Eicosapentaenoic acid (EPA) is an omega 3 fatty-acid essential to humans, who mainly rely on fish oil as a source of EPA. Unfortunately, this is not a sustainable source

Hernandez, E.

Vaccaro, N.M.

English

EPA and DHA. are essential fatty acids that humans need to live. Currently, they are usually obtained through fish oil, either from supplement or directly from oily fish. Unfortunately, fish farming is not sustainable and there is gathering interest in sourcing fatty acids from algae cells. The problem with algae growth comes from the varying lipid production per batch. Mid-production analysis is done on freeze dried samples after some time has passed, and fatty acid content cannot be measured until after the batch is complete. A real-time, on-line monitoring tool would greatly aid in algae production. Seven analytical techniques have been compared with Raman spectroscopy chosen as the most viable option to monitor omega-3 production in micro-algae

Canterbury Research and Theses Environment

Viability study on real-time monitoring of healthy omega-3 production in micro-algae

Research SpaceConference posterCLAIRE Real-time monitoring of healthy omega-3 production in micro-algae: A viability studyHernandez, E. and Vaccaro, N.M.Natasha Vaccaro and Dr. Ernesto Hernandez Bioinspired Engineering Research Group, Chemical Engineering, School of Engineering, Technology and Design, Canterbury Christ Church University, North Holmes Road, Canterbury, Kent CT1 1QU, UK (info@ernestohernandez.org)Raman Spectroscopy (RS)Can provide information on cell structure, phase and much more. It is an extremely fast, non destructive technique that can be used on any phase. Only basic training is needed to use Raman spectroscopy. Although the cost for the equipment can be moderate, there is also a very small risk of sample heating with this procedure potentially leading to the destruction of the sample. Fourier Transform Infrared Spectroscopy (FTIR)Relatively easy to perform procedure that determines the concentration of an analyte in a sample. Uses fluorescent properties of the sample, but not all compounds are fluorescent. Is also very expensive.Cell CytometryAlthough it has already been  successfully used for yeast and bacterial growth, it is very expensive and requires extensive training and on-going maintenance. Fluorescence SpectroscopyA relatively easy procedure to perform that can measure the compounds in a solution. It is highly sensitive and so only very small samples are needed for measurement. It is also non- destructive and non-invasive. It is similar to FTIR-spectroscopy but much cheaper, although it has the same disadvantage in which not all molecules are fluorescent.Nuclear Magnetic Resonance (NMR)Can give detailed information about the structure of a chemical and biological compounds. Beside from being very expensive, a high-magnetic-field is needed which can affect electronic monitors and computer-controlled devices. Mass Spectrometry (MS)Determines structural information of a sample, even from extremely small sample concentrations. On the down side, it can be quite expensive and time consuming. Differential Scanning Calorimetry (DSC)Can provide characteristic properties of a sample, however it is destructive and may struggle to analyse heterogeneous materials. There are also no real standards for DSC and so there has been difficulty in finding reproduceable results.Technique In-vivo Non-destructive Fast Cost TrainingCell Cytometry ✓ High ExtensiveDifferential Scanning Calorimetry ✓ Moderate ModerateFourier Transform Infrared Spectroscopy ✓ ✓ High ModerateFluorescence Spectroscopy ✓ ✓ ✓ Low BasicMass Spectrometry Low BasicNuclear Magnetic Resonance ✓ ✓ High ModerateRaman Spectroscopy ✓ ✓ ✓ Moderate BasicTheBackgroundEicosapentaenoic acid (EPA) is an omega 3 fatty-acid essential to humans, who mainly rely on fish oil as a source of