RESUMEN
The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.
Asunto(s)
Sulfolobus acidocaldarius , Extremófilos/fisiología , Adaptación Fisiológica/fisiología , Técnicas de Cultivo Celular por Lotes/métodos , Técnicas de Cultivo Celular por Lotes/instrumentaciónRESUMEN
Most pharmaceutical biotechnology companies use stirred-tank bioreactors (STR) for recombinant protein manufacturing. These bioreactors are used at a variety of different scales ranging from bench to production scales, with working volumes from 10 mL to 25,000 L. Bench-scale STRs are commonly used to culture mammalian cells for process development, to troubleshoot production scale bioreactors using scale-down models (SDM), or to conduct fundamental research. In this chapter, we describe the operations of a bench-scale STR for the production of recombinant proteins with suspension-adapted Chinese hamster ovary (CHO) cells. These operations include bioreactor setup and configuration, batching media, inoculation of the seed cell culture, production phase, and harvest of cell-free fluids.
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Reactores Biológicos , Cricetulus , Proteínas Recombinantes , Células CHO , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cricetinae , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo Celular por Lotes/métodos , Técnicas de Cultivo Celular por Lotes/instrumentaciónRESUMEN
The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high cell densities (HCD) and improved productivities. HCDs are achieved by cell retention and continuous medium exchange, which is often based on the cell-specific perfusion rate (CSPR). To obtain a cost-productive process the perfusion rate must be determined for each process individually. However, determining optimal operating conditions remain labor-intensive and time-consuming experiments, as investigations are performed in lab-scale perfusion bioreactors. Small-scale models such as microwell plates (MWPs) provide an option for screening multiple perfusion rates in parallel in a semi-perfusion mimic. This study investigated two perfusion rate strategies applied to the MWP platform operated in semi-perfusion. The CSPR-based perfusion rate strategy aimed to maintain multiple CSPR values throughout the cultivation and was compared to a cultivation with a perfusion rate of 1 RV d-1. The cellular performance was investigated with the dual aim (i) to achieve HCD, when inoculating at conventional and HCDs, and (ii) to maintain HCDs, when applying an additional manual cell bleed. With both perfusion rate strategies viable cell concentrations up to 50 × 106 cells mL-1 were achieved and comparable results for key metabolites and antibody product titers were obtained. Furthermore, the combined application of cell bleed and CSPR-based medium exchange was successfully shown with similar results for growth, metabolites, and productivities, respectively, while reducing the medium consumption by up to 50% for HCD cultivations.
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Reactores Biológicos , Cricetulus , Células CHO , Animales , Perfusión/métodos , Perfusión/instrumentación , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Recuento de Células , Técnicas de Cultivo Celular por Lotes/métodos , Técnicas de Cultivo Celular por Lotes/instrumentaciónRESUMEN
Model-based state estimators enable online monitoring of bioprocesses and, thereby, quantitative process understanding during running operations. During prolonged continuous bioprocesses strain physiology is affected by selection pressure. This can cause time-variable metabolic capacities that lead to a considerable model-plant mismatch reducing monitoring performance if model parameters are not adapted accordingly. Variability of metabolic capacities therefore needs to be integrated in the in silico representation of a process using model-based monitoring approaches. To enable online monitoring of multiple concentrations as well as metabolic capacities during continuous bioprocessing of spent sulfite liquor with Corynebacterium glutamicum, this study presents a particle filtering framework that takes account of parametric variability. Physiological parameters are continuously adapted by Bayesian inference, using noninvasive off-gas measurements. Additional information on current parameter importance is derived from time-resolved sensitivity analysis. Experimental results show that the presented framework enables accurate online monitoring of long-term culture dynamics, whereas state estimation without parameter adaption failed to quantify substrate metabolization and growth capacities under conditions of high selection pressure. Online estimated metabolic capacities are further deployed for multiobjective optimization to identify time-variable optimal operating points. Thereby, the presented monitoring system forms a basis for adaptive control during continuous bioprocessing of lignocellulosic by-product streams.
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Técnicas de Cultivo Celular por Lotes/métodos , Corynebacterium glutamicum , Azúcares/metabolismo , Técnicas de Cultivo Celular por Lotes/instrumentación , Teorema de Bayes , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Diseño de Equipo , Modelos Biológicos , Dinámicas no LinealesRESUMEN
By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have been improved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 1011 TCID50 /Lbioreactor /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.
