Influence of bioreactor hydraulic characteristics on a Saccharomyces cerevisiae fed-batch culture: hydrodynamic modelling and scale-down investigations

Yeast is a widely used microorganism at the industrial level because of its biomass and metabolite production capabilities. However, due to its sensitivity to the glucose effect, problems occur during scale-up to the industrial scale. Hydrodynamic conditions are not ideal in large-scale bioreactors, and glucose concentration gradients can arise when these bioreactors are operating in fed-batch mode. We have studied the effects of such gradients in a scale-down reactor, which consists of a mixed part linked to a non-mixed part by a recirculation pump, in order to mimic the hydrodynamic conditions encountered at the large scale. During the fermentation tests in the scale-down reactor, there was a drop in both biomass yield (ratio between the biomass produced and the glucose added) and trehalose production and an increase in both fermentation time (time between inoculation and beginning of stationary phase) and ethanol production. We have developed a stochastic model which explains these effects as the result of an induction process determined mainly by the hydrodynamic conditions. The concentration profiles experienced by the microorganisms during the scale-down tests were expressed and linked to the biomass yields of the scale-down tests.     

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.     

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.     

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.     

The effect of equipment scale and degree of mixing on continuous fermentation yield at low dilution rates

Previously, the degree of mixing was not felt to be an important consideration in fermentor design. In this study on the continuous propagation of Baker's yeast, it was found that at low dilution rates, i.e., 0.02hr^?1, the degree of mixing achieved does effect the cell yield. At low dilution rates, appreciable quantities of sugar can be utilized for endogenous respiration in comparison to that utilized for making cell mass. Poor distribution of the sugar aggravates the balance of sugar utilized for each process. Yields at these low dilution rates can be improved to a limited extent by using a multiple feed-distribution system and better mixing.     

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.     

Influence of invertase activity and glycerol synthesis and retention on fermentation of media with a high sugar concentration by Saccharomyces cerevisiae.

In the past, the fermentation activity of Saccharomyces cerevisiae in substrates with a high concentration of sucrose (HSuc), such as sweet bread doughs, has been linked inversely to invertase activity of yeast strains. The present work defines the limits of the relationship between invertase activity and fermentation in hyperosmotic HSuc medium. Fourteen polyploid, wild-type strains of S. cerevisiae with different invertase levels gave a similar ranking of fermentation activity in HSuc and in medium in which glucose and fructose replaced sucrose (HGF medium). Thus, invertase is unlikely to be the most important determinant of fermentation in sweet doughs. Yeasts produce the compatible solute-osmoprotective compound glycerol when exposed to hyperosmotic environments. Under low sugar concentrations (and nonstressing osmotic pressure), there was no correlation between glycerol and fermentation activities. However, there was a strong correlation between the ability of yeasts to ferment in HSuc or HGF medium and their capacity to produce and retain glycerol intracellularly. There was also a strong correlation between intracellular glycerol and fermentation activity of yeasts in a medium in which the nonfermentable sugar alcohol sorbitol replaced most of the sugars (HSor), but the ability to produce and retain glycerol was greater when yeasts were incubated in HGF medium under the same osmotic pressure. The difference between the amounts of glycerol produced and retained in HSor and in HGF media varied with strains. This implies that high fermentable sugar concentrations cause physiological conditions that allow for enhanced glycerol production and retention, the degree of which is strain dependent. In conclusion, one important prerequisite for yeast strains to ferment media with high concentrations of sugar is the ability to synthesize glycerol and especially to retain it.     

A scalable membrane gradostat reactor for enzyme production using Phanerochaete chrysosporium

This is the first demonstration of process scale-up of a membrane gradostat reactor for continuous enzyme production using Phanerochaete chrysosporium ME446. The fungus was immobilised by reverse filtration on to externally unskinned, ultrafiltration capillary membranes and then nutrient gradients were induced across the biofilm. A 10-fold scale-up from a single capillary bioreactor to a 2.4 l multi-capillary unit resulted in a 7-fold increase in enzyme productivity with a peak at 209 U l^–1 d^–1. Subsequent scale effects on the spore distribution, continuous manganese peroxidase production profile and biofilm development are discussed.     

