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A novel in situ product removal (ISPR) method that uses microcapsules to extract inhibitory products from the reaction suspension is introduced into fermentation technology. More specifically, L-phenylalanine (L-Phe) was transformed by Saccharomyces cerevisiae to 2-phenylethanol (PEA), which is inhibitory toward the yeast. In order to continuously remove PEA from the vicinity of the cells, the reaction suspension was brought into contact with capsules of 2.2-mm diameter that had a hydrophobic core of dibutyl sebacate and an alginate-based wall. This novel process combines the advantages of a normal in situ extraction process (fast mass transfer and simple process set-up) with the benefits of a membrane-based process (reduction of the solvent toxicity and avoidance of stable emulsions). In particular, the microbial cells are shielded from the phase toxicity of the organic solvent by a hydrogel layer surrounding the organic core. By placing the microcapsules into the fermenter, the final overall concentration of PEA in a fed-batch culture was increased from 3.8 to 5.6 g/L because a part of the inhibitory product dissolved in the dibutyl sebacate core. In another fermentation experiment, the capsules were placed in a fluidized bed that was connected via a loop to the fermenter. In addition, the fluidized bed was connected via a second loop to a back-extractor to regenerate the capsules. By alternating the extraction and back-extraction cycles, it was possible to limit the PEA concentration of the fed-batch culture in the fermenter to 2.4 g/L while producing important quantities of PEA that accumulated in an external reservoir.

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The bioconversion of L-phenylalanine (L-Phe) to 2-phenylethanol (PEA) by the yeast Saccharomyces cerevisiae is limited by the toxicity of the product. PEA extraction by a separate organic phase in the fermenter is the ideal in situ product recovery (ISPR) technique to enhance productivity. Oleic acid was chosen as organic phase for two-phase fed-batch cultures, although it interfered to some extent with yeast viability. There was a synergistic inhibitory impact toward S. cerevisiae in the presence of PEA, and therefore a maximal PEA concentration in the aqueous phase of only 2.1 g/L was achieved, compared to 3.8 g/L for a normal fed-batch culture. However, the overall PEA concentration in the fermenter was increased to 12.6 g/L, because the PEA concentration in the oleic phase attained a value of 24 g/L. Thus, an average volumetric PEA production rate of 0.26 g L(-1) h(-1) and a maximal volumetric PEA production rate of 0.47 g L(-1) h(-1) were achieved in the two-phase fed-batch culture. As ethanol inhibition had to be avoided, the production rates were limited by the intrinsic oxidative capacity of S. cerevisiae. In addition, the high viscosity of the two-phase system lowered the k(l)a, and therefore also the productivity. Thus, if a specific ISPR technique is planned, it consequently has to be remembered that the productivity of this bioconversion process is also quickly limited by the k(l)a of the fermenter at high cell densities.

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Modern bioprocesses are monitored by on-line sensing devices mounted either in situ or externally. In addition to sensor probes, more and more analytical subsystems are being exploited to monitor the state of a bioprocess on-line and in real time. Some of these subsystems deliver signals that are useful for documentation only, other, less delayed systems generate signals useful for closed loop process control. Various conventional and non-conventional monitoring instruments are evaluated; their usefulness, benefits and associated pitfalls are discussed.

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This contribution gives paradigms for static and dynamic effectors of microbial cultures. Macroscopic and physiological steady states are differentiated. The examples comprise: inhibitory and regulatory effects of medium components affecting growth and product formation, possible pitfalls in trying to speed-up operational procedures, responses to foam formation and supply of alternate carbon or oxygen sources, and cell cycle dependent events.

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This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101[pGEc47] can be related to yeast extract-enriched medium rather than plasmid properties. An optimal medium for growth of E. coli HB101[pGEc47] was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1). The yield coefficient for L-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for L-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation). Growth on defined medium after medium optimization is as rapid as on complex medium (0. 42-0.45 h-1). The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1.

