RESUMEN
The lactose autoinduction system for recombinant protein production was combined with enzymatic glucose release as a method to provide a constant feed of glucose instead of using glycerol as a carbon substrate. Bioreactor cultivation confirmed that the slow glucose feed does not prevent the induction by lactose. HPLC studies showed that with successful recombinant protein production only a very low amount of lactose was metabolized during glucose-limited fed-batch conditions by the Escherichia coli strain BL21(DE3)pLysS in well-aerated conditions, which are problematic for glycerol-based autoinduction systems. We propose that slow enzymatic glucose feed does not cause a full activation of the lactose operon. However recombinant PDI-A protein (A-domain of human disulfide isomerase) was steadily produced until the end of the cultivation. The results of the cultivations confirmed our earlier observations with shaken cultures showing that lactose autoinduction cultures based on enzymatic glucose feed have good scalability, and that this system can be applied also to bioreactor cultivations.
Asunto(s)
Reactores Biológicos , Glucosa/metabolismo , Lactosa/metabolismo , Proteínas Recombinantes/biosíntesis , Medios de Cultivo/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/farmacología , Humanos , Lactosa/farmacología , Procolágeno-Prolina Dioxigenasa/biosíntesis , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/biosíntesis , Proteína Disulfuro Isomerasas/genética , Proteínas Recombinantes/genéticaRESUMEN
This study was performed to examine the influence of the controlled glucose supply technology, EnBase(®) Flo, on growth and heavy metals uptake capacity of two Bacillus strains isolated from food industry wastewater. Bacillus sp. growth on EnBase Flo (mineral salt complex medium containing starch-derived polymer as substrate) was examined in 24 deep well plates, controlling the glucose amount release by adding two amyloglucosidase concentrations (3 and 6 UL(-1)). Adsorption of the heavy metals Zn(2+), Cd(2+) and Pb(2+) was assessed in a single component system using synthetic metal solutions and as a function of the initial concentration of adsorbate, equilibrium time and removal efficiency. The Langmuir and Freundlich adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants. A pseudo second-order model was applied to describe the uptake rate for two isolates. The EnBase(®) Flo technology improved the cells growth over ten times after 24 h of fed-batch cultivation. The EnBase(®) Flo technology improved the Cd(2+) and Pb(2+) uptake capacity of the bacterial strains by approximately 55 and 44 %, respectively. The biosorption of each metal was fairly rapid (within 30 min), which could be an advantage for large scale treatment of contaminated sites. This initial study may be a basis for future developments to apply EnBase Flo for the biomass production used further as biosorbent for heavy metal removal from aqueous solutions.
Asunto(s)
Bacillus/metabolismo , Contaminantes Ambientales/aislamiento & purificación , Glucosa/metabolismo , Metales Pesados/aislamiento & purificación , Adsorción , Bacillus/genética , Secuencia de Bases , Medios de Cultivo , Cartilla de ADN , Contaminantes Ambientales/metabolismo , Cinética , Metales Pesados/metabolismo , Modelos Teóricos , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Liquid perfluorochemicals (PFCs) are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD) for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase. RESULTS: In EnBase(®) cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 µM oxygen per ml) when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40% compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase. CONCLUSIONS: PFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen-enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation.
Asunto(s)
Escherichia coli/metabolismo , Fluorocarburos/química , Oxígeno/química , Proteínas Recombinantes/biosíntesis , Alcohol Deshidrogenasa/biosíntesis , Alcohol Deshidrogenasa/genética , Escherichia coli/crecimiento & desarrollo , Fluorocarburos/farmacología , Miniaturización , Oxígeno/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production. RESULTS: Cultivation of E. coli in the BIOSTAT CultiBag RM system in a conventional batch mode without control yielded an optical density (OD(600)) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD(600) of 60 (20 g L(-1) cell dry weight). By the use of an internal controlled glucose delivery system, EnBase Flo, OD(600) of 30 (10 g L(-1) cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor. CONCLUSIONS: Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.
