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Enrique Galindo - One of the best experts on this subject based on the ideXlab platform.
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molecular mass of poly 3 hydroxybutyrate p3hb produced by azotobacter vinelandii is determined by the ratio of synthesis and degradation under fixed Dissolved Oxygen Tension
Process Biochemistry, 2016Co-Authors: Modesto Millan, Enrique Galindo, Daniel Segura, Carlos PeñaAbstract:Abstract Poly-3-hydroxybutyrate (P3HB) is an intracellular polyester produced by numerous bacteria, including Azotobacter vinelandii . Thermo-mechanical properties and biomedical applications of P3HB depend on its molecular mass (MM), which in turn is controlled by the balance between synthesis and degradation of the polymer during its biosynthesis. The aim of this study was to determine the activity levels of enzymes involved in the synthesis and degradation of P3HB and their effect on the molecular mass of the polymer produced by A. vinelandii strain OP under the conditions of Dissolved Oxygen Tension of 1 and 15%. The results show that the MM of P3HB changed between the exponential and stationary growth phases, under both Oxygen conditions. During the exponential growth phase, the mean molecular mass (MMM) of P3HB was high (4800 kDa), coincident with a high activity P3HB synthase and with a low activity of P3HB depolymerase. In contrast, during the stationary phase, the P3HB MM decreased to 3600 kDa, because of the increased activity of the P3HB depolymerase.
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analysis of respiratory activity and carbon usage of a mutant of azotobacter vinelandii impaired in poly β hydroxybutyrate synthesis
Journal of Industrial Microbiology & Biotechnology, 2016Co-Authors: Lucero Jimenez, Enrique Galindo, Daniel Segura, Tania Castillo, Celia Flores, Carlos PeñaAbstract:In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-β-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific Oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed Dissolved Oxygen Tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).
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Oxygen transfer rate during the production of alginate by azotobacter vinelandii under Oxygen limited and non Oxygen limited conditions
Microbial Cell Factories, 2011Co-Authors: Esteban Lozano, Enrique Galindo, Carlos PeñaAbstract:Background The Oxygen transfer rate (OTR) and Dissolved Oxygen Tension (DOT) play an important role in determining alginate production and its composition; however, no systematic study has been reported about the independent influence of the OTR and DOT. In this paper, we report a study about alginate production and the evolution of the molecular mass of the polymer produced by a wild-type A. vinelandii strain ATCC 9046, in terms of the maximum Oxygen transfer rate (OTRmax) in cultures where the Dissolved Oxygen Tension (DOT) was kept constant.
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Oxygen transfer rate during the production of alginate by Azotobacter vinelandii under Oxygen-limited and non Oxygen-limited conditions
Microbial Cell Factories, 2011Co-Authors: Esteban Lozano, Enrique Galindo, Carlos F PeñaAbstract:Background The Oxygen transfer rate (OTR) and Dissolved Oxygen Tension (DOT) play an important role in determining alginate production and its composition; however, no systematic study has been reported about the independent influence of the OTR and DOT. In this paper, we report a study about alginate production and the evolution of the molecular mass of the polymer produced by a wild-type A. vinelandii strain ATCC 9046, in terms of the maximum Oxygen transfer rate (OTR_max) in cultures where the Dissolved Oxygen Tension (DOT) was kept constant. Results The results revealed that in the two Dissolved Oxygen conditions evaluated, strictly controlled by gas blending at 0.5 and 5% DOT, an increase in the agitation rate (from 300 to 700 rpm) caused a significant increase in the OTR_max (from 17 to 100 mmol L^-1 h^-1 for DOT of 5% and from 6 to 70 mmol L^-1 h^-1 for DOT of 0.5%). This increase in the OTR_max improved alginate production, as well as the specific alginate production rate (SAPR), reaching a maximal alginate concentration of 3.1 g L^-1 and a SAPR of 0.031 g _alg g _biom^-1 h^-1 in the cultures at OTR_max of 100 mmol L^-1 h^-1. In contrast, the mean molecular mass (MMM) of the alginate isolated from cultures developed under non-Oxygen limited conditions increased by decreasing the OTR_max, reaching a maximal of 550 kDa at an OTR_max of 17 mmol L^-1 h^-1 . However, in the cultures developed under Oxygen limitation (0.5% DOT), the MMM of the polymer was practically the same (around 200 kDa) at 300 and 700 rpm, and this remained constant throughout the cultivation. Conclusions Overall, our results showed that under Oxygen-limited and non Oxygen-limited conditions, alginate production and its molecular mass are linked to the OTR_max, independently of the DOT of the culture.
