Large-scale Bioreactors Research Articles

A dynamic model-based preparation of uniformly-13C-labeled internal standards facilitates quantitative metabolomics analysis of Penicillium chrysogenum

Targeted, quantitative metabolomics can, in principle, provide precise information on intracellular metabolite levels, which can be applied to accurate modeling of intracellular processes required in systems biology and metabolic engineering. However, quantitative metabolite profiling is often hampered by biased mass spectrometry-based analyses caused by matrix effects, the degradation of metabolites and metabolite leakage during sample preparation, and unexpected variation in instrument responses. Isotope Dilution Mass Spectrometry (IDMS) has been proven as the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key has been the acquisition of the corresponding fully uniformly (U) -13C-labeled metabolites to be measured. Here, we have prepared U-13C-labeled cell extracts by cultivating P. chrysogenum in a fed-batch fermentation with fully U-13C-labeled substrates. Towards this goal, a dynamic fed-batch model describing P. chrysogenum growth and penicillin production was used to simulate the fermentation process and design the fed-batch fermentation media. Further, a case study with extensive intracellular metabolomics data from glucose-limited cultivation of Penicillium chrysogenum under both single and repetitive glucose pulses was illustrated by using the IDMS methods with the prepared U-13C-labeled cell extracts as internal standards. In conclusion, the IDMS method can be incorporated into well-established fast sampling and quenching protocols to obtain dynamic quantitative in vivo metabolome data at the timescales of (tens of) seconds and elucidate the underlying regulatory architecture. The case study revealed gross differences between single and repeated pulses, which suggests that single pulse studies have limited value for understanding of metabolic responses in large-scale bioreactors. Instead, intermittent feeding should be favored.

Reproduction of Large-Scale Bioreactor Conditions on Microfluidic Chips.

Microbial cells in industrial large-scale bioreactors are exposed to fluctuating conditions, e.g., nutrient concentration, dissolved oxygen, temperature, and pH. These inhomogeneities can influence the cell physiology and metabolism, e.g., decelerate cell growth and product formation. Microfluidic systems offer new opportunities to study such effects in great detail by examining responses to varying environmental conditions at single-cell level. However, the possibility to reproduce large-scale bioreactor conditions in microscale cultivation systems has not yet been systematically investigated. Hence, we apply computational fluid dynamics (CFD) simulations to analyze and compare three commonly used microfluidic single-cell trapping and cultivation devices that are based on (i) mother machines (MM), (ii) monolayer growth chambers (MGC), and (iii) negative dielectrophoresis (nDEP). Several representative time-variant nutrient concentration profiles are applied at the chip entry. Responses to these input signals within the studied microfluidic devices are comparatively evaluated at the positions of the cultivated cells. The results are comprehensively presented in a Bode diagram that illustrates the degree of signal damping depending on the frequency of change in the inlet concentration. As a key finding, the MM can accurately reproduce signal changes that occur within 1 s or slower, which are typical for the environmental conditions observed by single cells in large-scale bioreactors, while faster changes are levelled out. In contrast, the nDEP and MGC are found to level out signal changes occurring within 10 s or faster, which can be critical for the proposed application.

Suspension culture of Marek’s disease virus and evaluation of its immunological effects

ABSTRACTMarek’s disease virus (MDV) is a cell-associated α-herpesvirus of chickens. It is difficult to grow MDV in suspension culture. Therefore, MDV vaccines are currently produced using adherent primary chicken embryo fibroblasts, and on a large scale this is labour-intensive and costly. In this study, the CVI988 strain of MDV was inoculated into chicken fibroblast cell line UMNSAH/DF-1 (DF-1) cultured by microcarrier suspension for the proliferation experiment. Moreover, the effects of culture conditions, such as inoculation method, multiplicity of infection (MOI), microcarrier concentration, and pH value, on the proliferation of MDV were investigated. The results demonstrated that the maximum viral load of 64.76 ± 2.64 × 106 PFU/flask in a working volume of 100 ml could be obtained using synchronous cell seeding and inoculation method at an MOI of 0.02 and a microcarrier concentration of 5 g/l at pH 7.2. At the same time, the CVI988/DF-1 vaccines prepared by the microcarrier culture process and the traditional adherent cell culture process (CVI988/Rispens) were compared through bird experiments. We found a protective rate of 94.4% using the CVI988/DF-1 vaccine with specific pathogen-free chickens that was equivalent to that of the commercial vaccine CVI988/Rispens (protection rate of 94.1%). In this study, the MDV CVI988/DF-1 vaccine prepared by the microcarrier suspension culture of DF-1 cells could provide effective immune protection for specific pathogen-free chickens, providing a reference for the prevention and control of MD and further development of a large-scale bioreactor for producing the MD vaccine.

