A methodology for the design of scale-down bioreactors by the use of mixing and circulation stochastic models

Scale-up is traduced in practice by an increase of the dimensions of the bioreactors, leading to a modification of the time scale and thus of the process dynamics. In the present work, a methodology to study the effect of scale-up on bioreactors hydrodynamics and to put in place scale-down reactors representative of the flow properties encountered in real scales bioreactors is detailed.In order to simplify the analysis, we have proposed the use of a stochastic model which is directly affected by the time scale. Indeed, to run simulations with such models, we have to specify the time taken to achieve a transition Δt. Stochastic models are thus reliable to study scale-up effect on stirred reactors hydrodynamics. In addition, these models permit to have an insight on the internal dynamic of the process.In the case of the circulation process, qualitative aspects have to be taken into account and induce a modification of the flow regions arrangement of the model. The stochastic analysis of large-scale bioreactors permits to propose a translating methodology into a scale-down context. Optimised scale-down reactors can be used further to carry out fermentation tests with the hydrodynamic conditions of the industrial scale. In a general rule, the performances of stochastic model allow to facilitate greatly the analysis of the scale-up effect and the hydrodynamic characteristics of both large-scale and scale-down reactors.     

Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

Comparison of the Baker's yeast process performance in laboratory and production scale

A 215 m³ industrial bubble column reactor for fedbatch production of Baker's yeast was sampled for sugar, to investigate the extent of concentration gradients. The results verify that such gradients exist: the concentration is higher closer to the feeding point. Effects of sugar heterogeneities at different scales were studied by 1)?performing a volumetric scale-down of the industrial process in a laboratory stirred tank reactor (STR); 2) performing the same scaled down process in a Scale-Down Reactor (SDR) with repeated short term exposure of the cells to high sugar concentrations. In this reactor about 10% of the Baker's yeast culture was intermittently exposed to high (0.45–1.9?g?l^?1) concentrations of sugar, for periods of 60 seconds. It was found that physiological parameters of glycolysis and respiration were affected by environmental heterogeneities: 1) A biomass yield reduction of about 6–7% was found, with both the production reactor and the SDR, as compared to the homogeneous reactor. The loss of yield is interpreted in terms of a metabolic by-pass via ethanol, where cells are consuming and producing ethanol with different yields. 2) The maximum respiration rate was higher in cells produced in the production unit and in the SDR. 3) The product quality, expressed as gassing power of the yeast in a dough, was increased for sweet and non-sugar doughs in the SDR, and for sweet doughs in the production reactor. Thus, the SDR, when run with defined glucose gradients, in some aspects resembles the large reactor. It could be regarded as a tool for scale-down and scale-up studies and may be useful in investigations on the scale-up sensitivity of a process.     

A scale-down two-compartment reactor with controlled substrate oscillations: Metabolic response of Saccharomyces cerevisiae

Substrate concentration gradients are likely to appear during large scale fermentations. To study effects of such gradients on microorganisms, an aerated scale-down reactor system was constructed. It consists of a plug flow reactor (PFR) and a stirred tank reactor (STR), between which the medium is circulated. The PFR, which is an aerated static mixer reactor, was characterized with respect to plug flow behaviour and oxygen transfer. A Bodenstein number of 15–220, depending on residence time and aeration rate, and a k_La of 500–1130 h^–1, depending mainly on aeration rate, were obtained. The biological test system used, was aerobic ethanol production by Saccharomyces cerevisiae, due to sugar excess. The ethanol concentration profile and the yield of biomass were compared in two fed-batch fermentations. In the first case, the feeding point of molasses was located at the inlet of the PFR. This simulates location of the feeding point in the segregated part of a heterogeneous reactor, with local high sugar concentrations. In the second mode of operation, as a control with good mixing conditions, the PFR was disconnected from the STR, into which the substrate was fed. Differences were found: Up to 6% less biomass was produced and a larger amount of ethanol was formed in the two-compartment reactor system, due to the uneven sugar concentration distribution. This emphasizes the importance of the location of, and the mixing conditions at, the feeding point in a bioreactor.     