Scale-up engineering in animal cell technology: Part I

‘Scale-up’ is sometimes loosely used to describe an increase in production capacity. The purpose of any scale-up procedure is to reproduce a given process on a larger scale and to achieve a predictable process result. Scale-up may, therefore, be defined as the ‘predictable’ (engineered) increase in production capacity. Ideally, what the scale-up engineer would like to do is simulate and predict the performance of a large-scale reactor (e.g. 5000 l reactor volume) using only data from a bench-top vessel (e.g. 5 l volume). In order to do this equations are required that correlate the performance of a reactor with its size.In this review we will address some of the current trends and approaches currently being pursued to establish the basis for such calculations. Most of the correlations in animal cell culture processes are based on laboratory models alone, and still await confirmation of their utility as practical industrial tools. Although development work of this kind is under way within the industry, commercial constraints impede publication.Neither the validity of the theoretical concepts underlying these models nor the various possible reactor designs will be reviewed. Rather we will address the approaches to establishing the numerical tools needed to determine the required reactor design, its subsequent optimization, scale-up and scale-down procedures.     

Bioprocess Scale-up/down

Cover illustration: Scale-up and scale-down strategies are a key for successful implementation of bioprocesses in industry. This issue of Biotechnology Journal, edited by Peter Neubauer (TU Berlin, Germany), features articles on methods for improved scale-up and -down strategies, e.g. measuring CO₂ gradients, drop breakage analysis and study of metabolic stress. One review article by Alvin Nienow presents strategies for improved beer production (http://dx.doi.org/10.1002/biot.201000414 ). Cover image: Outdoor fermentation vessels at a Spanish brewery. ©Olaf Hendel (Versuchs- und Lehranstalt für Brauerei in Berlin (VLB) e.V.).     

Continuous ethanol fermentation in a tower reactor with flocculating yeast recycle: scale-up effects on process performance, kinetic parameters and model predictions

An unsegregated and unstructured model developed for a small-scale process of ethanol production in a tower reactor with cell recycle was applied to describe the experimental data obtained in a large-scale process. The model was developed considering the following points: reactor hydrodynamic behavior analogous to that of ideal CSTR, substrate limitation, inhibition phenomena linked both to ethanol and to biomass, absence of fermentation in the settler, and no loss of cell viability. The scale-up criterion consisted in maintaining an identical relation settler volume/fermentor volume on the two scales. All large-scale experiments were carried out using a flocculating yeast strain IR-2, isolated from fermented food, and identified as Saccharomyces cerevisiae. Sugarcane juice was used as the substrate source with sugar concentrations of 150?g/l. Different values of dilution rate and recycle ratio were employed (D?=?0.11–0.33?h^?1, α?=?5.4–18.0) and the temperature was of 32?°C. The kinetic parameters were similar on both scales and the model predictions agreed well with the large-scale experimental data.     

Alcoholic fermentation by agar-immobilized yeast cells

Immobilized yeast cells in agar gel beads were used in a packed bed reactor for the production of ethanol from cane molasses at 30°C, pH 4.5. The maximum productivity, 79.5g ethanol/l.h was obtained with 195g/l reducing sugar as feed. Substrate (64.2%) was utilized at a dilution of 1.33h^-1. The immobilized cell reactor was operated continuously at a constant dilution rate of 0.67h^-1 for 100 days. The maximum specific ethanol productivity and specific sugar uptake rate were 0.610g ethanol/g cell.h and 1.275g sugar/g cell.h, respectively.     

Mortality of Rhagoletis indifferens exposed to hydrolyzed protein baits and spinosad in the absence and presence of yeast extract

Western cherry fruit fly, Rhagoletis indifferens Curran (Diptera: Tephritidae), is the major quarantine pest of sweet cherry, Prunus avium (L.) L. (Rosaceae), in the Pacific northwest of the USA and in British Columbia in Canada. Although spinosad bait (GF‐120 NF Naturalyte® Fruit Fly Bait) is used for the control of R. indifferens in this region, the effects of alternate food sources on fly responses to this bait have not been studied. In this study, experiments were conducted to determine mortalities of flies exposed to hydrolyzed protein baits in the presence of sugar only and sugar + yeast extract food. All baits contained Entrust® (termed ‘spinosad alone’). When flies were exposed to GF‐120 with or without added ammonia compounds (uric acid, ammonium acetate, and ammonium carbonate) for 48 h, mortalities were higher in the presence of sugar only than in the presence of sugar + yeast extract, but when flies were exposed to spinosad alone, mortalities were similar in presence of either of the two foods. In another experiment comparing GF‐120, Nu‐Lure, Mazoferm, Baker's yeast extract, and spinosad alone, mortalities in the GF‐120, Mazoferm, and Baker's yeast extract treatments were higher in the presence of sugar only than in the presence of sugar + yeast extract, but in the Nu‐Lure and spinosad alone treatments, mortalities were similar in the presence of either of the two foods. Overall results suggest that the indirect effects of yeast extract food on mortality are dependent on bait type and that mortalities caused by spinosad alone and baits are similar. Nu‐Lure and spinosad alone may have an advantage over other treatments for fly control, because their effects do not appear to be affected by the presence of nitrogenous food.     