A scaled-down model for the translation of bacteriophage culture to manufacturing scale.

Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment of antimicrobial-resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage is being investigated in preclinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimized and streamlined for efficient methods of scalable manufacture. Bacteriophage research is currently limited to laboratory scale studies ranging from 1-20 ml, emerging therapies include bacteriophage cocktails to increase the spectrum of infectivity and require multiple large-scale bioreactors (up to 50 L) containing different bacteriophage-bacterial host reactions. Scaling bioprocesses from the milliliter scale to multi-liter large-scale bioreactors is challenging in itself, but performing this for individual phage-host bioprocesses to facilitate reliable and robust manufacture of phage cocktails increases the complexity. This study used a full factorial design of experiments approach to explore key process input variables (temperature, time of infection, multiplicity of infection, agitation) for their influence on key process outputs (bacteriophage yield, infection kinetics) for two bacteriophage-bacterial host bioprocesses (T4 - Escherichia coli; Phage K - Staphylococcus aureus). The research aimed to determine common input variables that positively influence output yield and found that the temperature at the point of infection had the greatest influence on bacteriophage yield for both bioprocesses. The study also aimed to develop a scaled down shake-flask model to enable rapid optimization of bacteriophage batch bioprocessing and translate the bioprocess into a scale-up model with a 3 L working volume in stirred tank bioreactors. The optimization performed in the shake flask model achieved a 550-fold increase in bacteriophage yield and these improvements successfully translated to the large-scale cultures.

Aeration optimization of large-scale membrane bioreactors in a sewage treatment plant

Abstract Increasing stringency of environmental discharge standards has triggered an industry-wide inclination towards membrane bioreactors over conventional activated sludge processes to ensure fulfilment of environmental discharge criteria. Yet, despite its plentiful advantages, high aeration costs remain as a key deterrent to the widespread adoption of the MBR technology. This backdrop created an impetus for a wastewater treatment company to develop an efficient MBR air scouring protocol that can be realized in existing plants without retrofitting. Known as pulsed cyclic aeration, plant trial applications have demonstrated that fouling control and aeration savings can be improved by >30%, resulting in scouring energy consumptions that can be as low as 0.049 kWh/m3.

Effective bioreactor pH control using only sparging gases.

pH control is critical in bioreactor operations, typically realized through a two-sided control loop, where CO2 sparging and base addition are used in bicarbonate-buffered media. Though a common approach, base addition could compromise culture performance due to the potential impact from pH excursions and osmolality increase in large-scale bioreactors. In this study, the feasibility of utilizing control of sparge gas composition as part of the pH control loop was assessed in Chinese hamster ovary (CHO) fed-batch cultures. Fine pH control was evaluated in multiple processes at different setpoints in small-scale ambr®250 bioreactors. Desired culture pH setpoints were successfully maintained via air sparge feedback control. As part of the pH control loop, air sparging was increased to improve CO2 removal automatically, hence increase culture pH, and vice versa. The effectiveness of this pH control strategy was seamlessly transferred from ambr®250 to 200 L scale, demonstrating scalability of the proposed methodology. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2743, 2019.

Promising strategy to efficiently improve the kinsenoside and polysaccharide production of rhizome cultures of Anoectochilus roxburghii (Wall.) Lindl

Elicitation is an efficient strategy for enhancement of metabolite production during plant cell or organ culture. Salicylic acid (SA) and methyl jasmonate (MeJA) are the wildly used abiotic elicitors. To improve the accumulation of the kinsenoside and polysaccharides in rhizome cultures of Anoectochilus roxburghii (Wall.) Lindl., the present study added SA or MeJA to the culture medium after 30 days of rhizome suspension culture, and the effects of concentration and elicitation time of both elicitors on the kinsensode and polysaccharide accumulation were investigated. SA and MeJA efficiently enhanced contents of the bioactive compounds in concentration- and time-dependent manners. In SA groups, the maximum kinsenoside and polysaccharide contents were determined when rhizomes were treated with 500 μM SA for 12 days; in MeJA groups, kinsenoside and polysaccharide contents reached the highest levels when rhizomes were treated with 550 μM MeJA for 14 days and 16 days, respectively. In addition, the elicitation effects were compared in a large-scale bioreactor system. Kinsenoside and polysaccharide contents were markedly improved by elicitation with SA and MeJA compared with the un-elicited rhizomes and field-grown plants. The SA-treated group produced high kinsenoside (661.4 mg/g DW), which was 1.9-fold more than that from MeJA treatment. No significant difference was found in the polysaccharide content between SA and MeJA treatments. Thus, SA is a promising elicitor to economically improve the industrial production of kinsenoside and polysaccharides during rhizome culture of A. roxburghii.