Design of a tubular loop bioreactor for scale-up and scale-down of fermentation processes

Microorganisms traveling through circulation loops in large-scale bioreactors experience variations in their environment such as dissolved oxygen concentration and pH gradients. The same changes are not experienced in small bioreactors, and it is suggested that herein lies one of the major reasons for the problems encountered when translating fermentation data from one scale to another. One approach to study this problem is to look at the circulation loop itself. The present work concerns an attempt to simulate the circulation loops inside stirred tank reactors, using a tubular loop reactor specially constructed for the purpose. The reactor carries a number of ports and probes along its length for the determination of concentration gradients within. The broth is circulated around the loop by the use of peristaltic pumps, and the circulation time (t(c), s) is used as a measure of simulated reactor size. The reactor system has been evaluated using the citric acid fermentation by Aspergillus niger as a test process. Acid production and fungal morphology, in terms of the mean convex perimeter of mycelial clumps quantified by image analysis, were used as the parameters of evaluation for the two systems in comparison. From comparative experiments carried out in 10 and 200 L stirred tank bioreactors, it appears that the loop reactor simulates the corresponding stirred tank representing a valuable tool in scaling up and scaling down of fermentation process.     

Further studies related to the scale-up of high cell density Escherichia coli fed-batch fermentations: the additional effect of a changing microenvironment when using aqueous ammonia to control pH

In this work, we report on the further development of the scale-down, two-compartment (STR + PFR) experimental simulation model. For the first time, the effect on high cell density Escherichia coli fed-batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large-scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously. To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level. It was demonstrated that for E. coli W3110 under such conditions in a 20 m(3) industrial fed-batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well-mixed, 5L laboratory scale case. However, by using a combination of the well-mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m(3) reactor may be obtained. The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO(2) and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7.     

Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis

The establishment and the improvement of industrial bioprocesses calls for the selection of media compositions and process conditions in highly parallel experiments as well as for the intensified screening of new biocatalysts and improved production strains. This work presents for the first time the scale-down and the successful adaptation of an industrial riboflavin fed-batch production process with Bacillus subtilis to a fully automated setup with 48 parallel stirred bioreactors at a milliliter scale (10 mL). The feasibility of an intermittent feeding mode and a discontinuous at-line pH control for parallel cultivations over up to 53 h is demonstrated together with interlaced process analyses at a microliter scale for quasi-simultaneous at-line monitoring of biomass, substrate and product concentration. The discontinuous feeding mode necessitated an increased oxygen input, resulting in lower final biomass concentrations. However, the product yields and volumetric productivities in the milliliter setup were equivalent to the yields and productivities obtained during the reference cultivations at laboratory scale, which allows considering the automated system together with the developed schedule as a screening tool for high-throughput bioprocess design of the described production process.     

Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multiparameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration

Multiparameter flow cytometric techniques developed in our laboratories have been used for the "at-line" study of fed-batch bacterial fermentations. These fermentations were done at two scales, production (20 m(3)) and bench (5 x 10(-3) m(3)). In addition, at the bench scale, experiments were undertaken where the difficulty of achieving good mixing (broth homogeneity), similar to that found at the production scale, was simulated by using a two-compartment model. Flow cytometric analysis of cells in broth samples, based on a dual-staining protocol, has revealed, for the first time, that a progressive change in cell physiological state generally occurs throughout the course of such fermentations. The technique has demonstrated that a changing microenvironment with respect to substrate concentration (glucose and dissolved oxygen tension [DOT]) has a profound effect on cell physiology and hence on viable biomass yield. The relatively poorly mixed conditions in the large-scale fermentor were found to lead to a low biomass yield, but, surprisingly, were associated with a high cell viability (with respect to cytoplasmic membrane permeability) throughout the fermentation. The small-scale fermentation that most clearly mimicked the large-scale heterogeneity (i.e., a region of high glucose concentration and low DOT analogous to a feed zone) gave similar results. On the other hand, the small-scale well-mixed fermentation gave the highest biomass yield, but again, surprisingly, the lowest cell viability. The scaled-down simulations with high DOT throughout and locally low or high glucose gave biomass and viabilities between. Reasons for these results are examined in terms of environmental stress associated with an ever-increasing glucose limitation in the well-mixed case. On the other hand, at the large scale, and to differing degrees in scale-down simulations, cells periodically encounter regions of relatively higher glucose concentration.     