An effective and simplified scale-up strategy for acarbose fermentation based on the carbon source control

The scale-up strategy for acarbose fermentation by Actinoplanes sp. A56 was explored in this paper. The results obtained in shake-flask cultivation demonstrated that the ratio of maltose and glucose had significant effects on the biosynthesis of acarbose, and the feeding medium containing 3:1 (mass ratio) of maltose and glucose was favorable for acarbose production. Then the correlation of the carbon source concentration with acarbose production was further investigated in 100-l fermenter, and the results showed that 7.5–8.0 g of total sugar/100 ml and 4.0–4.5 g of reducing sugar/100 ml were optimal for acarbose production. Based on the results in 100-l fermenter, an effective and simplified scale-up strategy was successfully established for acarbose fermentation in a 30-m³ fermenter, by using total sugar and reducing sugar as the scale-up parameter. As a result, 4,327 mg of acarbose/l was obtained at 168 h of fermentation.     

Continuous ethanol production in an immobilized whole cell fermenter using untreated sugar cane bagasse as carrier

Summary Saccharomyces cerevisiae was immobilised by adsorption to untreated sugar cane bagasse in a packed bed reactor. Complete conversion of glucose to ethanol was obtained at a dilution rate of 0.19 h⁻¹. Continuous ethanol production was maintained for up to 57 days. Reactor productivity increased with increasing packing density of the bagasse. Plugging of void spaces due to cell overgrowth led to channelling of the feed and decreased reactor productivity. Increasing the average column temperature alleviated plugging and restored column performance over a short period; however prolonged exposure to the high temperature resulted in decreased ethanol production rates. Bagasse has advantages as a support material for ethanol production from sugar cane or beet, including negligible cost, ready availability and the capacity to support a high yeast population.     

Prelude to rational scale-up of penicillin production: a scale-down study

Penicillin is one of the best known pharmaceuticals and is also an important member of the β-lactam antibiotics. Over the years, ambitious yields, titers, productivities, and low costs in the production of the β-lactam antibiotics have been stepwise realized through successive rounds of strain improvement and process optimization. Penicillium chrysogenum was proven to be an ideal cell factory for the production of penicillin, and successful approaches were exploited to elevate the production titer. However, the industrial production of penicillin faces the serious challenge that environmental gradients, which are caused by insufficient mixing and mass transfer limitations, exert a considerably negative impact on the ultimate productivity and yield. Scale-down studies regarding diverse environmental gradients have been carried out on bacteria, yeasts, and filamentous fungi as well as animal cells. In accordance, a variety of scale-down devices combined with fast sampling and quenching protocols have been established to acquire the true snapshots of the perturbed cellular conditions. The perturbed metabolome information stemming from scale-down studies contributed to the comprehension of the production process and the identification of improvement approaches. However, little is known about the influence of the flow field and the mechanisms of intracellular metabolism. Consequently, it is still rather difficult to realize a fully rational scale-up. In the future, developing a computer framework to simulate the flow field of the large-scale fermenters is highly recommended. Furthermore, a metabolically structured kinetic model directly related to the production of penicillin will be further coupled to the fluid flow dynamics. A mathematical model including the information from both computational fluid dynamics and chemical reaction dynamics will then be established for the prediction of detailed information over the entire period of the fermentation process and thereby for the optimization of penicillin production, and subsequently also benefiting other fermentation products.     

Improved properties of baker's yeast mutants resistant to 2-deoxy-D-glucose

We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-D-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-D-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties.     

Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli

Industrial 20-m³-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated in a scale-down reactor with a controlled oxygen-limitation compartment. In this scale-down reactor similar results were obtained: i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid product formation (acetate, formate, d-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment. Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful for detecting local oxygen deficiency in large-scale E. coli processes. Received: 7 November 1998 / Received revision: 4 February 1999 / Accepted: 5 February 1999     

Engineering of a robust Escherichia coli chassis and exploitation for large-scale production processes

In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δm_s = 0.038 g_Glucose/g_CDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications.