Application of green fluorescent protein signal for effective monitoring of fermentation processes (original) (raw)
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Journal of Biotechnology, 2004
A high number of economically important recombinant proteins are produced in Escherichia coli based host/vector systems. The major obstacle for improving current processes is a lack of appropriate on-line in situ methods for the monitoring of metabolic burden and critical state variables. Here, a pre-evaluation of the reporter green fluorescent protein (GFP) was undertaken to assess its use as a reporter of stress associated promoter regulation. The investigation of GFP and its blue fluorescent variant BFP was done in model fermentations using E. coli HMS174(DE3)/pET11aGFPmut3.1and E. coli HMS174(DE3)/pET11aBFP host/vector systems cultured in fed-batch and chemostat regime. Our results prove the suitability of the fluorescent reporter proteins for the design of new strategies of on-line bioprocess monitoring. GFPmut3.1 variant can be detected after a short lag-phase of only 10 min, it shows a high fluorescence yield in relation to the amount of reporter protein, a good signal to noise ratio and a low detection limit. The fluorescence-signal and the amount of fluorescent protein, determined by ELISA, showed a close correlation in all fermentations performed. A combination of reporter technology with state of the art sensors helps to develop new strategies for efficient on-line monitoring needed for industrial process optimisation. The development of efficient monitoring will contribute to advanced control of recombinant protein production and accelerate the development of optimised production processes.
Biotechnology Progress, 1991
This article reports results on the application of fluorophore-based pH sensing to the monitoring of fermentation processes. Emphasis is placed on identifying potential strengths and limitations. A custom-built fluorometer, designed t o provide broad-band excitation (<420 nm) was used to monitor the emission of a pH-sensitive fluorophore (1,4-dihydroxyphthalonitrile) in solution a t two wavelengths. The overall sensing system was characterized with respect t o biocompatibility, response, and stability. The biocompatibility results revealed that the growth rate of Pseudomonas fluorescens (NRRL B-15132) was decreased by the fluorophore at high concentrations (1100 pM). Correlating the observed p H with the ratio of the two wavelengths (488 nm/434 nm) rendered the pH-sensing system immune to factors such as fluorophore oxidation, aeration, and reactor agitation. A comparison of response times for fluorescence and Ingold p H measurements revealed fluorescence sensing t o be slower. This was attributed to imperfect mixing and position differences between the two sensors in the fermentor. During the course of fermentation, on-line ratiometric pH measurements for NRRL B-15132 batch process were affected by changes in ionic strength and cell concentration. A p H difference of 0.66 was recorded (using an Ingold electrode as the standard) after 120 h of the fermentation. Recommendations for overcoming limitations were made.
Biotechnology progress
The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009
Application of Scanning Fluorometry for Monitoring of a Fermentation Process
Biotechnology Progress, 1996
Conventional observation of a fermentation process by using NAD(P)H-dependent fluorescence provides a mean for cell monitoring, but its practical application has been very limited due to many factors that affect culture fluorescence. In this work we studied correlation of scanning fluorometry signals with various process variables and with cell metabolic states and showed that scanning fluorometry is more informative than conventional NAD(P)H-dependent fluorescence. A stepwise multiple-regression procedure has been used to select meaningful signals from the whole spectrum, and we found that with three or four components of the excitation-emission plane the process variables can be portrayed with a rather good accuracy. The approach is demonstrated on the examples of wild-type or recombinant Escherichia coli and Saccharomyces cerevisiae fermentations.
A biosensor was developed for the determination of BOD value of fermentation industry effluent. e developed biosensor was fabricated by immobilizing the microbial consortium on cellulose acetate (CA) membrane in close proximity to a DO probe electrode. e microbial consortium was harvested from the fermentation industry effluent. e BOD biosensor was calibrated by using a solution containing the equivalent amount of glucose/glutamic acid (GGA) as a standard sample solution. e response time was optimized by immobilizing different concentrations of cell biomass on CA membrane. Once the response time was optimized, it was used for determination of BOD of fermentation industry effluent. For analysis of fermentation industry effluent, the response time was observed 7 minutes with detection limit 1 mg/L. Good linear range with GGA standard solution was observed, 2 0.99 with relative standard deviation (RSD) < 9%. e observed BOD value by biosensor showed a good comparison with the conventional method for the determination of BOD.
Engineering in Life Sciences, 2016
Rapid quantitation of product titer is a critical input for control of any bioprocess. This measurement, however, is marred by the myriad components that are present in the fermentation broth, often requiring extensive sample pretreatment before analysis. Spectroscopy techniques such as fluorescence spectroscopy are widely recognized as potential monitoring tools. Here, we investigate the possibility of using fluorescence of the culture supernatant as a potential at-line monitoring tool to measure the concentration of a recombinant therapeutic protein expressed in a Pichia pastoris fed-batch fermentation. We propose an integrated method wherein both the target protein and total protein concentrations are predicted using intrinsic riboflavin fluorescence and extrinsic fluorescence, respectively. The root mean square error for estimating the concentrations of the target protein (using riboflavin fluorescence) and total protein (using extrinsic fluorescence) have been estimated to be <0.1 and <0.2, respectively. The proposed approach has been validated for two different biotherapeutic products, human serum albumin and granulocyte colony stimulating factor, that were expressed using Mut + and Mut s strains of P. pastoris, respectively. The proposed approach is rapid (1 min analysis time, 10 min total with at line sampling) and thus could be a significant enabler for process analytical technology implementation in Pichia fermentation.
Biotechnology Progress, 2008
On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.
A Review of Dissolved Oxygen Concentration Measurement Methods for Biological Fermentations
2007 Minneapolis, Minnesota, June 17-20, 2007, 2007
Dissolved oxygen levels in biological processes depend on the biological, chemical, and physical properties of the process being monitored. The analysis of dissolved oxygen concentration is a key test for process control and optimization. A review of the measurement methods for dissolved oxygen concentrations will be presented in this paper. Included in this review are the chemical, volumetric, tubing, electrochemical electrode, and optode methods. Advantages and disadvantages of these methods are discussed and key considerations for their use are summarized.
Microbial Cell Factories, 2009
Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cellbased reporting of oxygen limitation. Because there are greater than 10 9 cells in a fermentor, one can outfit a vessel with more than 10 9 sensors. Our concept was tested in high density, lab-scale (5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency -one verses four impellers.