EPA. Unfortunately, this is not a sustainable sourceThe ProblemAlthough algae is a much more sustainable and environmentally friendly source of EPA, there are limitations. Total lipid production can vary dramatically from batch to batch, from 20-50% of their total dry weight1. ASolution?To aid in the production of fatty acids, and in particular EPA, a real time, in-vivo, online monitoring tool would be advantageous2, to allow fast decision to be made to help boost the final yield of EPA. PotentialTechniques• Cell cytometry• Differential Scanning Calorimetry• Fourier Transform Infrared Spectroscopy• Fluorescence Spectroscopy• Mass Spectrometry• Nuclear Magnetic Resonance• Raman Spectroscopy1Wu, H., Volponi, J., Oliver, A., Parikh, A., Simmons, B. and Singh, S., 2011. In vivo lipidomics using single-cell Raman spectroscopy. Proceedings of the National Academy of Sciences, 108(9), pp.3809-3814.2M. Sa, N. Ferrer-Ledo, R. Wijffels, J. G. Crespo, M. Barbosa and C. F. Galinha (2019). Monitoring of eicosapentaenoic acid (EPA) production in the microalgae T Nannochloropsis oceanica. Algal Research [online], 45(2020), . [27/07/21]. 3Pedro L. Fale, Ali Altharawi, K.L. Andrew Chan (2015). In situ Fourier transform infrared analysis of live cells' response to doxorubicin, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. Volume 1853, Issue 10, Part A, 2015, Pp 2640-2648)4Mehendale, N., Jenne, F., Joshi, C., Sharma, S., Masakapalli, S.K. and MacKinnon, N., 2020. A Nuclear Magnetic Resonance (NMR) Platform for Real-Time Metabolic Monitoring of Bioprocesses. Molecules, [online] 25(20), p.4675. 5Burlacot, A., Burlacot, F., Li-Beisson, Y. and Peltier, G., 2020. Membrane Inlet Mass Spectrometry: A Powerful Tool for Algal Research. Frontiers in Plant Science, 11.6Schalk, R., Heintz, A., Braun, F., Iacono, G., Rädle, M., Gretz, N., Methner, F.-J. and Beuermann, T. 2019. Comparison of Raman and Mid-Infrared Spectroscopy for Real-Time Monitoring of Yeast Fermentations: A Proof-of-Concept for Multi-Channel Photometric Sensors. Applied Sciences 9(12), p. 2472. Available at: http://dx.doi.org/10.3390/app91224727Lagarto, J.L., Villa, F., Tisa, S. et al. Real-time multispectral fluorescence lifetime imaging using Single Photon Avalanche Diode arrays. Sci Rep 10, 8116 (2020). https://doi.org/10.1038/s41598-020-65218-3A Flow Cytometer (CC-BY-SA-3.0)Conceptual diagram of a real-time NMR using flow-through capillary system* ( Mehendale et al., 2020)4Differential Scanning Calorimeter and scanning results (Nick Birse, Wikimedia Commons) Schematic of Membrane Inlet Mass Spectrometry*  (Burlacot et al., 2020)5Schematic of of cell culture set up for live cells’ FTIR measurement* (Fale et al., 2015)3ReferencesAlthough each of the techniques have advantages and disadvantages, the two most promising for the scope of this research are Raman spectroscopy and Fluorescence spectroscopy. There are already many case studies that have used these two techniques to study the make-up of algae cells in real time with promising results. ConclusionFluorescence Spectrophotometer lifetime imagining layout*  (Lagarto et al., 2020)7Measurement schematic used for yeast fermentation monitoring with Raman spectroscopy* (Schalk et al., 2019)6An example of Raman Spectra (FBI, Public Domain) Flow-through capillary systemReal-time monitoring of healthy omega-3 production in micro-algaeA viability study*Image use granted by CC-BY 4.0.AcknowledgementsSpecial thanks to sponsors ‘The Biotechnology and Biological Sciences Research Council’ (BBSRC) and ‘Algae-UK’, and to ‘AlgaeCytes’ and the  ‘Bioinspired Engineering Research Group’.

Real-time monitoring of healthy omega-3 production in micro-algae: A viability study

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Real-time monitoring of healthy omega-3 production in micro-algae: A viability study

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