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The stirred and aerated tank fermentor is still the most important type of bioreactor for industrial production processes. The agitator or agitators are required to perform a wide range of functions: adequate momentum, heat and mass transfer, and mixing as well as gas-dispersion and homogenization of suspensions. As these various operations make different demands, optimization of the individual tasks would result in different design of the impellers. Therefore, agitators used in practice always reflect compromises. Conventional impellers used in fermentation are typically classified into axial and radial flow impellers. Examples of the two groups are illustrated in Fig. 7.1. Of the many impeller geometries, the six-blade disk impeller (Rushton turbine) with gas sparging below the impeller is most often used in standard configurations. However, an interest in using high-flow, low-power-number agitators such as Intermig (Fig. 7.2a), Lightnin A315 (Fig. 7.2b), Prochem Maxflow T (Fig. 7.2c), and Scaba 6SRGT (Fig. 7.2d) has been developed and improved performance of fermentation processes has been reported with such alternative designs [1-3].

To investigate the intracellular concentrations of adenosine phosphates in Escherichia coli, especially during bioreactor cultivations, a method that enables reproducible determination of adenosine phosphates in culture solutions containing at least 0.25 g dry cell weight/L has been developed. The detection limits of AMP, ADP, and ATP were found to be as low as 1 pmol. The method involves fast sampling, instantaneous inactivation of cell metabolism, extraction of nucleotides, and quantitative analysis by ion-pair reversed-phase HPLC.

Während des Zweiten Weltkrieges hat die Produktion und Verarbeitung von 0,8 Millionen Tonnen Trinitrotoluol (TNT) im damaligen Deutschen Reich an vielen Rüstungsstandorten zu Bodenkontaminationen geführt, die heute die Trinkwasserressourcen anliegender Gemeinden gefährden. Bei der Suche nach geeigneten Sanierungsverfahren wurden in den vergangenen Jahren auch mikrobiologische Verfahren geprüft. Dabei wurde eine vollständige Verwertung des TNT als Kohlenstoff- oder Energiequelle bislang nicht nachgewiesen. Bei Zugabe eines leicht verfügbaren Substrates wie Glucose sind aber viele Mikroorganismen in der Lage, die Nitrogruppen cometabolisch über Nitroso- und Hydroxylamino- zu Aminogruppen zu reduzieren.

The nonaqueous lipase-catalyzed kinetic resolution of racemic 2-methyl-1-pentanol in a continuously operated fixed bed reactor was theoretically and experimentally studied. A 2-dimensional mathematical model was developed to predict the performance of the heterogeneous enantioselective biotransformation and to optimize the productivity and effective stereoselectivity of the process by taking pore diffusion, solid(SINGLEBOND)liquid mass transfer, convection, and axial dispersion into account. Experimental investigations were conducted with lipase from Pseudomonas sp. immobilized by ionic binding in the pores of the anion exchange resin Duolite A 568. As determined in prior initial rate experiments, the specific activities of the immobilized lipase were maximal at a water activity of approximately a(w) = 0.67 and revealed a significant dependence on the amount of enzyme bound to the carrier material, with enantiomeric ratios slightly increasing with increasing water activities. Continuous resolution processes were carried out at a wide range of enzyme loadings. By controlled immobilization according to the theoretically evaluated optimal enzyme loading, the continuous racemic resolution could be optimized to obtain (R)-2-methyl-1-pentanol at high productivity and enantiopurity (ee > 95%). The steady-state characteristics of the system could be generally predicted by the model, despite the necessity to reevaluate the kinetic properties of the immobilized lipase to account for the complex non-aqueous microkinetics in a heterogeneous environment. Further model extension introducing competitive inhibition of free water in solutions as well as water diffusion and adsorption to the biocatalyst were useful in providing a more accurate description of the experimental results. (c) 1996 John Wiley & Sons, Inc.

We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells. The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection. This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection. The cellfree based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins). The infection is abolished when RNase A or trypsin treatment is included before addition of cells. Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity. These findings suggest the formation of ‘minimal infectious units’, simple complexes of MS2 RNA and maturation protein. Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system. A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B12. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.

TNT is found as a major soil contaminant at the sites of armament plants from World War I and II. While no complete biological mineralization of TNT has yet been found, many bacteria can co-metabolically reduce the nitro groups of TNT to amino groups during anaerobic fermentation of readily available substrates. In a soil slurry, the final reduction product (TAT) irreversibly reacts with soil components. The biological treatment of TNT-contaminated soil with complete reduction of TNT to TAT and immobilization of TAT is a potential remediation process that is currently being tested at a technical scale. The large-scale experiment is accompanied by investigations on the immobilization of TAT and the toxicity of the treated soil.

The aim of this contribution is to discuss some new aspects in the design of structured models for stirred tank bioreactors. The modelling strategy starts from a simple multiphase compartment model, which is based on an aggregation of well-mixed multiphase compartiments including mass transfer between the phases. In order to incorporate the available information of intrinsic fluid dynamics it is necessary to put finite volume elements in the place of these compartments. The fluxes from and into those elements can be estimated from the real fluid dynamics. Numerical solutions of the set of momentum equations for gas and liquid phase are shown along with application examples for a biological model (oxygen sensitive culture of Bacillus subtilis) and a physico-chemical model for pH-calculation.

Although research on the development of on-line HPLC systems has been conducted for many years, there is still no satisfactory solution available in the area of fully automatic sampling and sample preparation during fermentation processes. Sample preparation, which is often complicated, is of great importance for a successful application. A particular problem, for example, is still the reliable and robust dilution of the sample. The proposed system fulfills the requirement of fully automatic sampling and sample preparation by use of the biofiltrator and an autosampler with microrobotic-system. In addition to applications for on-line-HPLC, the same configuration can be applied to on-line FIA (Flow Injection Analysis). This double function leads to a new name: High-Pressure-Injection Analysis (HPIA). The selection of the individual components was largely based on convenient use and industrial suitability. Because of the modular structure of the system, it is possible to include other components at any time. Furthermore, it is even possible to use this application in other areas such as environmental analysis and process chromatography.

CD95 is a plasma membrane receptor that can mediate apoptosis after activation by its specific ligand CD95L. Upon stimulation the death receptor recruits various proteins that form the so-called death inducing complex (DISC). The DISC contains the protease procaspase-8 that becomes active by dimerization and self-processing. This process is thought to require high local concentration, that can be achieved by CD95 and DISC clustering. Several mechanisms have been proposed to achieve this clustering, including CD95 self-association, lipid raft localization and oligomerization of the DISC protein FADD.Our goal is to understand how DISC clusters correlate with CD95 spatial organization on the membrane and more generally how this organization can regulate cell sensitivity to CD95-induced apoptosis.We visualize the distribution of CD95 by immunofluorescence and GFP fusion, using confocal microscopy, stimulated emission depletion microscopy and total internal reflection microscopy. As this distribution appears as a dotty pattern (fig.), we determine the dot density in different cell types. We investigate the influence of system perturbations such as cholesterol depletion and actin depolymerization on the CD95 distribution and on apoptosis sensitivity. We also investigate the dependence of clustering on the receptor amount.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Thiobacillus ferrooxidans could be used to regenerate ferrous sulphate solution produced in a process to remove H2S from waste gas if the reaction rate could be increased. The aim of the present study was to increase the volumetric productivity by using immobilized cells. Kinetic data of ferrous iron oxidation were determined in fixed-bed and fluidized-bed reactor configurations with different support materials in order to find the most practical system for scale-up. By using a fixed-bed reactor the iron oxidation rate can be increased to 3.6 g l−1 h−1, fivefold higher than suspended cells, and results in a bioreactor of reasonable size. With the kinetic data obtained, the biological reaction is no longer a limiting factor for industrial-scale application.

Kinetic data of ferrous iron oxidation by Thionacillus ferrooxidans were determined. The aim was to remove H2S (

Theoretical studies of the synthesis of isoamylacetate catalyzed by immobilized lipase are presented. The reaction equation and kinetic parameters have been used to analyze internal and external diffusion effects for varying process conditions. Effectiveness factors have been shown to be strongly dependent on the ratios of concentrations and diffusivities between substrates and products. The influence of backmixing on the overall conversion and reactor efficiency have been studied by coupling the pore diffusion model with a macroscopic tanks-in-series model. Furthermore, the performance of the multi-stage reactor can be enhanced by means of distributed substrate feeding to overcome substrate inhibition problems.