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Reactores Biológicos/virología , ADN Viral/metabolismo , Virus Vaccinia , Cultivo de Virus , Animales , Técnicas de Cultivo Celular por Lotes/instrumentación , Técnicas de Cultivo Celular por Lotes/métodos , Recuento de Células , Línea Celular , Cromatografía en Gel , Costos y Análisis de Costo , Patos , Diseño de Equipo , Virus Vaccinia/aislamiento & purificación , Virus Vaccinia/metabolismo , Cultivo de Virus/instrumentación , Cultivo de Virus/métodosRESUMEN
OBJECTIVE: The novel engineered bioprocess, which was designed and modeled to provide the clinically relevant cell numbers for different therapies in our previous work (Kaleybar et al. Food Bioprod Process 122:254-268, https://doi.org/10.1016/j.fbp.2020.04.012 , 2020), was evaluated by using U937 as hematopoietic model cells. RESULTS: The culture system showed a 30-fold expansion of U937 cells in one-step during a 10-day culture period. The cell growth profile, the substrate and oxygen consumptions, and byproduct formations were all in agreement with the model predications during 7 days. The cell proliferation decrease after 7 days was attributed to optional oxygen limiting condition in the last days of culture. The bioreactor culture system revealed also a slight enhancement of lactate dehydrogenase (LDH) production as compared to the 2D conventional culture system, indicating the low impact of shear stress on cellular damage in the dynamic system. CONCLUSIONS: The results demonstrated that the conceptual bioprocess for suspended stem cell production has a great potential in practice although additional experiments are required to improve the system.
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Técnicas de Cultivo Celular por Lotes/métodos , Células Madre Hematopoyéticas/citología , Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos , Proliferación Celular , Supervivencia Celular , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Modelos Biológicos , Oxígeno/análisis , Células U937RESUMEN
A continuous Chinese hamster ovary (CHO) cell culture process comprised of a highly proliferative N-1 perfusion bioreactor utilizing a hydrocyclone as a cell retention device linked to a production continuous-flow stirred tank reactor (CSTR) is presented. The overflow stream from the hydrocyclone, which is only partially depleted of cells, provides a continuous source of high viability cells from the N-1 perfusion bioreactor to the 5-20 times larger CSTR. Under steady-state conditions, this linked-bioreactor system achieved a peak volumetric productivity of 0.96 g/L/day, twofold higher than the optimized fed-batch process. The linked bioreactor system using a hydrocyclone was also shown to be 1.8-3.1 times more productive than a dual, cascading CSTR system without cell retention.
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Técnicas de Cultivo Celular por Lotes/instrumentación , Técnicas de Cultivo Celular por Lotes/métodos , Reactores Biológicos , Animales , Células CHO , Cricetinae , Cricetulus , Diseño de Equipo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismoRESUMEN
Syngas fermentation is a potential player for future emission reduction. The first demonstration and commercial plants have been successfully established. However, due to its novelty, development of syngas fermentation processes is still in its infancy, and the need to systematically unravel and understand further phenomena, such as substrate toxicity as well as gas transfer and uptake rates, still persists. This study describes a new online monitoring device based on the respiration activity monitoring system for cultivation of syngas fermenting microorganisms with gaseous substrates. The new device is designed to online monitor the carbon dioxide transfer rate (CO2 TR) and the gross gas transfer rate during cultivation. Online measured data are used for the calculation of the carbon monoxide transfer rate (COTR) and hydrogen transfer rate (H2 TR). In cultivation on pure CO and CO + H2 , CO was continuously limiting, whereas hydrogen, when present, was sufficiently available. The maximum COTR measured was approximately 5 mmol/L/h for pure CO cultivation, and approximately 6 mmol/L/h for cultivation with additional H2 in the gas supply. Additionally, calculation of the ratio of evolved carbon dioxide to consumed monoxide, similar to the respiratory quotient for aerobic fermentation, allows the prediction of whether acetate or ethanol is predominantly produced. Clostridium ljungdahlii, a model acetogen for syngas fermentation, was cultivated using only CO, and CO in combination with H2 . Online monitoring of the mentioned parameters revealed a metabolic shift in fermentation with sole CO, depending on COTR. The device presented herein allows fast process development, because crucial parameters for scale-up can be measured online in small-scale gas fermentation.