An analysis of organism lifelines in an industrial bioreactor using Lattice-Boltzmann CFD

Euler-Lagrange CFD simulations, where the biotic phase is represented by computational particles (parcels), provide information on environmental gradients inside bioreactors from the microbial perspective. Such information is highly relevant for reactor scale-down and process optimization. One of the major challenges is the computational intensity of CFD simulations, especially when resolution of dynamics in the flowfield is required. Lattice-Boltzmann large-eddy simulations (LB-LES) form a very promising approach for simulating accurate, dynamic flowfields in stirred reactors, at strongly reduced computation times compared to finite volume approaches. In this work, the performance of LB-LES in resolving substrate gradients in large-scale bioreactors is explored, combined with the inclusion of a Lagrangian biotic phase to provide the microbial perspective. In addition, the hydrodynamic performance of the simulations is confirmed by verification of hydrodynamic characteristics (radial velocity, turbulent kinetic energy, energy dissipation) in the impeller discharge stream of a 29 cm diameter stirred tank. The results are compared with prior finite volume simulation results, both in terms of hydrodynamic and biokinetic observations, and time requirements.     

Screening of phenylpyruvic acid producers and optimization of culture conditions in bench scale bioreactors

Alpha keto acids are deaminated forms of amino acids that have received significant attention as feed and food additives in the agriculture and medical industries. To date, their production has been commonly performed at shake-flask scale with low product concentrations. In this study, production of phenylpyruvic acid (PPA), which is the alpha keto acid of phenylalanine was investigated. First, various microorganisms were screened to select the most efficient producer. Thereafter, growth parameters (temperature, pH, and aeration) were optimized in bench scale bioreactors to maximize both PPA and biomass concentration in bench scale bioreactors, using response surface methodology. Among the four different microorganisms evaluated, Proteus vulgaris was the most productive strain for PPA production. Optimum temperature, pH, and aeration conditions were determined as 34.5 °C, 5.12, and 0.5 vvm for PPA production, whereas 36.9 °C, pH 6.87, and 0.96 vvm for the biomass production. Under these optimum conditions, PPA concentration was enhanced to 1,054 mg/L, which was almost three times higher than shake-flask fermentation concentrations. Moreover, P. vulgaris biomass was produced at 3.25 g/L under optimum conditions. Overall, this study demonstrated that optimization of growth parameters improved PPA production in 1-L working volume bench-scale bioreactors compared to previous studies in the literature and was a first step to scale up the production to industrial production.     

Influence of scale-up on the quality of recombinant human growth hormone

The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer.     

A model-based framework for parallel scale-down fed-batch cultivations in mini-bioreactors for accelerated phenotyping

Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small-scale parallel cultivation devices. In this study, we combine mechanistic growth models with a parallel mini-bioreactor system to create a high-throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled-down approach, a model-based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large-scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale-down scheme, compared with the reference cultivations. In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale-down studies to select the most robust strains for bioprocess scale-up.     

Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses

The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed‐acid fermentation metabolism was studied in a two‐compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini‐plug‐flow reactor (PFR: BioScope, 3.5 mL volume). Such a system can mimic the situation often encountered in large‐scale, fed‐batch bioreactors. The STR represented the zones of a large‐scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen‐poor and glucose‐rich regions can occur in large‐scale bioreactors. The response of E. coli to these large‐scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D = 0.1 h^?1) into the mini‐PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92 s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accumulation of acetate and formate was detected after E. coli cells had been exposed for only 2 s to the glucose‐rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and succinate, which were also detected as soon as 2 s after of exposure of E. coli cells to the glucose and O₂ gradients. This approach (STR‐mini‐PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large‐scale bioreactors and for the design of modified strains with an improved behavior under large‐scale conditions. Biotechnol. Bioeng. 2009; 104: 1153–1161. © 2009 Wiley Periodicals, Inc.     