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E. coli HB101[pGEc47], which is able to convert octane to octanoate, but cannot oxidize octanoate further, was grown on defined medium with glucose as carbon source in batch and continuous culture. The biomass yield on glucose decreased from 0.32 +/- 0.02 g g-1 in aqueous cultivations to 0.25 +/- 0.02 g g-1 in the presence of octane. Maximal octanoate productivities of 0.6 g L-1 h-1 were the same as found in cultivations on complex medium. The glucose-based carbon recovery in these experiments was 99 +/- 4% (in extreme, between 90% and 105%). An increase of the octane feed from 1% to 2% (v/v) or more led to washout of cells. This effect was reversible when the octane feed was decreased to its initial value of 1%. Analysis of experimental data by model simulation strongly suggested that washout was due to inhibition by octanoate only. Pulses of octanoate to a continuous culture grown on aqueous media were applied to analyze the inhibition further. Inhibition by acetate was not significant, but its presence in the medium reflected a physiological state that made the cells more sensitive to octanoate inhibition. Model simulation with linear inhibition kinetics could perfectly predict glucose consumption and the resulting glucose concentration. The linear type of inhibition was confirmed by a variety of batch experiments in the presence of different concentrations of octanoate. The glucose-based specific growth rate, mu, decreased linearly with increasing concentrations of octanoate and became zero at a threshold concentration pmax of 5.25 +/- 0.25 g L-1.

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The oxidation of medium chain length alkanes and alkenes (C6 to C12) by Pseudomonas oleovorans and related, biocatalytically active recombinant organisms, in two-liquid phase cultures can be used for the biochemical production of several interesting fine chemicals. The volumetric productivities that can be attained in two-liquid phase systems can be, in contrast to aqueous fermentations, limited by the transport of substrates from an apolar phase to the cells residing in the aqueous phase and by toxic effects of apolar solvents on microbial cells. We have assessed the impact of these possible limitations on attainable productivities in two-liquid phase fermentations operated with mcl-alkanes. Pseudomonas oleovorans grows well in two-liquid phase media containing a bulk n-octane phase as the sole carbon source. However, cells are also damaged, typically resulting in a cell lysis rate of about 0.08 to 0. 10 h-1. These rates could be lowered by 50 to 70% to 0.03 h-1 and substrate yields increased from 0.55 to 0.85 g g-1 by diluting octane in non-metabolizable long-chain hydrocarbon solvents. Transfer rates of medium chain length (mcl) alkanes from the apolar phase to the cells were determined by following growth and the rate at which carbon-containing metabolites accumulated in the different phases of the cultures. mcl-Alkane solvent-cell transfer rates of at least 79, 64, and 18 mmol per liter of aqueous medium per hour were determined for n-heptane, n-octane, and n-decane, respectively. Rates of up to 30 mmol L-1 h-1 were observed under octane-limiting conditions in systems where the apolar substrate was dissolved to concentrations below 3% (v/v) in hexadecene. Based on low power input experiments, we estimated the maximum obtainable mass transfer rates in large scale processes to be in the range of 13 mmol L-1 h-1 for decane and higher than 45 mmol L-1 h-1 for octane and heptane. The results indicate that high solvent to cell mass transfer rates and minimized cell damage will enable high production rates in two-liquid phase bioprocesses, justifying ongoing efforts to attain high densities of catalytically, highly active cells in such systems. Copyright 1998 John Wiley & Sons, Inc.

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When a continuously grown yeast culture was allowed to rest at the low dilution rate for an extended time period and was then challenged by increasing the dilution rate, applied as a step function, an overshoot of the concentration of residual glucose occurred reproducibly. A structural extension of the bottleneck model describing another intracellular bottleneck in glucose consumption allowed to predict such overshoots quantitatively. The model assumes that an intracellular enzymatic pool increases in response to a challenge by excess substrate supply, and experimentally, the relaxation time was determined to be on the order of 1 h. When the culture is reset to more limiting conditions, the enzymatic pool shrinks with a relaxation time determined by the reciprocal of the current specific growth rate. Generalized, microbial populations do memorize their (recent) history by adapting their metabolic outfit.

