombinant Escherichia coli strain producing tryptophan synthetase has been simulated by a back-propagation neural network. Data for the network were generated through known kinetics applied to a reactor model with an adjustable degree of macromixing of the broth. A network with just one hidden layer performed satisfactorily for both poor and good macromixing. The best performance was at an intermediate level of mixing, in the region of maximum productivity of the recombinant protein.     

Simple sets for digital microphotography used and tested in the study of microorganisms

Four simple sets for digital microphotography are described that have been tested with the Carl Zeiss Jena, Meopta Prague, Lambda Prague, and LOMO Sankt Petersburg microscopes and with DSLR Nikon D 70 and Nikon D 300 cameras. They permit precise image focusing in the camera using a prism Zeiss. The sets make use of commonly available extensions Zeiss, Praktica and reductions Nikon??Praktica manufactured by ROWI (without a lens) or HAMA (with a lens). An extension has further been designed and manufactured for connecting the DSLR Nikon D 300 camera fitted with the HAMA reduction (only with a lens) and a focusing extensible prism with Zeiss Jena light measurement. It permits a precise image focusing of low light intensity objects (autofluorescence or low-contrast or moving objects when using positive or negative phase contrast). The sets are applicable to all microscopes constructed according to German DIN industrial standards.