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design and characterization of a one compartment scale down system for simulating Dissolved Oxygen Tension gradients
Journal of Chemical Technology & Biotechnology, 2010Co-Authors: Antonio De Leonrodriguez, Enrique Galindo, Octavio T RamirezAbstract:BACKGROUND: A laboratory scale one-compartment scale-down system (1-CSDS), used to generate Dissolved Oxygen Tension (DOT) gradients was designed and characterized. The system consists of a 1.5-L stirred-tank bioreactor coupled to an automatic DOT controller that changes the Oxygen partial pressure in the inlet gas through a feedback proportional–integral–derivative algorithm, while maintaining the hydrodynamic conditions constant. Oscillatory control of DOT was achieved by employing time-dependent square wave or sinusoidal setpoints. RESULTS: The 1-CSDS can be modeled as a first-order dynamic system, but showing a permanent lag between the system response and the setpoint. The 1-CSDS had a faster response rate for generating oscillating DOT when a square wave setpoint was used rather than a sinusoidal setpoint. The 1-CSDS generated symmetric DOT oscillations at periods above of 100 s. CONCLUSION: The 1-CSDS is suited to investigate the responses of microorganisms and cells, of biotechnological importance, to oscillatory DOT conditions. It was found that the response of the 1-CSDS was limited by the kLa. Copyright © 2010 Society of Chemical Industry
Octavio T Ramirez - One of the best experts on this subject based on the ideXlab platform.
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design and characterization of a one compartment scale down system for simulating Dissolved Oxygen Tension gradients
Journal of Chemical Technology & Biotechnology, 2010Co-Authors: Antonio De Leonrodriguez, Enrique Galindo, Octavio T RamirezAbstract:BACKGROUND: A laboratory scale one-compartment scale-down system (1-CSDS), used to generate Dissolved Oxygen Tension (DOT) gradients was designed and characterized. The system consists of a 1.5-L stirred-tank bioreactor coupled to an automatic DOT controller that changes the Oxygen partial pressure in the inlet gas through a feedback proportional–integral–derivative algorithm, while maintaining the hydrodynamic conditions constant. Oscillatory control of DOT was achieved by employing time-dependent square wave or sinusoidal setpoints. RESULTS: The 1-CSDS can be modeled as a first-order dynamic system, but showing a permanent lag between the system response and the setpoint. The 1-CSDS had a faster response rate for generating oscillating DOT when a square wave setpoint was used rather than a sinusoidal setpoint. The 1-CSDS generated symmetric DOT oscillations at periods above of 100 s. CONCLUSION: The 1-CSDS is suited to investigate the responses of microorganisms and cells, of biotechnological importance, to oscillatory DOT conditions. It was found that the response of the 1-CSDS was limited by the kLa. Copyright © 2010 Society of Chemical Industry
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transcriptional and metabolic response of recombinant escherichia coli to spatial Dissolved Oxygen Tension gradients simulated in a scale down system
Biotechnology and Bioengineering, 2006Co-Authors: Alvaro R Lara, Lidia Leal, Noemi Flores, Guillermo Gosset, Francisco Bolivar, Octavio T RamirezAbstract:Escherichia coli, expressing recombinant green fluorescent protein (GFP), was subjected to Dissolved Oxygen Tension (DOT) oscillations in a two-compartment system for simulating gradients that can occur in large-scale bioreactors. Cells were continuously circulated between the anaerobic (0% DOT) and aerobic (10% DOT) vessels of the scale-down system to mimic an overall circulation time of 50 s, and a mean residence time in the anaerobic and aerobic compartments of 33 and 17 s, respectively. Transcription levels of mixed acid fermentation genes (ldhA, poxB, frdD, ackA, adhE, pflD, and fdhF), measured by quantitative RT-PCR, increased between 1.5- to over 6-fold under oscillatory DOT compared to aerobic cultures (constant 10% DOT). In addition, the transcription level of fumB increased whereas it decreased for sucA and sucB, suggesting that the tricarboxylic acid cycle was functioning as two open branches. Gene transcription levels revealed that cytrochrome bd, which has higher affinity to Oxygen but lower energy efficiency, was preferred over cytochrome bO3 in oscillatory DOT cultures. Post-transcriptional processing limited heterologous protein production in the scale-down system, as inferred from similar gfp transcription but 19% lower GFP concentration compared to aerobic cultures. Simulated DOT gradients also affected the transcription of genes of the glyoxylate shunt (aceA), of global regulators of aerobic and anaerobic metabolism (fnr, arcA, and arcB), and other relevant genes (luxS, sodA, fumA, and sdhB). Transcriptional changes explained the observed alterations in overall stoichiometric and kinetic parameters, and production of ethanol and organic acids. Differences in transcription levels between aerobic and anaerobic compartments were also observed, indicating that E. coli can respond very fast to intermittent DOT conditions. The transcriptional responses of E. coli to DOT gradients reported here are useful for establishing rational scale-up criteria and strain design strategies for improved culture performance at large scales. © 2005 Wiley Periodicals, Inc.
