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.     

Scale-down model to simulate spatial pH variations in large-scale bioreactors

For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2. The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme.     

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.     

Influence of dissolved oxygen concentration on the oxygen kinetics of Gluconobacter oxydans

Summary As shown in earlier studies in production scale bioreactors oxygen limited zones occur. Microorganisms in these reactors are therefore subjected to concentrations of oxygen varying with time. To simulate these conditions, the effect of low oxygen concentrations upon product formation and kinetics of oxygen of Gluconobacter oxydans are studied at laboratory scale.Under these oxygen limited conditions comparable kinetic parameters for oxygen are observed as under normally aerated conditions.So, a saturation constant for oxygen K _{O 2}=6.9 mol/l is observed, which is equivalent to a DOT value of about 3% of air saturation.For optimization purposes of production scale conditions, gassing with oxygen enriched air or with pure oxygen is one of the possibilities.To study the effect of high oxygen concentrations upon kinetics and product formation, the organisms are also cultivated under these extreme conditions. Although at oxygen concentrations larger then 60% saturation with pure oxygen, still growth was observed, the growth rate and also the product formation rate were strongly diminished.From these experiments it can be concluded that gassing with pure oxygen to achieve higher oxygen transfer rates at production scale will be restricted.     

Relationships of Phenylobacterium immobile and purple nonsulfur bacteria on the basis of surface antigens

Abstract Indirect immunofluorescence tests with antisera against whole cells of Phenylobacterium immobile strains revealed a serological relationship to Pseudomonas vesicularis, Aquaspirillum itersonii and Rhodospirillum rubrum , three members of the purple nonsulfur bacteria (group I) and also to Gluconobacter oxydans and Azotobacter vinelandii . Antisera against whole cells of Gluconobacter oxydans and Pseudomonas vesicularis reacted positively with the Phenylobacterium immobile strains, tested. Furthermore, a serological relationship of Gluconobacter oxydans to Acetobacter aceti , and of Pseudomonas vesicularis to Pseudomonas diminuta and Aquaspirillum itersonii could be demonstrated.     

Influence of constant and oscillating dissolved oxygen concentrations on keto acid production by Gluconobacter oxydans subsps. melanogenum.

Gluconobacter species are known to oxidise glucose via a direct oxidation pathway which is distinct from the pentose phosphate pathway. In the present communication results of an investigation on the influence of different dissolved oxygen concentrations (DO) on the production of 2,5-diketogluconic acid in batch and chemostat cultures are given. DO of 30% relative to air at 1 bar was found as a threshold level for optimum productivity. The positive influence of continuous availability of dissolved oxygen on the process of rapid glucose oxidation was unambiguously shown as the result of induction of membrane bound dehydrogenases involved in direct glucose oxidation. Furthermore data of scale-down experiments in which the organism was cultivated under oscillations of dissolved oxygen, are given. The influences of such oscillations of DO in the region of the established threshold (30% saturation) were found to result in a prolonged lag phase for growth and product formation. The data obtained in this study revealed critical residence times at low DO that could be employed as a criterion for scale up of this aerobic process.     

Characterization of production of free gluconic acid by Gluconobacter suboxydans adsorbed on ceramic honeycomb monolith

Gluconobacter suboxydans IFO 3290 was immobilized by adsorption on ceramic honeycomb monolith and continuous production of free gluconic acid from glucose was performed in an aerated reactor. The effects of reactor residence time, aeration rate, and glucose concentration were investigated on the gluconic acid yield. Observation of SEM photographs revealed that the cells were adsorbed with a high density not only on the outer surface of the support but also on the inner surface of large pores. From measurement of the number of the adsorbed cells, it was elucidated that the biofilm comprised a monolayer or bilayer of the cells. Maximum specific rate of growth was estimated for the free and adsorbed cells, and the adsorbed cells were found to grow at a fast rate compared with the free cells. In the continuous fermentation performed for one month at the glucose concentration of 100 kg/m(3), reactor residence time of 3.5 h and aeration rate of 900 cm(3)/min, the activity of the adsorbed cells was appreciably stable. The high productivity of 26.3 kg/(m(3)-reactor . h) was attained with the gluconic acid yield of 84.6% and glucose conversion of 94%.     

Development of a predictive description of an immobilized-cell, three-phase, fluidized-bed bioreactor

A Large bioreactor is an inhomogenous system with concentration gradients which depend on the fluid dynamics and the mass transfer of the reactor, the feeding strategy, the saturation constant, and the cell density. The responses of Escherichia coli cells to short-term oscillations of the carbon/energy substrate in glucose limited fed-batch cultivations were studied in a two-compartment reactor system consisting of a stirred tank reactor (STR) and an aerated plug flow reactor (PFR) as a recycle loop. Short-term glucose excess or starvation in the PFR was simulated by feeding of glucose to the PFR or to the STR alternatively. The cellular response to repeated short-term glucose excess was a transient increase of glucose consumption and acetate formation. But, there was no accumulation of acetate in the culture, because it was consumed in the STR part where the glucose concentration was growth limiting. However, acetate accumulated during the cultivation if the oxygen supply in the PFR was insufficient, causing higher acetate formation. The biomass yield was then negatively influenced, which was also the case if the PFR was used to simulate a glucose starvation zone. The results suggest that short-term heterogeneities influence the cellular physiology and growth, and can be of major importance for the process performance. (c) 1995 John Wiley & Sons, Inc.     

Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain.

We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.     

Kinetics of escherichia coli hydrogen production during short term repeated aerobic-anaerobic fluctuations

A coupled reactor system, consisting of one aerated stirred reactor and one anaerobic plug flow reactor was constructed. Circulation of microbial cells in this system, is chosen to represent the insufficient mixing conditions, present in a large scale vessel. Hereby it is shown, that oscillating oxygen concentrations, give effects on E.coli metabolism. The hydrogen production, resulting from mixed acid fermentation of anaerobically grown cells, is used to indicate the metabolic changes. These changes are measured as hydrogen concentration in the gas outlet of the aerated fermenter, with a Pd-MOS sensor. The dependency of the hydrogen evolution on the anaerobic residence time is shown, and the relevance of this model system for studies on the bioreactor performance, are discussed.     

固定化细胞分布对动力学影响的模型化研究

通过对固定化Gluconobacter oxydans和Bacillus cereus的活细胞系统的研究,提出了固定化细胞不均匀分布模型,将此类分布与均匀分布(最劣分布模型)进行比较,分析了它们对固定化细胞内部的基质浓度分布、有效速率因子和选择率的影响,指出不均匀分布和均匀分布对于该固定化活细胞系统的动力学行为无显著影响,这一模型分析结果在实验中得以验证。通过无因次化分析,建立了适用于固定化生物催化剂动力学研究的模型方法。     

Gluconobacter oxydans: its biotechnological applications

Gluconobacter oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae. G. oxydans is an obligate aerobe, having a respiratory type of metabolism using oxygen as the terminal electron acceptor. Gluconobacter strains flourish in sugary niches e.g. ripe grapes, apples, dates, garden soil, baker's soil, honeybees, fruit, cider, beer, wine. Gluconobacter strains are non-pathogenic towards man and other animals but are capable of causing bacterial rot of apples and pears accompanied by various shades of browning. Several soluble and particulate polyol dehydrogenases have been described. The organism brings about the incomplete oxidation of sugars, alcohols and acids. Incomplete oxidation leads to nearly quantitative yields of the oxidation products making G. oxydans important for industrial use. Gluconobacter strains can be used industrially to produce L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose; and dihydroxyacetone from glycerol. It is primarily known as a ketogenic bacterium due to 2,5-diketogluconic acid formation from D-glucose. Extensive fermentation studies have been performed to characterize its direct glucose oxidation, sorbitol oxidation, and glycerol oxidation. The enzymes involved have been purified and characterized, and molecular studies have been performed to understand these processes at the molecular level. Its possible application in biosensor technology has also been worked out. Several workers have explained its basic and applied aspects. In the present paper, its different biotechnological applications, basic biochemistry and molecular biology studies are reviewed.     

Incapability of Gluconobacter oxydans to produce tartaric acid

The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that trataric acid could only be produced in a medium containing vanadate (NH(4)VO(3)). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5-ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5-ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L-(+)-type. Oxalic acid was formed from 5-ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.     

A comparison of high cell density fed-batch fermentations involving both induced and non-induced recombinant Escherichia coli under well-mixed small-scale and simulated poorly mixed large-scale conditions

In this work, multi-parameter flow cytometric techniques, coupled with dual colour fluorescent staining, have been used to study the metabolic consequences of inclusion body formation in high cell density fed-batch cultures of the recombinant E. coli strain MSD3735, producing the IPTG inducible model mammalian protein, AP50. Further, we report on the development of the scale-down, two compartment (STR + PFR) experimental simulation model to study, for the first time, the effect of a changing microenvironment with respect to three of the major spatial heterogeneities that may be associated with large-scale bioprocessing (pH, glucose and dissolved oxygen concentration) on a recombinant bacterial system. Using various time points for induction and various scale-down configurations, it has been shown that inclusion body formation is followed immediately by a detrimental progressive change in individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability, resulting in a lower final biomass yield. However, the extent of this change was found to be dependent on whether the AP50 protein was induced or not, on the time of induction and on which combination of heterogeneities was being simulated. From this and previous work, it is clear that the scale-down two-compartment model can be used to study the impact of genetically modifying an organism to produce inclusion bodies and any range and combination of potential heterogeneities known to exist at the large scale.     

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 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.     

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.     

Vc 二步发酵中的微生物生态调控

研究了Vc二步混合菌发酵中氧化葡萄糖酸杆菌与巨大芽孢杆菌的生长和相互作用。结果表明,2株混合菌在发酵中可形成一种协同共生,促进2－酮基－L－古龙酸产生;二菌协同共生的过程及条件不同,促进产酸能力亦不同。环境因子影响二菌协同共生,优化环境因子可显著改善二菌协同共生效率,并提高醇到发酵转化率。