This investigation was undertaken in order to examine the factors affecting mechanical damage to micro-organisms in stirred bioreactors. Growth of the mould Rhizopus nigricans was studied in stirred bioreactors at different geometrical and operating parameters. Since experimental results cannot be described by conventional key parameters, a new concept has been suggested. It is based on the analogy to processes of mechanical disintegration. It is shown that the same key parameter, i.e. the ratio of power input to flow rate, can be used for a satisfactory correlation of experimental data on mechanical damage to micro-organisms, which is an important step in the recovery of intracellular products in biotechnology.

In einer okonomischen Betrachtung wird Methanol als Substrat fur die Produktion von Einzeller-Protein (SCP) mit anderen Rohstoffen an Hand von publizierten Daten verglichen. Die Kriterien sind einerseits der Preis des Substrates und andererseits die Substrat-Zellausbeute, der Sauerstoff-Bedarf und die Warme-Entwicklung im Fermentationsprozes. Weiterhin wird ein Uberblick uber methanol-verwertende Mikroorganismen und uber die Kriterien fur ihre Eignung zur SCP-Produktion gegeben. Bei der Hefe Candida boidinii wurde der Methanol-Stoffwechsel untersucht und mit dem von Bakterien verglichen. Methanol wird wie bei Bakterien uber Formaldehyd und Formiat zu CO2 oxidiert. Das C1-Substrat scheint bei Hefen uber einen Zucker-phosphat-Weg assimiliert zu werden, der ahnlich dem Ribose-phosphat-Weg in Bakterien ist. Das Problem der Prozestechnik wird unter dem Gesichtspunkt der Prozesfuhrung, Reaktortechnik und mathematischen Modellierung der Prozesse diskutiert.

In recent years considerable emphasis has been placed on the application of computer control to substrate feeding in baker's yeast production. Many investigators have studied the dynamics of different control loops and also first steps have already been taken to optimize the control algorithms. As far as the experimental verification of different strategies is concerned, the published work is, however, restricted to laboratory scale only. From current practice in baker's yeast industry which is most often based on open loop control with fixed schedule for carbon substrate addition, it is well known that mixing of substrate is playing an important role. The effects of the dynamics of mixing process in mechanicaly stirred tanks upon the performance of baker's yeast fermentation with fixed schedule have been investigated earlier by using a circulation model (Bajpai and Reuss, 1982). In the present contribution this framework is used in conjunction with a closed computer control of carbon substrate feeding based on exhaust air analysis. From experimental results for computer controlled fermentation in 10 and 2 000 l scale a decrease of productivity with increasing circulation times of liquid is observed. These results can be confirmed with the model calculations.

The major bottleneck for computer-coupling of fermentation processes is the lack of reliable, sterilisable and robust sensors for process key variables like biomass, substrate and product concentrations. Principally there are two ways to overcome this difficulty: a) construction of new sensors like product specific electrodes for in situ application or b) automatic coupling of powerful analyser systems like HPLC. This work is aimed to present a new system for coupling automatically an HPLC-System to fermentors. Furthermore this system is able to measure automatically filtration characteristies of the fermentation broth. The relationship between filtration characteristics, the height of the filter cake and its density was applied to the determination of morphology parameters of Penicillium by Nestaas and Wang (1981). Similar to other attempts for coupling autoanalyser systems, their concept was based on a filter unit with backflushed filter media. Problems in the use of such concepts for industrial media had been described several times in literature. Therefore in this work the concept of renewable filters.is chosen.The filter media is a band of filter paper or membrane filter (dependinq on the process), which lies between the media support and the'filter body.The sequence of the different operations : sterilisation, sampling, filtration, HPLC-analysis of the filtrate cleaning and finally transportation of the filter media is computer controlled. During filtration the computer registers the data for weight of the filtrate (load cell) filtration time and height of the filter cake from a couple of reflection light barriers. Using this information! the computer predicts filtration and morphology parameters. Examples for the application of this automatic system will be discussed with the aid of fermentations with a mycelial organism and yeasts. In all of these examples the emphasis of discussion is placed on both filtration parameters and HPLC-coupling.