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production of β galactosidase by kluyveromyces marxianus under oscillating Dissolved Oxygen Tension
Process Biochemistry, 2005Co-Authors: Gabriela Gaxiola Cortes, Octavio T Ramirez, Mauricio A Trujilloroldan, Enrique GalindoAbstract:Abstract The effect of oscillating Dissolved Oxygen Tension (DOT) on the metabolism of Kluyveromyces marxianus was investigated, particularly on the productivity of the enzyme β-galactosidase or lactase (EC 3.2.1.23). Sinusoidal DOT oscillations were established by manipulating the inflowing gases (nitrogen and Oxygen), at wave periods of 300, 600 and 1200 s and DOT ranging from 0 to 20%. Cultures carried out at different constant DOT ranging from 0 to 60% of air saturation were used for comparison. Oscillations at the shortest period tested (i.e., 300 s) resulted in higher specific and volumetric enzyme activity (2800 U/g and 31,700 U/l, respectively) and ethanol concentration (12.3 g/l) compared with cultures carried out at longer wave periods (600 and 1200 s) and those cultures carried out at constant DOT (0, 1, 5 and 20%). Final biomass concentration was not affected by oscillating DOT (12.2±0.7 g/l) and specific growth rate was slightly affected. These findings suggest that some non-ideal DOT control conditions can be advantageous for the case of lactase production, leading to increases in the enzyme volumetric or specific activity, without affecting maximum biomass concentration.
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effects of Dissolved Oxygen Tension on the production of recombinant penicillin acylase in escherichia coli
Enzyme and Microbial Technology, 2003Co-Authors: Antonio De Leon, Enrique Galindo, Vanessa Hernandez, Octavio T RamirezAbstract:Abstract The effect of Dissolved Oxygen Tension (DOT) on over-expression of penicillin acylase (PA) by a recombinant Escherichia coli was studied. Relevant kinetic and stoichiometric parameters were evaluated in batch cultures performed in 1-l bioreactors maintained at constant DOT values in the range of 0–40%. Specific growth rate, biomass concentration, and biomass yield from glucose increased in a Monod-type fashion, whereas acetate and formate concentration decreased as DOT increased. As DOT decreased, specific PA activity at glucose depletion increased from 0.09 to 0.16 U mg −1 dry cell weight (DCW), whereas volumetric PA activity decreased in a Monod-type behavior from 0.25 to 0 U ml −1 . The PA activity increased 1.5–3.3-fold when prolonging the culture for 10–25 h after growth cessation. Maximum PA activity was obtained at 1% DOT. Kinetics of PA activity followed a combined growth-associated and non-growth-associated behavior. It is proposed that the Luedeking–Piret coefficients can be expressed as a function of DOT. The growth-associated production coefficient increased with DOT following a Monod-type behavior, whereas the non-growth-associated production coefficient showed a maximum between 1 and 5% of DOT. Moreover, the non-growth-associated coefficient was importantly affected by DOT. Novel control and optimization strategies for DOT can be proposed based in the determined Luedeking–Piret coefficients.