Operational experience of large-scale membrane bioreactors in an underground sewage treatment plant

Abstract With rapid urbanization, great strains are not only being placed on the production of clean water; there is also an equal, if not, more important need to treat the increasing quantities of wastewaters being produced. With urbanization driving up land prices drastically, it makes good sense to leverage on technologies with smaller plant footprints, like the membrane bioreactor (MBR), as well as to adopt an innovative underground installation of the sewage treatment plant (STP), allowing a non-obnoxious co-existence with nearby residences. Being one of the largest underground STPs, the Jingxi Underground STP in Guangzhou is a 100,000 m3/day MBR-based treatment plant that was commissioned in 2010. The case study described here demonstrates the operational excellence of the STP by illustrating how an optimized internal recirculation for the A2O-MBR process reduced aeration energy demands by 18% while simultaneously improving Total Nitrogen (TN) removal by 24%. Furthermore, the success of a plant trial elucidating conditions that stabilized higher flux operations (to handle surges in water production demands) were also discussed.

PH excursions impact CHO cell culture performance and antibody N-linked glycosylation.

pH excursions exist due to frequent base addition and environmental heterogeneity in large-scale bioreactors. Such excursions could lead to suboptimal culture performance. Here we investigated the impact of pH excursions on cell culture performance and N-linked glycosylation for three MAb-producing Chinese hamster ovary cell lines. Frequent pH excursions were introduced by bolus base addition (in total 2-6% of initial volume, fixed bolus addition distributed from day 2 to 8) into small-scale bioreactors. Base addition led to increase in osmolality, pCO2, and lactate production. Lactate production increase was mainly caused by increased culture pH due to base addition, and bolus addition led to higher glucose and lactate metabolic rates than continuous addition. For the three cell lines studied, antibody galactosylation increased with the increase in cultivating pH, correlating to the decrease in cell-specific productivity. Interestingly, pH excursions led to significantly higher galactosylation for one cell line, which also had a higher response to different cultivating pHs. On the other hand, there was no such substantial impact of pH excursions on galactosylation for the other two cell lines, both of which also had minimal response to cultivating pH. This suggests that the impact of pH excursions on antibody N-linked glycosylation is cell line specific and is closely related to cell line response to cultivating pH.

Quality, safety and efficacy profiling of ginseng adventitious roots produced in vitro.

Ginseng (Panax ginseng C. A. Meyer, Family Araliaceae) is one of the major medicinal and nutraceutical plants, which is native to oriental region. It is used worldwide as a popular herbal medicine because of its pharmacological effects like anti-oxidative, anti aging, anti-cancer, adaptogenic, and other health-improving activities. Chief components of ginseng identified till date are ginsenosides, a group of saponins with triterpenoid structure. Ginseng is cultivated under controlled conditions, and for harvesting of fully grown roots of the plant, the cultivation takes long duration of about 5-7years and cultivated ginseng roots are inferior in quality and ginsenoside content. Wild Mountain ginseng is superior in quality and ginsenoside content but is scarce in nature. Therefore, for obtaining the useful compounds of this plant at commercial scale, cell and organ cultures especially adventitious roots have been established by using superior clones of wild mountain ginseng, ginseng biomass is produced by applying large scale bioreactors. In this paper, an effort has been made to shed light on the scientific literature and to decipher the evidences for quality, safety, and efficacy of ginseng adventitious roots produced from in vitro cultures.