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Técnicas de Cultivo Celular por Lotes/instrumentación , Técnicas de Cultivo Celular por Lotes/métodos , Monóxido de Carbono , Fermentación/fisiología , Hidrógeno , Monóxido de Carbono/análisis , Monóxido de Carbono/metabolismo , Clostridium/metabolismo , Hidrógeno/análisis , Hidrógeno/metabolismoRESUMEN
The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.
Asunto(s)
Agrobacterium tumefaciens/crecimiento & desarrollo , Agrobacterium tumefaciens/metabolismo , Reactores Biológicos/microbiología , Agricultura Molecular/métodos , Proteínas Bacterianas/biosíntesis , Técnicas de Cultivo Celular por Lotes/instrumentación , Técnicas de Cultivo Celular por Lotes/métodos , ProteómicaRESUMEN
Pathogenic bacterial biofilms can be life-threatening, greatly decrease patient's quality of life, and are a substantial burden on the healthcare system. Current methods for evaluation of antibacterial treatments in clinics and in vitro systems used in drug development and screening either do not facilitate biofilm formation or are cumbersome to operate, need large reagent volumes, and are costly, limiting their usability. To address these issues, this work presents the development of a robust in vitro cell culture platform compatible with confocal microscopy. The platform shaped as a compact disc facilitates long-term bacterial culture without external pumps and tubing and can be operated for several days without additional liquid handling. As an example, Pseudomonas aeruginosa biofilm is grown from single cells, and it is shown that (1) the platform delivers reproducible and reliable results; (2) growth is dependent on flow rate and growth medium composition; and (3) efficacy of antibiotic treatment depends on the formed biofilm. This platform enables biofilm growth, quantification, and treatment as in a conventional flow setup while decreasing the application barrier of lab-on-chip systems. It provides an easy-to-use, affordable option for end users working with cell culturing in relation to, e.g., diagnostics and drug screening.
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Antibacterianos/farmacología , Técnicas de Cultivo Celular por Lotes/métodos , Biopelículas/efectos de los fármacos , Dispositivos Laboratorio en un Chip , Microscopía Confocal/métodos , Pseudomonas aeruginosa , Técnicas de Cultivo Celular por Lotes/instrumentación , Biopelículas/crecimiento & desarrollo , Biomasa , Pseudomonas aeruginosa/fisiologíaRESUMEN
The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.
Asunto(s)
Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos/virología , Filtración/instrumentación , Tamaño de la Partícula , Perfusión/métodos , Virus/crecimiento & desarrollo , Línea Celular , Membranas Artificiales , Virus/aislamiento & purificaciónRESUMEN
In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20-25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.
Asunto(s)
Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Recuento de Células , Separación Celular , Supervivencia Celular , Cricetulus , Diseño de Equipo , Hidrodinámica , Perfusión/instrumentaciónRESUMEN
The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.
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Técnicas de Cultivo Celular por Lotes/instrumentación , Vacunas Neumococicas/inmunología , Streptococcus pneumoniae/inmunología , Animales , Técnicas de Cultivo Celular por Lotes/métodos , Biomasa , Reactores Biológicos , Diseño de Equipo , Femenino , Humanos , Inmunización , Ratones Endogámicos C57BL , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/uso terapéutico , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/prevención & control , Streptococcus pneumoniae/citologíaRESUMEN
The critical process parameters cell density and viability during mammalian cell cultivation are assessed by UV/VIS spectroscopy in combination with multivariate data analytical methods. This direct optical detection technique uses a commercial optical probe to acquire spectra in a label-free way without signal enhancement. For the cultivation, an inverse cultivation protocol is applied, which simulates the exponential growth phase by exponentially replacing cells and metabolites of a growing Chinese hamster ovary cell batch with fresh medium. For the simulation of the death phase, a batch of growing cells is progressively replaced by a batch with completely starved cells. Thus, the most important parts of an industrial batch cultivation are easily imitated. The cell viability was determined by the well-established method partial least squares regression (PLS). To further improve process knowledge, the viability has been determined from the spectra based on a multivariate curve resolution (MCR) model. With this approach, the progress of the cultivations can be continuously monitored solely based on an UV/VIS sensor. Thus, the monitoring of critical process parameters is possible inline within a mammalian cell cultivation process, especially the viable cell density. In addition, the beginning of cell death can be detected by this method which allows us to determine the cell viability with acceptable error. The combination of inline UV/VIS spectroscopy with multivariate curve resolution generates additional process knowledge complementary to PLS and is considered a suitable process analytical tool for monitoring industrial cultivation processes.