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effect of oscillating Dissolved Oxygen Tension on the production of alginate by azotobacter vinelandii
Biotechnology Progress, 2001Co-Authors: Mauricio A Trujilloroldan, Carlos Peña, Octavio T Ramirez, Enrique GalindoAbstract:The effect of oscillating Dissolved Oxygen Tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the Oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW max ) of the alginate produced. The higher the amplitude, the lower the MMW max . As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW max increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW max of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.
Carlos Peña - One of the best experts on this subject based on the ideXlab platform.
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molecular mass of poly 3 hydroxybutyrate p3hb produced by azotobacter vinelandii is determined by the ratio of synthesis and degradation under fixed Dissolved Oxygen Tension
Process Biochemistry, 2016Co-Authors: Modesto Millan, Enrique Galindo, Daniel Segura, Carlos PeñaAbstract:Abstract Poly-3-hydroxybutyrate (P3HB) is an intracellular polyester produced by numerous bacteria, including Azotobacter vinelandii . Thermo-mechanical properties and biomedical applications of P3HB depend on its molecular mass (MM), which in turn is controlled by the balance between synthesis and degradation of the polymer during its biosynthesis. The aim of this study was to determine the activity levels of enzymes involved in the synthesis and degradation of P3HB and their effect on the molecular mass of the polymer produced by A. vinelandii strain OP under the conditions of Dissolved Oxygen Tension of 1 and 15%. The results show that the MM of P3HB changed between the exponential and stationary growth phases, under both Oxygen conditions. During the exponential growth phase, the mean molecular mass (MMM) of P3HB was high (4800 kDa), coincident with a high activity P3HB synthase and with a low activity of P3HB depolymerase. In contrast, during the stationary phase, the P3HB MM decreased to 3600 kDa, because of the increased activity of the P3HB depolymerase.
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analysis of respiratory activity and carbon usage of a mutant of azotobacter vinelandii impaired in poly β hydroxybutyrate synthesis
Journal of Industrial Microbiology & Biotechnology, 2016Co-Authors: Lucero Jimenez, Enrique Galindo, Daniel Segura, Tania Castillo, Celia Flores, Carlos PeñaAbstract:In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-β-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific Oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed Dissolved Oxygen Tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).
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Oxygen transfer rate during the production of alginate by azotobacter vinelandii under Oxygen limited and non Oxygen limited conditions
Microbial Cell Factories, 2011Co-Authors: Esteban Lozano, Enrique Galindo, Carlos PeñaAbstract:Background The Oxygen transfer rate (OTR) and Dissolved Oxygen Tension (DOT) play an important role in determining alginate production and its composition; however, no systematic study has been reported about the independent influence of the OTR and DOT. In this paper, we report a study about alginate production and the evolution of the molecular mass of the polymer produced by a wild-type A. vinelandii strain ATCC 9046, in terms of the maximum Oxygen transfer rate (OTRmax) in cultures where the Dissolved Oxygen Tension (DOT) was kept constant.
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Production of alginate by Azotobacter vinelandii in a stirred fermentor simulating the evolution of power input observed in shake flasks
Process Biochemistry, 2008Co-Authors: Carlos Peña, Modesto Millan, Enrique GalindoAbstract:By simulating the evolution of the actual power input observed in shake flasks it was possible to produce, in a fermentor, alginates having a very similar mean molecular mass (1700 kDa) to that obtained in the cultures developed in shake flasks (1800 kDa). Similar profiles of Dissolved Oxygen Tension and Oxygen transfer rate could be the reason.
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the Oxygen transfer rate influences the molecular mass of the alginate produced by azotobacter vinelandii
Applied Microbiology and Biotechnology, 2007Co-Authors: A Diazbarrera, Carlos Peña, Enrique GalindoAbstract:The influence of Oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without Dissolved Oxygen Tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were Oxygen-limited. The OTR reached different maximum levels (OTRmax) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTRmax decreased. The molecular mass obtained at 3.0 mmol l−1 h−1 was 7.0 times higher (1,560 kDa) than at 9.0 mmol l−1 h−1 (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as Oxygen limitation.
Jochen Buchs - One of the best experts on this subject based on the ideXlab platform.
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combined Dissolved Oxygen Tension and online viscosity measurements in shake flask cultivations via infrared fluorescent Oxygen sensitive nanoparticles
Biotechnology and Bioengineering, 2019Co-Authors: Tobias Ladner, David Flitsch, Mihaly Lukacs, Michaela Sieben, Jochen BuchsAbstract:: Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient Oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the Dissolved Oxygen Tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent Oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the Oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the Oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric Oxygen transfer coefficient (kL a) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris.