A Framework for the Development of Integrated and Computationally Feasible Models of Large-Scale Mammalian Cell Bioreactors

Industrialization of bioreactors has been achieved by applying several core concepts of science and engineering. Modeling has deepened the understanding of biological and physical phenomena. In this paper, the state of existing cell culture models is summarized. A framework for development of dynamic and computationally feasible models that capture the interactions of hydrodynamics and cellular activities is proposed. Operating conditions are described by impeller rotation speed, gas sparging flowrate, and liquid fill level. A set of admissible operating states is defined over discretized process parameters. The burden on a dynamic solver is reduced by assuming hydrodynamics at its fully developed state and implementation of compartmental modeling. A change in the conditions of operation is followed by hydrodynamics switching instantaneously to the steady state that would be reached under new conditions. Finally, coupling the model with optimization solvers leads to improvements in operation.

Methodical Aspects of Biogas Production in Small-Volume Bioreactors in Laboratory Investigations

The aim of this study was to develop a methodology to investigate the biofermentation process in small-volume fermenters. Dark serum bottles with a volume of 100–120 mL, tightly sealed with a rubber septum, were used as bioreactors. The optimum measurement conditions in this type of bioreactor comprise: (i) filling two-thirds of the maximum volume with a suspension; (ii) a 2% bioreactor loading (on a dry basis) and; (iii) the daily equalization of pressure by removing the biogas through the septum pierced with a syringe needle and the intensive mixing of the remaining suspension. The methane yield (quantity and dynamics) obtained in this type of bioreactor is analogous to that of industrial bioreactors or large-scale laboratory bioreactors. The use of small-volume bioreactors that can be incubated will facilitate the preliminary selection of analysed systems and provide an indication of those that should be investigated in large-scale bioreactors.

Importance of the cultivation history for the response of Escherichia coli to oscillations in scale-down experiments.

Large-scale bioreactors are inhomogeneous systems, in which the fluid phase expresses concentration gradients. They depend on the mass transfer and fluid dynamics in the reactor, the feeding strategy, the cell-specific substrate uptake parameters, and the cell density. As high cell densities are only obtained at low specific growth rates, it is necessary to investigate the cellular responses to oscillations in particular under such conditions, an issue which is mostly neglected. Instead, the feed oscillations are often started directly after the batch phase, when the specific growth rate is close to the maximum. We show here that the cultivation mode before oscillations are started has a tremendous effect on the metabolic responses. In difference to cells, which were pre-grown under batch conditions at a high growth rate, Escherichia coli cells that were pre-grown under glucose limitation at a low growth rate accumulate short-chain fatty acids (acetate, lactate, succinate) and glycolysis-related amino acids to a higher extent in a two-compartment scale-down bioreactor. Thus, cells which enter oscillations from a lower specific growth rate seem to react more sensitive to oscillations than cells that are subjected to oscillations directly after a batch phase. These results are interesting in designing reliable scale-down systems, which better reflect large-scale bioprocesses.

Computational studies of a novel magnetically driven single-use-technology bioreactor: A comparison of mass transfer models

This work applies computational fluid dynamics (CFD) modelling to a novel 1000 L design of single-use-technology (SUT) bioreactor, with a magnetically driven floor-mounted impeller and spargers distributed across the tank floor. A two-phase Euler-Euler model using the k-ε turbulence model and population balance is presented alongside the use of immersed solid method for modelling the impeller motion. This work also provides the first CFD analysis of a large-scale SUT bioreactor, identifying key flow characteristics of the non-standard design at different operating conditions. Five models for the mass transfer coefficient, kL, are compared, with kLa values compared to experimental measurements. The slip velocity model is found to be the best prediction of the mass transfer coefficient for this SUT system. Separating the influence of the mass transfer coefficient and specific area,a shows that the latter is the dominant driving force behind changes in kLa that occur at different operating conditions. Comparing the present work to previous studies for traditional stirred tanks highlights the need for understanding the hydrodynamics of non-standard reactor designs when identifying suitable mass transfer models in gas–liquid flow systems.

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized.

Characterizing the preparation of a concentrated nutrient feed solution for a large-scale cell culture process

Fed-batch mammalian cell culture processes frequently employ concentrated and complex media and feeds during the production phase. Though significant efforts are undertaken to characterize the large-scale bioreactor with respect to its control parameters, similar efforts are not commonly undertaken to characterize the feed preparation process. During the characterization of a commercial cell culture process using elements of Quality-by-Design (QbD) principles, we identified the complex nutrient feed solution preparation as an important step and temperature, mixing time, and mixing intensity were identified as factors impacting this unit operation via a systematic risk assessment. To best simulate the large-scale feed preparation process, a 3L model was developed to determine the impact of the feed preparation conditions. Feed preparation parameters were characterized and the solutions generated were analyzed using various analytical methods, a filterability assessment, and cell culture use tests that included evaluation of culture performance and relevant product quality attributes. Based on the data from the characterization studies, appropriate ranges for an allowable multivariate operating space for large scale feed preparation were determined. These studies demonstrated how the preparation of complex nutrient feed solutions could be modeled and characterized to ensure the success of a cell culture manufacturing campaign.