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Recuento de Células , Supervivencia Celular , Espectrofotometría Ultravioleta/instrumentación , Animales , Técnicas de Cultivo Celular por Lotes/instrumentación , Células CHO , Cricetulus , Diseño de Equipo , Análisis de los Mínimos CuadradosRESUMEN
The aim of this work is to establish a large volume batch production system to produce sufficient volumes of ghost cells to facilitate hemolysis testing of mechanical circulatory support devices. A volume of more than 405 mL with a hematocrit of at least 28% is required to perform in vitro hemolysis testing of mechanical circulatory support devices according to international standards. The established ghost cell production method performed at the institute is limited to 3.1 mL of concentrated cells, that is, cells with 100% hematocrit, due to predominantly manual process steps. Through semi-automation of the existing method by using the large volume batch production system, productivity is increased 60-fold to 188 mL while almost doubling process efficiency to 23.5%. Time-consuming manual work such as pipetting is now supported by sensor-based process engineering. With the help of the large volume batch production system, the objective of producing large quantities of ghost cells is successfully achieved. Thus, this work lays the foundation for spatially resolved hemolysis evaluation of mechanical circulatory support devices in combination with the small-scale fluorescent hemolysis detection method.
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Técnicas de Cultivo Celular por Lotes/métodos , Hemólisis , Técnicas In Vitro/instrumentación , Técnicas In Vitro/métodos , Técnicas de Cultivo Celular por Lotes/instrumentación , Biotecnología/instrumentación , Biotecnología/métodos , Eritrocitos , Colorantes Fluorescentes , Hematócrito/métodosRESUMEN
OBJECTIVES: The metabolic pathway related to uridine production was modified in Bacillus subtilis in order to increase the production of uridine. RESULTS: Decreasing the relative transcriptional level of pur operon in Bacillus subtilis TD300 to 80%, and the production of the derived strain TD312 was increased to 11.81 g uridine/l and the yield was increased to 270 mg uridine/g glucose. The expression of pucR gene in situ by PccpA resulting in a 194.01-fold increase in the relative transcriptional level of pucR gene and 349.71-fold increase in the relative transcriptional level of ure operon, respectively. Furthermore, the production of TD314 reached 13.06 g uridine/l, while the yield reached 250 mg uridine/g glucose. CONCLUSION: This is the first report that more than 13 g uridine/l with a yield of 250 mg uridine/g glucose is produced in shake flask fermentation of genetically engineered Bacillus subtilis.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Regulación hacia Abajo , Redes y Vías Metabólicas , Uridina/biosíntesis , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Técnicas de Cultivo Celular por Lotes/instrumentación , Fermentación , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Glucosa/metabolismo , Mutagénesis Sitio-Dirigida , OperónRESUMEN
OBJECTIVES: To isolate a novel cis-epoxysuccinate hydrolase (CESH)-producing fungus for production of L( +)-tartaric acid, before this, all strains were selected from bacteria. RESULTS: A CESH-producing fungus was first isolated from soil and identified as Aspergillus niger WH-2 based on its morphological properties and ITS sequence. The maximum activity of hyphaball and fermentation supernatants was 1278 ± 64 U/g and 5.6 ± 0.3 U/mL, respectively, in a 5 L fermenter based on the conditions optimized on the flask. Almost 70% of CESH was present in hyphaball, which maintained 40% residual activity at pH 4.0 and showed a good acid stability (pH 3.0-10.0), high conversion rate (> 98%), and enantioselectivity (EE > 99.6%). However, the reported CESHs from bacteria can't be catalyzed under acidic conditions. CONCLUSIONS: The Aspergillus niger WH-2 was the first reported CESH-producing fungus, which could biosynthesize L ( +)-tartaric acid under acidic conditions and provide an alternative catalyst and process.