A two-compartment bioreactor system made of commercial parts for bioprocess scale-down studies: impact of oscillations on Bacillus subtilis fed-batch cultivations

This study describes an advanced version of a two-compartment scale-down bioreactor that simulates inhomogeneities present in large-scale industrial bioreactors on the laboratory scale. The system is made of commercially available parts and is suitable for sterilization with steam. The scale-down bioreactor consists of a usual stirred tank bioreactor (STR) and a plug flow reactor (PFR) equipped with static mixer modules. The PFR module with a working volume of 1.2 L is equipped with five sample ports, and pH and dissolved oxygen (DO) sensors. The concept was applied using the non-sporulating Bacillus subtilis mutant strain AS3, characterized by a SpoIIGA gene knockout. In a fed-batch process with a constant feed rate, it is found that oscillating substrate and DO concentration led to diminished glucose uptake, ethanol formation and an altered amino acid synthesis. Sampling at the PFR module allowed the detection of dynamics at different concentrations of intermediates, such as pyruvic acid, lactic acid and amino acids. Results indicate that the carbon flux at excess glucose and low DO concentrations is shifted towards ethanol formation. As a result, the reduced carbon flux entering the tricarboxylic acid cycle is not sufficient to support amino acid synthesis following the oxaloacetic acid branch point.     

An industrial perspective on bioreactor scale-down: what we can learn from combined large-scale bioprocess and model fluid studies

For industrial bioreactor design, operation, control and optimization, the scale-down approach is often advocated to efficiently generate data on a small scale, and effectively apply suggested improvements to the industrial scale. In all cases it is important to ensure that the scale-down conditions are representative of the real large-scale bioprocess. Progress is hampered by limited detailed and local information from large-scale bioprocesses. Complementary to real fermentation studies, physical aspects of model fluids such as air-water in large bioreactors provide useful information with limited effort and cost. Still, in industrial practice, investments of time, capital and resources often prohibit systematic work, although, in the end, savings obtained in this way are trivial compared to the expenses that result from real process disturbances, batch failures, and non-flyers with loss of business opportunity. Here we try to highlight what can be learned from real large-scale bioprocess in combination with model fluid studies, and to provide suitable computation tools to overcome data restrictions. Focus is on a specific well-documented case for a 30-m(3) bioreactor. Areas for further research from an industrial perspective are also indicated.     

Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans

To simulate production-scale conditions of gluconic acid fermentation by Gluconobacter oxydans, different experimental setups are presented in this study. From the determination of the time constants of a production-scale reactor, it can be concluded that mixing and oxygen transfer are the rate-limiting mechanisms. This results in oxygen concentration gradients which were simulated in a one-compartment reactor in which the oxygen concentration was fluctuated by a fluctuated gassing with air and nitrogen. It could be concluded that only very long periods of absence of oxygen (ca. 180 s) results in lower specific oxygen uptake rates by Gluconobacter oxydans. From scale-down studies carried out in a two-compartment system to simulate a production-scale reactor more accurately, it could be concluded that not only the residence time in the aerated part of the system is important, but the liquid flow in between the different parts of the reactor is also an essential parameter. It could also be concluded that the microorganisms are not influenced negatively by the fluctuated oxygen concentrations with respect to their maximal oxidation capacity. The two-compartment system can also be used for optimization experiments in which the "aerated" compartment was gassed with pure oxygen. From these experiments it was concluded that also a short residence of the cells at high oxygen concentrations diminished the growth and product formation rates. These experiments show the necessity of the scale-down experiments if optimization is carried out. The two-compartment system presented in this study is a very attractive tool for reliable scale-down experiments.     

Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system: metabolic response and production of recombinant protein

A significant problem of large-scale cultures, but scarcely studied for recombinant E. coli, is the presence of gradients in dissolved oxygen tension (DOT). In this study, the effect of DOT gradients on the metabolic response of E. coli and production of recombinant pre-proinsulin, accumulated as inclusion bodies, was determined. DOT gradients were simulated in a two-compartment scale-down system consisting of two interconnected stirred-tank bioreactors, one maintained at anoxic conditions and the other at a DOT of at least 6%. Cells were continuously circulated between both vessels to simulate circulation times (tc) of 20, 50, 90, and 180 sec. A complete kinetic and stoichiometric characterization was performed in the scale-down system as well as in control cultures maintained at constant DOT in the range of 0-20%. The performance of E. coli cultured under oscillating DOT was significantly affected, even at a tc of 20 sec corresponding to transient exposures of only 13.3 sec to anaerobic conditions. Specific growth rate decreased linearly with tc to a maximum reduction of 30% at the highest tc tested. The negative effect of DOT gradients was even more pronounced for the overall biomass yield on glucose and the maximum concentration and yield of pre-proinsulin. In these cases, the losses were 9%, 27%, and 20%, respectively, at tc of 20 sec and 65%, 94%, and 87%, respectively, at tc of 180 sec. Acetic, lactic, formic, and succinic acids accumulated during oscillatory DOT cultures, indicating that deviation of carbon flow to anaerobic metabolism was responsible for the observed losses. The results of this study indicate that even very short exposures to anaerobic conditions, typical of large-scale operations, can substantially reduce recombinant protein productivity. The information presented here is useful for establishing improved rational scale-up strategies and understanding the behavior of recombinant E. coli exposed to DOT gradients.     