Probing lactate metabolism variations in large-scale bioreactors.

Lactate metabolism variations are frequently encountered in mammalian cell culture processes, especially during process scale-up. In this study, we took a multipronged approach to investigate the impact of pH, pCO2 , osmolality, base addition, and mixing conditions on the observed lactate variations in a Chinese Hamster Ovary (CHO) fed-batch process at 2,000 L scale. Two cultivating methods, CO2 -controlled and pH-controlled, were used to decouple the individual and synergistic effects from those factors. The individual effects from pH, pCO2 , and osmolality on lactate consumption/reproduction in the stationary phase were insignificant in the ranges studied though the initial lactate production rates varied. In contrast, lactate metabolism was found to be impacted by an interaction between mixing conditions and CO2 accumulation. High CO2 accumulation and poor mixing led to lactate reproduction, whereas either low CO2 or improved mixing were sufficient to result in lactate consumption. Base addition was not required for pH control in the low CO2 conditions, and therefore lactate reproduction was correlated with base addition under poor mixing conditions. Under good mixing conditions, CO2 -triggered base addition did not significantly impact lactate reproduction. It is reasonable to postulate that increased mixing times further promoted lactate production during base addition. As lactate reproduction results in more base addition to maintain pH, a cycle could be formed between lactate production and base addition. As a remediation, we showed that such lactate reproduction could be eliminated by improving CO2 removal at 2,000 L scale. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:756-766, 2018.

Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors.

Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants. Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (PAOX1 ), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol dependent expression strains into methanol free systems. While constitutive overexpression showed only marginal or detrimental effects, derepressed expression (activated when the repressing carbon source is depleted) showed that three transcription factors (TFs) are single handedly suitable to strongly activate PAOX1 in P. pastoris without relying on any specifically engineered host strains. Transcriptome analyses demonstrated that Mxr1, Mit1, and Prm1 regulate partly overlapping and unique sets of genes. Derepressed overexpression of a single TF was sufficient to retrofit existing PAOX1 based expression strains into glucose/glycerol regulated, methanol-free systems. Given the wide applicability of carbon source regulated promoters, the simplicity and low cost of controlling carbon source feed rates in large scale bioreactors, similar approaches as in P. pastoris may also be useful in other organisms.

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale-down cultivations.

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large-scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production-scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two- and three-compartment scale-down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab-scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale-down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three-compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single-cell size distribution was observed under oscillatory growth conditions with three-dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single-cell-based analysis provides useful information about the homogeneity of responses among the population.

Microparticle-enhanced polygalacturonase production by wild type Aspergillus sojae.

Polygalacturonases (PGs), an important industrial enzyme group classified under depolymerases, catalyze the hydrolytic cleavage of the polygalacturonic acid chain through the introduction of water across the oxygen bridge. In order to produce and increase the concentration of this enzyme group in fermentation processes, a new approach called microparticle cultivation, a promising and remarkable method, has been used. The aim of this study was to increase the PG activity of Aspergillus sojae using aluminum oxide (Al2O3) as microparticles in shake flask fermentation medium. Results indicated that the highest PG activity of 34.55±0.5 U/ml was achieved with the addition of 20g/L of Al2O3 while the lowest activity of 15.20±0.2 U/mL was obtained in the presence of 0.1g/L of Al2O3. In fermentation without microparticles as control, the activity was 15.64±3.3 U/mL. Results showed that the maximum PG activity was 2.2-fold higher than control. Additionally, smaller pellets formed with the addition of Al2O3 where the lowest pellet diameter was 955.1µm when 10g/L of the microparticle was used. Also, it was noticed that biomass concentration gradually increased with increasing microparticle concentration in the fermentation media. Consequently, the PG activity was significantly increased in microparticle-enhanced shake flask fermentation. In fact, these promising preliminary data can be of significance to improve the enzyme activity in large-scale bioreactors.