Asunto(s)
Alcohol Deshidrogenasa/biosíntesis , Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula/instrumentación , Escherichia coli/crecimiento & desarrollo , Proteínas Recombinantes/biosíntesis , Alcohol Deshidrogenasa/genética , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Recombinantes/genéticaRESUMEN
We studied differences between phagocytic responses to nanoparticles (NPs) versus microparticles in the pulmonary region by synthesizing magnetite of different sizes and instilling suspensions of these particles intratracheally into rats' lungs. Ten and 50 nm particles caused a greater increase in cell counts of the bronchoalveolar lavage fluid (BALF) than the instillation of microparticles. The response to 10 nm particles was weaker than to 50 nm ones, and the smaller NPs were more cytotoxic; both were more cytotoxic than the microparticles. Phagocytic activity was also studied using optical and atomic force microscopy. Phagocytes were more "loaded" in the lungs instilled with 10 nm particles as compared with those instilled with 50 nm particles; NPs of both sizes were engulfed more avidly than microparticles. We found in a separate comparative experiment that magnetite NPs were more cytotoxic than titanium dioxide and quartz suspensions having particle size distribution typical of industrial dusts.
Asunto(s)
Líquido del Lavado Bronquioalveolar/citología , Compuestos Férricos/efectos adversos , Nanopartículas de Magnetita/efectos adversos , Animales , Femenino , Compuestos Férricos/administración & dosificación , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Nanopartículas de Magnetita/administración & dosificación , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Tamaño de la Partícula , Fagocitosis , RatasRESUMEN
A new method is described to remove and separate biosurfactants from complex mixtures by compressing and harvesting the liquid surface layer. This method was applied to Bacillus subtilis cultures, in which the lipopeptide antibiotic fengycin as well as the polyketide antibiotic bacillaene were produced. The automated harvesting and collection in a custom-built glass body called 'flounder' was repeated several hundred times. The fengycin concentration in the fractions was found to be four times higher than in the culture centrifugate. Of the overall fengycin, 50% (w/w) were recovered after 300 cycles, 95% (w/w) after 800 harvesting cycles. A separation of fengycin from the less surface-active bacillaene could be achieved due to stronger surface activity of fengycin. The ratio of partition coefficients of fengycin and bacillaene was nine times higher compared to foam fractionation. A stepwise increase of the equilibrium surface tension in the centrifugate from 29 to 33 mN/m indicated a fractionated separation of different surface-active substances. The utilization of cell containing culture broth instead of centrifugate had only slight effects on separation efficiency. These results demonstrate the possibility to separate biosurfactants directly from cultivation without the use of extraction solvents or foam formation.
Asunto(s)
Microbiología Industrial , Polienos/química , Polienos/aislamiento & purificación , Tensoactivos/química , Tensoactivos/aislamiento & purificación , Adsorción , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Centrifugación , Fraccionamiento Químico , Lipopéptidos , Lipoproteínas/química , Lipoproteínas/aislamiento & purificación , Tensión SuperficialRESUMEN
Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of 200-400 U (gx h)-1 were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of D = 0.01-0.35 with smooth change of D at a rate of 0.003 h-2. EPG production was found to be comparable with a qp of 400 U (gx h)-1 at D = 0.27 h-1. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.
RESUMEN
The enzyme controlled substrate delivery cultivation technology EnBase(®) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.
Asunto(s)
Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos/microbiología , Análisis de Inyección de Flujo/instrumentación , Glucosa Oxidasa/metabolismo , Glucosa/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Análisis de Inyección de Flujo/métodos , Glucosa/química , Proyectos PilotoRESUMEN
The dissolved oxygen concentration is a crucial parameter in aerobic bioprocesses due to the low solubility of oxygen in water. The present study describes a new method for determining the oxygen transfer rate (OTR) in shaken-culture systems based on the sodium sulfite method in combination with an electrochemical oxygen sensor. The method replaces the laborious titration of the remaining sulfite by an on-line detection of the end point of the reaction. This method is a two-step procedure that can be applied in arbitrary flasks that do not allow the insertion of electrodes. The method does not therefore depend on the type of vessel in which the OTR is detected. The concept is demonstrated by determination of the OTR for standard baffled 1-L shake flasks and for opaque Ultra Yield™ flasks. Under typical shaking conditions, k(L) a values in the standard baffled flasks reached values up to 220 h(-1) , whereas the k(L) a values of the Ultra Yield flasks were significantly higher (up to 422 h(-1) ).