Improvement of bioprocess monitoring: development of novel concepts

The advancement of bioprocess monitoring will play a crucial role to meet the future requirements of bioprocess technology. Major issues are the acceleration of process development to reduce the time to the market and to ensure optimal exploitation of the cell factory and further to cope with the requirements of the Process Analytical Technology initiative. Due to the enormous complexity of cellular systems and lack of appropriate sensor systems microbial production processes are still poorly understood. This holds generally true for the most microbial production processes, in particular for the recombinant protein production due to strong interaction between recombinant gene expression and host cell metabolism. Therefore, it is necessary to scrutinise the role of the different cellular compartments in the biosynthesis process in order to develop comprehensive process monitoring concepts by involving the most significant process variables and their interconnections. Although research for the development of novel sensor systems is progressing their applicability in bioprocessing is very limited with respect to on-line and in-situ measurement due to specific requirements of aseptic conditions, high number of analytes, drift, and often rather low physiological relevance. A comprehensive survey of the state of the art of bioprocess monitoring reveals that only a limited number of metabolic variables show a close correlation to the currently explored chemical/physical principles. In order to circumvent this unsatisfying situation mathematical methods are applied to uncover "hidden" information contained in the on-line data and thereby creating correlations to the multitude of highly specific biochemical off-line data. Modelling enables the continuous prediction of otherwise discrete off-line data whereby critical process states can be more easily detected. The challenging issue of this concept is to establish significant on-line and off-line data sets. In this context, online sensor systems are reviewed with respect to commercial availability in combination with the suitability of offline analytical measurement methods. In a case study, the aptitude of the concept to exploit easily available online data for prediction of complex process variables in a recombinant E. coli fed-batch cultivation aiming at the improvement of monitoring capabilities is demonstrated. In addition, the perspectives for model-based process supervision and process control are outlined

On-Line Monitoring of Biological Parameters in Microalgal Bioprocesses Using Optical Methods

Microalgae are promising sources of fuels and other chemicals. To operate microalgal cultivations efficiently, process control based on monitoring of process variables is needed. On-line sensing has important advantages over off-line and other analytical and sensing methods in minimizing the measurement delay. Consequently, on-line, in-situ sensors are preferred. In this respect, optical sensors occupy a central position since they are versatile and readily implemented in an on-line format. In biotechnological processes, measurements are performed in three phases (gaseous, liquid and solid (biomass)), and monitored process variables can be classified as physical, chemical and biological. On-line sensing technologies that rely on standard industrial sensors employed in chemical processes are already well-established for monitoring the physical and chemical environment of an algal cultivation. In contrast, on-line sensors for the process variables of the biological phase, whether biomass, intracellular or extracellular products, or the physiological state of living cells, are at an earlier developmental stage and are the focus of this review. On-line monitoring of biological process variables is much more difficult and sometimes impossible and must rely on indirect measurement and extensive data processing. In contrast to other recent reviews, this review concentrates on current methods and technologies for monitoring of biological parameters in microalgal cultivations that are suitable for the on-line and in-situ implementation. These parameters include cell concentration, chlorophyll content, irradiance, and lipid and pigment concentration and are measured using NMR, IR spectrophotometry, dielectric scattering, and multispectral methods. An important part of the review is the computer-aided monitoring of microalgal cultivations in the form of software sensors, the use of multi-parameter measurements in mathematical process models, fuzzy logic and artificial neural networks. In the future, software sensors will play an increasing role in the real-time estimation of biological variables because of their flexibility and extendibility

Sensors and Techniques for On-Line Determination of Cell Viability in Bioprocess Monitoring

In recent years, the bioprocessing industry has experienced significant growth and is increasingly emerging as an important economic sector. Here, efficient process management and constant control of cellular growth are essential. Good product quality and yield can only be guaranteed with high cell density and high viability. Whereas the on-line measurement of physical and chemical process parameters has been common practice for many years, the on-line determination of viability remains a challenge and few commercial on-line measurement methods have been developed to date for determining viability in industrial bioprocesses. Thus, numerous studies have recently been conducted to develop sensors for on-line viability estimation, especially in the field of optical spectroscopic sensors, which will be the focus of this review. Spectroscopic sensors are versatile, on-line and mostly non-invasive. Especially in combination with bioinformatic data analysis, they offer great potential for industrial application. Known as soft sensors, they usually enable simultaneous estimation of multiple biological variables besides viability to be obtained from the same set of measurement data. However, the majority of the presented sensors are still in the research stage, and only a few are already commercially available