Oxygen supply is a critical factor for many aerobic fermentation processes. It is no wonder then, that the study of oxygen transfer and reaction has attracted many researchers. There is a tendency, however, to treat these two subjects in isolation. The present contribution will emphasize the concept of interaction of these two topics because both of them individually and jointly affect the overall performance of an aerobic process. The choice of an appropriate mathematical model with which effects of oxygen supply on microbial activities can be quantitatively explained is dictated by the nature of biomass suspension, reactor configuration and scale of operation. Some examples of practical interest will be presented to throw light upon the role of oxygen transfer gas-liquid, liquid-biomass, diffusion and reaction. Special emphasis is given to problems of feed back from biomass to oxygen transfer via physical properties of the broth. Because physical environment that biomass encounters in production scale can be drastically different to that in a pilot plant a mechanistic framework is developed in which the concept of diffusion depending oxygen consumption in mycelial broths is used in conjunction with local energy dissipation and circulation models for the liquid.

Die zukunftige Entwicklung der computergekoppelten Fermentationen wird im entscheidenden Mase durch die intensive Bearbeitung der folgenden Problemkreise bestimmt werden: (1.) Weiterentwicklung bereits existierender sowie Neuentwicklung von Messonden und Mesverfahren, die insbesondere auch Mesgrosen beinhalten, die bisher nicht zuganglich sind. (2.) Entwicklung brauchbarer mathematischer Modelle. Dabei sollte ein besonderes Augenmerk darauf gerichtet sein, das innerhalb dieser Modelle die physikalischen Transportprozesse von Wachstum und Produktkinetik explizit getrennt werden. Dies ist notwendig, um die Struktur der Modelle bei den unterschiedlichen Reaktoren beizubehalten und die veranderte Hydrodynamik durch mesbare und physikalisch sinnvolle Grosen in den Modellen zu berucksichtigen. (3.) Ausarbeitung ubergeordneter prozesunspezifischer Methoden, um den Zeitraum zwischen Stammselektionierung und Prozesoptimierung rapide zu verkurzen.

Residence time distributions of gas phase have been measured in fermentation broths and in simulated media of low as well as high viscosity (1 × 10 −3 − 0.5 Pas) using a step-function-Helium-tracer technique. On the basis of these, simple mathematical models have been developed and their parameters are qualitatively correlated with operating conditions. It was shown that even in low viscosity Newtonian fluids, gas phase does not behave as well mixed. Differences between gas flow patterns in low and high viscosity fluids, already reported in literature, have been verified.

The effect of the dynamics of the mixing process in mechanically stirred bioreactors upon their performance has been investigated by using a circulation-model for flow of fluid and a two-environment-model to account for the extent of micromixing in the vessel. With the help of an empirical kinetic model to depict the oxidative and the fermentative growth of bakers' yeast, experimentally observed effects of initial sugar concentrations upon critical dilution rates in continuous cultures of this system could be explained. Effect of different scales of operation upon productivities of fed-batch cultures of bakers' yeast could also be satisfactorily predicted. A discrete simulation procedure was found to be a convenient tool in coupling the two-environment circulation-model of mixing with kinetics of microbial reactions. On a etudie l'effet de la dynamique du processus de melange, dans les bioreacteurs agites mecaniquement, sur leurs performances; on a employe a cette fin un modele de circulation pour l'ecoulement du fluide et un modele comportant deux milieux pour representer le degre de micromelange dans le recipient. On a pu expliquer, grǎce a l'emploi d'un modele cinetique empirique de la croissance de la levure de boulangerie par oxydation et fermentation, les effets observes de la concentration initiale du sucre sur les vitesses de dilution critiques dans les cultures continues du systeme en question. On a pu aussi prevoir d'une maniere satisfaisante l'effet de l'operation a differentes echelles, sur les productivites des cultures de levure de boulangerie alimentees de maniere discontinue. On a trouve qu'une procedure de simulation discrete constituait un moyen approprie pour associer le modele de melange impliquant deux milieux, a la cinetique des reactions microbiennes.