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The glucose content of the culture liquid during shift experiments and synchronized cultures of Saccharomyces cerevisiae H1022 (ATCC 32167) was monitored using a greatly improved and highly precise FIA. During shift-up experiments on the dilution rate, an overshoot of the glucose-concentration was observed. The amplitude of the overshoot showed a dependency on the duration of undisturbed cultivation before application of the shift. Mutarotational non-equilibrium was excluded as the cause of the observed overshoot. For the first time glucose measurements of oscillating cultures of Saccharomyces cerevisiae are demonstrated with high accuracy and reproducibility. The data strongly support the proposals by Münch et al. (1992a, b) that faint oscillations in glucose concentration are responsible for the persistence of the synchronization. Analytical subsystems prove to be a powerful tool for investigation of the dynamics of metabolic pathways of microbial organisms. Accurate glucose measurements at low concentrations point out the limits and allow refinements of commonly used models.

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The aerobic thermophilic treatment process of sewage sludge was studied at different bioreactor scales in a pilot plant installation. Since, for a satisfactory sludge disinfection, the Swiss legislation requires minimal incubation times of all volume elements, the bioreactors were operated in repetitive batch mode (draw and fill). Different retention times and frequencies of the volume changes were applied in order to prove the capability of the particular operation modes in assuring high degradative potential. The main enzymatic activity involved during the aerobic treatment was proteolysis: the RQ values ranged between 0.8 and 0.9 depending on the applied operating conditions. Although not in a linear manner, the efficiency of the microflora decreased as the bioreactor scale increased, when this increase corresponded with a reduction of the specific power input. The sludge oxidation rates can be tuned by some process operating conditions such as the volume change frequency, the changed volume quantities and the retention times. It was possible to improve the microbial degradative efficiency by an increased frequency of the changes, while the mean retention time influenced in particular the ultimate product quality, described as residual organic matter content of the sludge. The microflora present was also satisfactorily active at mean hydraulic retention times of less than 10 h. The organic matter concentration of the inlet sewage sludge plays an important role: it influences the aerobic degradation process positively.

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The performance of the ATS process depends essentially on the oxygen transfer efficiency. Improvement of the mass transfer capacity of a bioreactor allowed to reduce the incubation time necessary to attain sludge stabilization. It is important to use equipment with a high aeration efficiency such as an injector aeration system. The ratio between the total oxygen consumption and the organic matter degradation (delta COD) ranged between 0.4 and 0.8 in the pilot plant, whereas 1.23 was found in completely mixed bioreactors (Bomio, 1990). No significant improvement of the bacterial degradation efficiency was attained with a specific power input exceeding 6-8 kW m-3. A mean residence time of less than 1 d allowed organic matter removals up to 40% with specific power consumption of 10 kWh kg-1 COD oxidized. The sludge hygienization is one of the objectives and benefits of the thermophilic treatment: not only temperature but also the total solids content were important factors affecting inactivation of pathogens. The inactivation rate was promoted by the increase of temperature, while the residual colony forming units decreased with reducing the total solids content of sewage sludge. It is concluded that continuous operation mode would not affect the quality of the hygienization but could display the high degradation potential of the aerobic system.

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The recent investigations in our high performance bioreactors have shown that living cells can be extremely sensitive to physical-chemical environmental conditions and their changes. Consequently, the relationship bioreactor-living cell must thoroughly be investigated in order to discuss both: whether bioreactor characteristics are limiting/dominating during cultivation and to what extent controlled changes of the cellular environment can lead the cells to a desired physiological state. For these investigations, a generally accepted biological test organism would be helpful, of which the requirements and reactions under certain conditions are well known. Saccharomyces cerevisiae is a well known, very robust but nevertheless sensitive organism, eligible for this purpose. In this article a typical batch cultivation on glucose is presented, collected from approx. 300 experiments. Regarding metabolite production and consumption, seven different phases are distinguished on the basis of approx. 20 sensor signals and their metabolic background is discussed. Prerequisite, however, was an exhaustive knowledge upon extracellular conditions, a task which could successfully be fulfilled with the highly automated equipment introduced in the preceding articles of this series.