Comparison of Raman and Mid-Infrared Spectroscopy for Real-Time Monitoring of Yeast Fermentations: A Proof-of-Concept for Multi-Channel Photometric Sensors

Raman and mid-infrared (MIR) spectroscopy are useful tools for the specific detection of molecules, since both methods are based on the excitation of fundamental vibration modes. In this study, Raman and MIR spectroscopy were applied simultaneously during aerobic yeast fermentations of Saccharomyces cerevisiae. Based on the recorded Raman intensities and MIR absorption spectra, respectively, temporal concentration courses of glucose, ethanol, and biomass were determined. The chemometric methods used to evaluate the analyte concentrations were partial least squares (PLS) regression and multiple linear regression (MLR). In view of potential photometric sensors, MLR models based on two (2D) and four (4D) analyte-specific optical channels were developed. All chemometric models were tested to predict glucose concentrations between 0 and 30 g L−1, ethanol concentrations between 0 and 10 g L−1, and biomass concentrations up to 15 g L−1 in real time during diauxic growth. Root-mean-squared errors of prediction (RMSEP) of 0.68 g L−1, 0.48 g L−1, and 0.37 g L−1 for glucose, ethanol, and biomass were achieved using the MIR setup combined with a PLS model. In the case of Raman spectroscopy, the corresponding RMSEP values were 0.92 g L−1, 0.39 g L−1, and 0.29 g L−1. Nevertheless, the simple 4D MLR models could reach the performance of the more complex PLS evaluation. Consequently, the replacement of spectrometer setups by four-channel sensors were discussed. Moreover, the advantages and disadvantages of Raman and MIR setups are demonstrated with regard to process implementation

Perspectives of fluorescence spectroscopy for online monitoring in microalgae industry

OSR‐2016‐CPF‐2907‐05 UIDB/50006/2020 UIDP/50006/2020 SFRH/BD/108894/2015Microalgae industrial production is viewed as a solution for alternative production of nutraceuticals, cosmetics, biofertilizers, and biopolymers. Throughout the years, several technological advances have been implemented, increasing the competitiveness of microalgae industry. However, online monitoring and real-time process control of a microalgae production factory still require further development. In this mini-review, non-destructive tools for online monitoring of cellular agriculture applications are described. Still, the focus is on the use of fluorescence spectroscopy to monitor several parameters (cell concentration, pigments, and lipids) in the microalgae industry. The development presented makes it the most promising solution for monitoring up-and downstream processes, different biological parameters simultaneously, and different microalgae species. The improvements needed for industrial application of this technology are also discussed.publishersversioninpres

Optimization of insect cell based protein production processes - online monitoring, expression systems, scale-up

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale-up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes

Development of a monitoring tool based on fluorescence and climatic data for pigments profile estimation in Dunaliella salina

When growing microalgae for biorefinery processes, a high product yield is desired. For that reason, monitoring the concentration of the desired products during growth and products induction procedure is of great interest. 2D Fluorescence spectroscopy is a fingerprinting technique, used in situ and at real time, with a high potential for online monitoring of biological systems. In this work, Dunaliella salina pigment content was monitored using fluorescence data coupled with chemometric tools. Climatic parameters were also used as input variables due to their impact on the pigments profile in outdoor cultivations. Predictive models were developed for chlorophyll content (a, b, and total) with variance captured between 50 and 90%, and R2 varying between 0.6 and 0.9 for both training and validation data sets. Total carotenoids models captured 70 to 80% of variance, and R2 between 0.7 and 0.9, for training and validation. Models for specific carotenoids (zeaxanthin, α-carotene, all-trans-β-carotene, and 9-cis-β-carotene) captured variance between 60 and 90%, with validation and training R2 between 0.6 and 0.9. With this methodology, it was possible to calibrate a monitoring tool for pigments quantification, as a bulk and as individual compounds, proving that 2D fluorescence spectroscopy and climatic data combined with chemometric tools can be used to assess simultaneously and at real time different pigments in D. salina biomass production.info:eu-repo/semantics/publishedVersio