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glucose containing polymer rings enable fed batch operation in microtiter plates with parallel online measurement of scattered light fluorescence Dissolved Oxygen Tension and ph
Biotechnology and Bioengineering, 2019Co-Authors: Tobias Habicher, Vroni Czotscher, Tobias Klein, Andreas Daub, Timm Keil, Jochen BuchsAbstract:: Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, Oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fed-batch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the Dissolved Oxygen Tension and pH could be avoided by choosing appropriate polymer-ring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, Dissolved Oxygen Tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development.
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easy to use and reliable technique for online Dissolved Oxygen Tension measurement in shake flasks using infrared fluorescent Oxygen sensitive nanoparticles
Microbial Cell Factories, 2016Co-Authors: David Flitsch, Tobias Ladner, Mihaly Lukacs, Jochen BuchsAbstract:Despite the progressive miniaturization of bioreactors for screening purposes, shake flasks are still widespread in biotechnological laboratories and industry as cultivation vessels. Shake flasks are often applied as the first or second step in applications such as strain screening or media optimization. Thus, there are ongoing efforts to develop online measurement techniques for shake flasks, to gain as much information as possible about the cultured microbial system. Since Dissolved Oxygen Tension (DOT) is a key experimental parameter, its accurate determination during the course of experiment is critical. Some of the available DOT measurement techniques can lead to erroneous measurements or are very difficult to handle. A reliable and easy to use DOT measurement system, based on suspended Oxygen-sensitive nanoparticles, is presented in this work. In a cultivation of Kluyveromyces lactis, a new DOT measurement technique via suspended Oxygen-sensitive nanoparticles was compared with the conventional DOT measurement via fixed sensor spots. These experiments revealed the main disadvantage of applying sensor spots. With further cultivations of Escherichia coli and Hansenula polymorpha, the new measurement technique was successfully validated. In combination with a RAMOS device, kLa values were determined during the presented cultivations. The determined kLa values are in good agreement with a correlation recently found in the literature. The presented DOT measurement technique via suspended Oxygen-sensitive nanoparticles in shake flasks turned out to be easy to use, robust and reliable under all applied combinations of shaking frequencies and filling volumes. Its applicability as an online monitoring system for cultivations was shown by means of four examples. Additionally, in combination with a RAMOS device, the possibility of experimental kLa determination was successfully demonstrated.
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online monitoring of Dissolved Oxygen Tension in microtiter plates based on infrared fluorescent Oxygen sensitive nanoparticles
Microbial Cell Factories, 2015Co-Authors: Tobias Ladner, David Flitsch, Tino Schleputz, Jochen BuchsAbstract:During the past years, new high-throughput screening systems with capabilities of online monitoring turned out to be powerful tools for the characterization of microbial cell cultures. These systems are often easy to use, offer economic advantages compared to larger systems and allow to determine many important process parameters within short time. Fluorescent protein tags tremendously simplified the tracking and observation of cellular activity in vivo. Unfortunately, interferences between established fluorescence based Dissolved Oxygen Tension (DOT) measurement techniques and fluorescence-based protein tags appeared. Therefore, the applicability of new Oxygen-sensitive nanoparticles operated within the more suitable infrared wavelength region are introduced and validated for DOT measurement. The biocompatibility of the used dispersed Oxygen-sensitive nanoparticles was proven via RAMOS cultivations for Hansenula polymorpha, Gluconobacter oxydans, and Escherichia coli. The applicability of the introduced DOT measurement technique for online monitoring of cultivations was demonstrated and successfully validated. The nanoparticles showed no disturbing effect on the online measurement of the fluorescence intensities of the proteins GFP, mCherry and YFP measured by a BioLector prototype. Additionally, the DOT measurement was not influenced by changing concentrations of these proteins. The kLa values for the applied cultivation conditions were successfully determined based on the measured DOT. The introduced technique appeared to be practically as well as economically advantageous for DOT online measuring in microtiter plates. The disadvantage of limited availability of microtiter plates with immobilized sensor spots (optodes) does not apply for this introduced technique. Due to the infrared wavelength range, used for the DOT measurement, no interferences with biogenic fluorescence or with expressed fluorescent proteins (e.g. YFP, GFP or mCherry) occur.