The major bottleneck for computer-coupling of fermentation processes is the lack of reliable, sterilisable and robust sensors for process key variables like biomass, substrate and product concentrations. Principally there are two ways to overcome this difficulty: a) construction of new sensors like product specific electrodes for in situ application or b) automatic coupling of powerful analyser systems like HPLC. This work is aimed to present a new system for coupling automatically an HPLC-system to fermentors. Furthermore this system is able to measure automatically filtration characteristics of the fermentation broth. The relationship between filtration characteristics, the height of the filter cake and its density was applied to the determination of morphology parameters of Penicillium by Nestaas and Wang (1981). Similar to other attempts for coupling autoanalyser systems, their concept was based on a filter unit with backflushed filter media. Problems in the use of such concepts for industrial media had been described several times in literature. Therefore in this work the concept of renewable filters is chosen. The filter media is a band of filter paper or membrane filter (depending on the process), which lies between the media support and the filter body. The sequence of the different operations:sterilisation, sampling, filtration, HPLC-analysis of the filtrate cleaning and finally transportation of the filter media is computer controlled. During filtration the computer registers the data for weight of the filtrate (load cell) filtration time and height of the filter cake from a couple of reflection light barriers. Using this information the computer predicts filtration and morphology parameters. Examples for the application of this automatic system will be discussed with the aid of fermentations with a mycelial organism and yeasts. In all of these examples the emphasis of discussion is placed on both filtration parameters and HPLC-coupling.

In recent years considerable emphasis has been placed on the application of computer control to substrate feeding in baker's yeast production. Many investigators have studied the dynamics of different control loops and also first steps have already been taken to optimize the control algorithms. As far as the experimental verification of different strategies is concerned, the published work is, however, restricted to laboratory scale only. From current practice in baker's yeast industry which is most often based on open loop control with fixed schedule for carbon substrate addition, it is well known that mixing of substrate is playing an important role. The effects of the dynamics of mixing process in mechanicaly stirred tanks upon the performance of baker's yeast fermentation with fixed schedule have been investigated earlier by using a circulation model (Bajpai and Reuss, 1982). In the present contribution this framework is used in conjunction with a closed computer control of carbon substrate feeding based on exhaust air analysis. From experimental results for computer controlled fermentation in 10 and 20001 scale a decrease of productivity with increasing circulation times of liquid is observed. These results can be confirmed with the model calculations.

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

Background The liver plays a major role in metabolism and performs a number of vital functions in the body. Therefore, the determination of hepatic metabolite dynamics and the analysis of the control of the respective biochemical pathways are of great pharmacological and medical importance. Extra- and intracellular time-series data from stimulus-response experiments are gaining in importance in the identification of in vivo metabolite dynamics, while dynamic network models are excellent tools for analyzing complex metabolic control patterns. This is the first study that has been undertaken on the data-driven identification of a dynamic liver central carbon metabolism model and its application in the analysis of the distribution of metabolic control in hepatoma cells. Results Dynamic metabolite data were collected from HepG2 cells after they had been deprived of extracellular glucose. The concentration of 25 extra- and intracellular intermediates was quantified using HPLC, LC-MS-MS, and GC-MS. The in silico metabolite dynamics were in accordance with the experimental data. The central carbon metabolism of hepatomas was further analyzed with a particular focus on the control of metabolite concentrations and metabolic fluxes. It was observed that the enzyme glucose-6-phosphate dehydrogenase exerted substantial negative control over the glycolytic flux, whereas oxidative phosphorylation had a significant positive control. The control over the rate of NADPH consumption was found to be shared between the NADPH-demand itself (0.65) and the NADPH supply (0.38). Conclusions Based on time-series data, a dynamic central carbon metabolism model was developed for the investigation of new and complex metabolic control patterns in hepatoma cells. The control patterns found support the hypotheses that the glucose-6-phosphate dehydrogenase and the Warburg effect are promising targets for tumor treatment. The systems-oriented identification of metabolite dynamics is a first step towards the genome-based assessment of potential risks posed by nutrients and drugs.