Computational fluid dynamics simulation improves the design and characterization of a plug-flow-type scale-down reactor for microbial cultivation processes

The scale-up of bioprocesses remains one of the major obstacles in the biotechnology industry. Scale-down bioreactors have been identified as valuable tools to investigate the heterogeneities observed in large-scale tanks at the laboratory scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here, we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor was connected to a stirred-tank bioreactor. With the help of CFD using the realizable k-ε model, the mixing time difference between a 20 and 4000 L bioreactor was evaluated and used as scale-down criterion. CFD simulations using a shear stress transport (SST) k-ω turbulence model were used to characterize the plug-flow reactor in more detail, and the model was verified using experiments. Additionally, the model was used to simulate conditions where experiments technically could not be performed due to sensor limitations. Nevertheless, verification is difficult in this case as well. This was the first time a scale-down setup was tested on high-cell-density Escherichia coli cultivations to produce industrially relevant antigen-binding fragments (Fab). Biomass yield was reduced by 11% and specific product yield was reduced by 20% during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The flexibility of the introduced scale-down setup in combination with CFD simulations makes it a valuable tool for investigating scale effects at the laboratory scale. More information about the large scale is still necessary to further refine the setup and to speed up bioprocess scale-up in the future.     

Performing in spite of starvation: How Saccharomyces cerevisiae maintains robust growth when facing famine zones in industrial bioreactors

In fed-batch operated industrial bioreactors, glucose-limited feeding is commonly applied for optimal control of cell growth and product formation. Still, microbial cells such as yeasts and bacteria are frequently exposed to glucose starvation conditions in poorly mixed zones or far away from the feedstock inlet point. Despite its commonness, studies mimicking related stimuli are still underrepresented in scale-up/scale-down considerations. This may surprise as the transition from glucose limitation to starvation has the potential to provoke regulatory responses with negative consequences for production performance. In order to shed more light, we performed gene-expression analysis of Saccharomyces cerevisiae grown in intermittently fed chemostat cultures to study the effect of limitation-starvation transitions. The resulting glucose concentration gradient was representative for the commercial scale and compelled cells to tolerate about 76 s with sub-optimal substrate supply. Special attention was paid to the adaptation status of the population by discriminating between first time and repeated entry into the starvation regime. Unprepared cells reacted with a transiently reduced growth rate governed by the general stress response. Yeasts adapted to the dynamic environment by increasing internal growth capacities at the cost of rising maintenance demands by 2.7%. Evidence was found that multiple protein kinase A (PKA) and Snf1-mediated regulatory circuits were initiated and ramped down still keeping the cells in an adapted trade-off between growth optimization and down-regulation of stress response. From this finding, primary engineering guidelines are deduced to optimize both the production host's genetic background and the design of scale-down experiments.     

Optimization of fermentation conditions for gluconic acid production using Aspergillus niger immobilized on cellulose microfibrils

The optimisation of gluconic acid fermentation using immobilized Aspergillus niger on a highly porous cellulose support is described. Experimental results showing the effects of variations in oxygen partial pressure, glucose concentration and biomass concentration have been obtained with a continuous recirculation reactor. Levels of dissolved oxygen and glucose concentrations during fermentation significantly affect the production and fermentation time. The optimum biomass requirement on a porous cellulose support has been estimated to be 0.234 mg cm⁻² for efficient bioconversion. Increasing the quantum of biomass beyond this value resulted in an overgrown biofilm, which affected productivity adversely. Morphological characteristics of immobilized A. niger have also been investigated.