Tobias Ladner - One of the best experts on this subject based on the ideXlab platform.
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combined Dissolved Oxygen Tension and online viscosity measurements in shake flask cultivations via infrared fluorescent Oxygen sensitive nanoparticles
Biotechnology and Bioengineering, 2019Co-Authors: Tobias Ladner, David Flitsch, Mihaly Lukacs, Michaela Sieben, Jochen BuchsAbstract:: Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient Oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the Dissolved Oxygen Tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent Oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the Oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the Oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric Oxygen transfer coefficient (kL a) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris.
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easy to use and reliable technique for online Dissolved Oxygen Tension measurement in shake flasks using infrared fluorescent Oxygen sensitive nanoparticles
Microbial Cell Factories, 2016Co-Authors: David Flitsch, Tobias Ladner, Mihaly Lukacs, Jochen BuchsAbstract:Despite the progressive miniaturization of bioreactors for screening purposes, shake flasks are still widespread in biotechnological laboratories and industry as cultivation vessels. Shake flasks are often applied as the first or second step in applications such as strain screening or media optimization. Thus, there are ongoing efforts to develop online measurement techniques for shake flasks, to gain as much information as possible about the cultured microbial system. Since Dissolved Oxygen Tension (DOT) is a key experimental parameter, its accurate determination during the course of experiment is critical. Some of the available DOT measurement techniques can lead to erroneous measurements or are very difficult to handle. A reliable and easy to use DOT measurement system, based on suspended Oxygen-sensitive nanoparticles, is presented in this work. In a cultivation of Kluyveromyces lactis, a new DOT measurement technique via suspended Oxygen-sensitive nanoparticles was compared with the conventional DOT measurement via fixed sensor spots. These experiments revealed the main disadvantage of applying sensor spots. With further cultivations of Escherichia coli and Hansenula polymorpha, the new measurement technique was successfully validated. In combination with a RAMOS device, kLa values were determined during the presented cultivations. The determined kLa values are in good agreement with a correlation recently found in the literature. The presented DOT measurement technique via suspended Oxygen-sensitive nanoparticles in shake flasks turned out to be easy to use, robust and reliable under all applied combinations of shaking frequencies and filling volumes. Its applicability as an online monitoring system for cultivations was shown by means of four examples. Additionally, in combination with a RAMOS device, the possibility of experimental kLa determination was successfully demonstrated.
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online monitoring of Dissolved Oxygen Tension in microtiter plates based on infrared fluorescent Oxygen sensitive nanoparticles
Microbial Cell Factories, 2015Co-Authors: Tobias Ladner, David Flitsch, Tino Schleputz, Jochen BuchsAbstract:During the past years, new high-throughput screening systems with capabilities of online monitoring turned out to be powerful tools for the characterization of microbial cell cultures. These systems are often easy to use, offer economic advantages compared to larger systems and allow to determine many important process parameters within short time. Fluorescent protein tags tremendously simplified the tracking and observation of cellular activity in vivo. Unfortunately, interferences between established fluorescence based Dissolved Oxygen Tension (DOT) measurement techniques and fluorescence-based protein tags appeared. Therefore, the applicability of new Oxygen-sensitive nanoparticles operated within the more suitable infrared wavelength region are introduced and validated for DOT measurement. The biocompatibility of the used dispersed Oxygen-sensitive nanoparticles was proven via RAMOS cultivations for Hansenula polymorpha, Gluconobacter oxydans, and Escherichia coli. The applicability of the introduced DOT measurement technique for online monitoring of cultivations was demonstrated and successfully validated. The nanoparticles showed no disturbing effect on the online measurement of the fluorescence intensities of the proteins GFP, mCherry and YFP measured by a BioLector prototype. Additionally, the DOT measurement was not influenced by changing concentrations of these proteins. The kLa values for the applied cultivation conditions were successfully determined based on the measured DOT. The introduced technique appeared to be practically as well as economically advantageous for DOT online measuring in microtiter plates. The disadvantage of limited availability of microtiter plates with immobilized sensor spots (optodes) does not apply for this introduced technique. Due to the infrared wavelength range, used for the DOT measurement, no interferences with biogenic fluorescence or with expressed fluorescent proteins (e.g. YFP, GFP or mCherry) occur.