Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganisms
Simona DzurendováPernille Margrethe OlsenDana ByrtusováValeria TafintsevaVolha ShapavalSvein Jarle HornAchim KöhlerMartin SzotkowskiIvana MárováBoris Zimmermann
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Abstract Background Monitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups. Results The system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R 2 ) in the 0.94–0.99 and 0.89–0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles. Conclusions The FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.Abstract Modern bioprocess control requires instantaneous data acquisition and in‐time evaluation of bioprocess variables. Optical sensor systems are of increasing interest in bioprocess monitoring because they are very sensitive and offer the possibility of on‐line, in situ , and noninvasive process monitoring. In this regard, on‐line fluorescence spectroscopy is a useful tool in the development of methods for bioprocess monitoring because many cellular substances and media components are biogenic fluorophors. This technique and the application of chemometric methods offer a deeper insight into biological processes and thus process monitoring, optimization, and control is accessible. In this review, an overview of the developments and applications of on‐line fluorescence techniques for bioprocess monitoring over the last 25 years is given with emphasis on 2D fluorescence spectroscopy.
Monitoring and control
Process Analytical Technology
Bioprocess engineering
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Claviceps purpurea
Process Analytical Technology
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Microorganisms utilizing glycerol as a carbon source were isolated from various soils for glycerol/O2bio-fuel cell system. Among 19 locations at the campus of Osaka Prefecture University Collage of Technology and Uchiagegawa flood-control green space in Neyagawa city, the microorganisms isolated from three points (sample numbers 1-2, 1-3 and 1-8) were able to grow in the medium containing 15% glycerol at 37°C and ones from ten locations could proliferate in the same concentration of glycerol at 25°C. In the case of the microorganisms growing at 37°C, they were similar in appearance and seemed to form biofilm. Among ten isolation bacteria at 25°C, three samples (sample number 2-2, 2-3 and 2-5) showed good proliferation in the condition of 15% glycerol medium. The growth profiles of the microorganism which showed the most significant proliferation (sample number 2-5) were also investigated at various glycerol concentrations, resulting that maximum cell density was suppressed at higher glycerol concentration.
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Carbon fibers
Growth medium
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In this investigation, the fermentation step of a standard mammalian cell-based industrial bioprocess for the production of a therapeutic protein was studied, with particular emphasis on the evolution of cell viability. This parameter constitutes one of the critical variables for bioprocess monitoring since it can affect downstream operations and the quality of the final product. In addition, when the cells experiment an unpredictable drop in viability, the assessment of this variable through classic off-line methods may not provide information sufficiently in advance to take corrective actions. In this context, Process Analytical Technology (PAT) framework aims to develop novel strategies for more efficient monitoring of critical variables, in order to improve the bioprocess performance. Thus, in this work, a set of chemometric tools were integrated to establish a PAT strategy to monitor cell viability, based on fluorescence multiway data obtained from fermentation samples of a particular bioprocess, in two different scales of operation. The spectral information, together with data regarding process variables, was integrated through chemometric exploratory tools to characterize the bioprocess and stablish novel criteria for the monitoring of cell viability. These findings motivated the development of a multivariate classification model, aiming to obtain predictive tools for the monitoring of future lots of the same bioprocess. The model could be satisfactorily fitted, showing the non-error rate of prediction of 100%.
Process Analytical Technology
Critical quality attributes
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Extracellular vesicles (EVs) are membrane vesicles that are produced by cells to be released into their microenvironment. In this study, we present the EV concentration as a new factor for optimization of industrial bioprocess control. The release of EVs depends on many cell properties, including cell activation and stress status, and cell death. Therefore, the EV concentration might provide a readout for identification of the cell state and the conditions during a bioprocess. Our data show that the EV concentration increased during the bioprocess, which indicated deteriorating conditions in the bioreactor. This increase in EV concentration in the fermentation broth was the consequence of two different processes: cell activation, and cell death. However, the release of EVs from activated living cells had a much weaker impact on EV concentration in the bioreactor than those released during cell death. EVs and cells in the bioprocess environment were quantified by flow cytometry. The most accurate data were obtained directly from unprocessed samples, making the monitoring of the EV concentration a rapid, easy, and cheap method. These EV concentrations reflect the conditions in the bioreactor and provide new information regarding the state of the bioprocess. Therefore, we suggest EV concentration as a new and important parameter for the monitoring of industrial bioprocesses.
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格林荧光灯蛋白质(GFP ) 和它的变体 / 相当或相同的事物蛋白质通常作为象 GFP 一样被叫荧光灯蛋白质(FP ) 它是广泛地作为可见分子的工具使用了由于他们简单、精确、实时的 quantification 的能力监视大量生物过程。基于 FPs 的分子、可见的 quantification 工具在 bioprocess 工程上正在给更多的影响，启用 biomolecule 水平与过程级的事件要连接的动态信息。在这评论，在有快速的分子的 bioprocess quantification 上的强调的生物工程的 FP 的不同应用例如抄写效率的 quantification，蛋白质生产，合拢效率的蛋白质，房间集中，细胞内部的微型环境等等，将首先被介绍。包括 FP 和熔化搭挡蛋白质， FP 的成熟，内部过滤器效果和察觉到的技术的相互作用为精确 quantification 关于实际 bioprocess 应用使用 FP 的挑战然后被讨论。最后，为在分子的 bioprocess quantification 使用的 FP 的未来发展将被建议。
Biomolecule
Biological fluids
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