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Experimental programs are the basis for the development of new processes as well as for basic biological research. Hence, an unbiased approach is essential, otherwise the interpretation of results will be misleading. The complex chemical composition of cultivation media in combination with the impedded measuring under monoseptic conditions are ideal circumstances for misinterpretations and unsuited experimental approaches. In this article, practical examples for such pitfalls are given. They are undervalued in mass transfer and mixing effects, error propagation during RQ determination and possible influence of the medium preparation on the time evolution of the growth process. Further, the difficulty of sound interpretation is demonstrated by a batch cultivation carried out under sinusoidal changes of the stirrer speed. Summary conclusions close this series about equipment, methodology and benefits of bioprocess automation.

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The reasons for and historical backgrounds of biomass determination are discussed under the aspects of theoretical and practical importance, usefulness and representativity. Off-line methods are evaluated and compared with on-line methods; constraints of applications and conclusiveness of results are rated. Special emphasis is given to the fact that mere knowledge of a bio-mass concentration is not sufficiently valuable to learn more about physiology nor to determine the effectiveness of a biotechnological process. A combination of several different alternative measuring principles in parallel as well as the exploitation of software sensors is proposed as a promising future solution.

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Bioprocesses are generally ill controlled. This is due to the fact that the measurement of relevant variables is difficult. Therefore, fundamental knowledge of metabolic interrelations is, at least in vivo, limited. In this article, some of the most important measurement techniques are reviewed in order to provide an evaluation of their current state. Emphasis is given to the underlying principles and on-line capability which allow to judge their importance and potential for exploitation resulting in well (maybe entirely) controlled bioprocesses in the future.

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Sound data biologically relevant are prerequisites when developing high-performance bioprocesses. Understanding of physiological regulation as well as sophisticated control strategies are highly dependent on the observability of the culture, i.e. the generation and exploitation of suited signals even under complex environmental measurement conditions. Against this background, the increasing number of analytical systems is very supportive and, accordingly, an appropriate handling of sensors and measured data is of decisive importance. This article reports on practical experience with routines for maintenance, service and calibration of hardware sensors which improve the quality of measurements significantly. Verification and validation of signals is outlined in order to make the value of data exploitation tools obvious. A method for the characterization of information is introduced by practical examples of Saccharomyces cerevisiae cultures when explaining the specific properties of extracting biological information from raw data. Finally, examples for advantageous exploitation of on-line data are given.

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The dynamic behaviour of the cell cycle and the physiology of Saccharomyces cerevisiae was monitored in transient experiments. Frequent flow cytometric analyses of the DNA (nuclear phase state) and the cell size enabled us to characterize the proliferation properties of yeast cells under well controlled and undisturbed cultivation conditions. Preliminarily, the correlation between flow cytometric light scattering measurements and the cell size was attested for yeasts. These flow cytometric results are compared with the physiological behaviour of the culture that was detected by high resolution on-line analyses and off-line measurements. The presented results focus on the importance of the yeast cell cycle behaviour for the dynamic growth characterization. Any kind of transients in yeast cultures induced partial synchronization. The characteristics and the time course of the yeast cell cycle were found to be strongly dependent on the physiological environment.

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Quantitation is a characteristic property of natural sciences and technologies and is the background for all kinetic and dynamic studies of microbial life. This presentation concentrates therefore on materials and methods as tools necessary to accomplish a sound, quantitative and mechanistic understanding of metabolism. Mathematical models are the software, bioreactors, actuators and analytical equipment are the hardware used. Experiments must be designed and performed in accordance with the relaxation times of the biosystem investigated; some of the respective consequences are discussed and commented in detail. Special emphasis is given to the required density, accuracy and reproducibility of data as well as their validation.

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