Asunto(s)
Aspergillus niger/crecimiento & desarrollo , Aspergillus niger/aislamiento & purificación , Tartratos/metabolismo , Ácidos/química , Aspergillus niger/clasificación , Técnicas de Cultivo Celular por Lotes/instrumentación , Fermentación , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Hidrolasas/metabolismo , Filogenia , Microbiología del SueloRESUMEN
OBJECTIVES: Acetyl-CoA is a precursor for phloroglucinol (PG), and pyruvate is one of the sources of intracellular acetyl-CoA. Therefore, enhancing intracellular pyruvate levels may help to improve the anabolic pathway of PG. RESULTS: In this study, the effects of phosphoenolpyruvate carboxykinase (PckA, encoded by pckA) or triosephosphate isomerase (TpiA, encoded by tpiA) overexpression on the production of PG were studied. Overexpression of pckA or tpiA could enhance the pyruvate anabolic pathway in shake-flask culture compared to the control strain, and the concentration of PG also increased by 44% and 92%, respectively. In addition, the acetate levels were all down regulated by the overexpression of the two genes to some extent and lower acetate level resulted in lower ATP pool and higher survival rate. CONCLUSIONS: These results indicate that overexpression of pckA or tpiA can enhance the pyruvate "pool" and PG production in Escherichia coli, which provides a new reference for further increasing the production of PG.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Floroglucinol/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Ácido Pirúvico/metabolismo , Triosa-Fosfato Isomerasa/metabolismo , Técnicas de Cultivo Celular por Lotes/instrumentación , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentación , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Plásmidos/genética , Transformación Bacteriana , Triosa-Fosfato Isomerasa/genéticaRESUMEN
In a biotechnological process, standard monitored process variables are pH, partial oxygen pressure (pO2), and temperature. These process variables are important, but they do not give any information about the metabolic activity of the cell. The ISICOM is an in situ combi-sensor that is measuring the cell-specific oxygen uptake rate (qOUR) online. This variable allows a qualitative judgement of metabolic cell activity. The measuring principle of the ISICOM is based on a volume element enclosed into a small measuring chamber. Inside the measuring chamber, the pO2 and the scattered light is measured. Within a defined measuring interval, the chamber closes, and the oxygen supply for the cells is interrupted. The decreasing oxygen concentration is recorded by the pO2 optode. This measuring principle, known as the dynamic method, determines the oxygen uptake rate (OUR). Together with the scattered light signal, the cell concentration is estimated and the qOUR is available online. The design of the ISICOM is focused on functionality, sterility, long-term stability, and response time behavior so the sensor can be used in bioprocesses. With the ISICOM, measurement of online and in situ measurement of the OUR is possible. The OUR and qOUR online measurement of an animal cell batch cultivation is demonstrated, with maximum values of OUR = 2.5 mmol L-1 h-1 and a qOUR = 9.5 pmol cell-1 day-1. Information about limitation of the primary and secondary substrate is derived by the monitoring of the metabolic cell activity of bacteria and yeast cultivation processes. This sensor contributes to a higher process understanding by offering an online view on to the cell behavior. In the sense of process analytical technology (PAT), this important information is needed for bioprocesses to realize a knowledge base process control.
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Técnicas Biosensibles/instrumentación , Oxígeno/metabolismo , Animales , Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos , Células CHO , Cricetulus , Diseño de Equipo , Escherichia coli/metabolismo , Oxígeno/análisis , Saccharomycetales/metabolismoRESUMEN
Real-time measurements and adjustments of critical process parameters are essential for the precise control of fermentation processes and thus for increasing both quality and yield of the desired product. However, the measurement of some crucial process parameters such as biomass, product, and product precursor concentrations usually requires time-consuming offline laboratory analysis. In this work, we demonstrate the in-line monitoring of biomass, penicillin (PEN), and phenoxyacetic acid (POX) in a Penicilliumchrysogenum fed-batch fermentation process using low-cost microspectrometer technology operating in the near-infrared (NIR). In particular, NIR reflection spectra were taken directly through the glass wall of the bioreactor, which eliminates the need for an expensive NIR immersion probe. Furthermore, the risk of contaminations in the reactor is significantly reduced, as no direct contact with the investigated medium is required. NIR spectra were acquired using two sensor modules covering the spectral ranges 1350-1650 nm and 1550-1950 nm. Based on offline reference analytics, partial least squares (PLS) regression models were established for biomass, PEN, and POX either using data from both sensors separately or jointly. The established PLS models were tested on an independent validation fed-batch experiment. Root mean squared errors of prediction (RMSEP) were 1.61 g/L, 1.66 g/L, and 0.67 g/L for biomass, PEN, and POX, respectively, which can be considered an acceptable accuracy comparable with previously published results using standard process spectrometers with immersion probes. Altogether, the presented results underpin the potential of low-cost microspectrometer technology in real-time bioprocess monitoring applications. Graphical abstract.