United Arab Emirates University Scholarworks@UAEU Electrical Engineering Theses Electrical Engineering 4-2018 VISUALIZATION AND QUANTIFICATION OF LIPID AND PROTEIN IN SINGLE MICROALGAL CELL Hina Laghari Mohammad Shakeel Follow this and additional works at: https://scholarworks.uaeu.ac.ae/electric_theses Part of the Engineering Commons Recommended Citation Shakeel, Hina Laghari Mohammad, "VISUALIZATION AND QUANTIFICATION OF LIPID AND PROTEIN IN SINGLE MICROALGAL CELL" (2018). Electrical Engineering Theses. 13. https://scholarworks.uaeu.ac.ae/electric_theses/13 This Thesis is brought to you for free and open access by the Electrical Engineering at Scholarworks@UAEU. It has been accepted for inclusion in Electrical Engineering Theses by an authorized administrator of Scholarworks@UAEU. For more information, please contact [email protected]. iii Copyright Copyright © 2018 Hina Laghari Mohammad Shakeel Laghari All Rights Reserved vi Abstract Microalgae have the potential to be used as a main source of renewable energy in the future due to their lipid contents. Microalgae are comprised of several components including carbohydrates, lipids and proteins. This lipid and protein composition needs to be quantified so the amount of usable energy can be measured. Therefore, the main objective of this thesis is to develop an image processing technique for the quantification of lipid and protein contents in single microalgal cell. This image processing technique has made lipid quantification an easier process as compared to previous methods used to quantify lipid content. The technique is based on staining microalgal cells with several dyes for the visualization of the lipid and protein content in a microalgal cell. The quantification is then carried out by applying image processing methods. To enhance the lipid accumulation and simultaneously reduce the protein, these microalgal cells are put under stress conditions such as nitrogen starvation. The nitrogen starvation stress condition results in alteration of the lipid and protein content inside the cells. This will significantly influence the ex vivo optimization of microalgal growth conditions for enhanced oil productivity. The image processing-based approach is then used to analyze the time course of lipid accumulation and protein reduction patterns inside the corresponding-stained microalgal cells. There are many different types of strains of microalgae. In this study, Nannochloropsis microalgal strain was used and cultivated in a nitrogen deficient medium. The intracellular Nannochloropsis cell content was estimated by mathematically evaluating the lipid and protein volume inside the corresponding-stained cell. This mathematical evaluation was made vii possible with the image processing technique that has been developed in this thesis. This image processing approach allows the quick characterization of microalgal cells. It also takes precedence over any techniques or methods that have been developed in the past to quantify lipid content of microalgal cells. This will be explored further in the subsequent studies. Keywords: Lipid, Microalgal cell, Protein, Quantification, Visualization. viii Title and Abstract (in Arabic) ﺗﺤﺪﻳﺪ ﻣﻜﻮﻧﺎﺕ ﺍﻟﻬﻦ ﻭ ﺍﻟﺒﺮﻭﺗﻴﻦ ﺑﺪﺍﺧﻞ ﺧﻠﻴﺔ ﻃﺤﻠﺒﻴﺔ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺗﻘﻨﻴﺔ ﺍﻟﺘﺼﻮﻳﺮ ﺍﻟﻤﻠﺨﺺ ﺍﻟﻄﺤﺎﻟﺐ ﺍﻟﺪﻗﻴﻘﺔ ﺑﺸﻜﻞ ﻋﺎﻡ ﻫﻲ ﻭﺍﺣﺪﺓ ﻣﻦ ﺍﻫﻢ ﺍﻟﻤﺼﺎﺩﺭ ﺍﻟﺮﺋﻴﺴﻴﺔ ﺍﻟﻤﺜﻴﺮﺓ ﻟﻼﻫﺘﻤﺎﻡ ﻓﻲ ﻣﺠﺎﻝ ﺍﻟﻄﺎﻗﺔ ﺍﻟﻤﺘﺠﺪﺩﺓ ﺍﻟﺨﻀﺮﺍء. ﻭﺑﺎﻟﺘﺎﻟﻲ ﻣﻦ ﺍﻟﻤﻬﻢ ﻭ ﺍﻟﻤﺘﻮﻗﻊ ﺃﻥ ﻳﻜﻮﻥ ﻟﺪﻳﻨﺎ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﻓﻬﻢ ﺩﻭﺭﻣﺤﺘﻮﻳﺎﺗﻬﺎ ﻓﻲ ﻋﻠﻢ ﻭﻇﺎﺋﻒ ﺍﻟﺨﻠﻴﺔ. ﻭﻟﺬﻟﻚ، ﻓﺈﻥ ﺍﻟﻬﺪﻑ ﺍﻟﺮﺋﻴﺴﻲ ﻣﻦ ﻫﺬﻩ ﺍﻷﻃﺮﻭﺣﺔ ﻫﻮ ﺗﻄﻮﻳﺮ ﺗﻘﻨﻴﺔ ﻣﺒﻨﻴﺔ ﻋﻠﻰ ﻣﻌﺎﻟﺠﺔ ﺍﻟﺼﻮﺭ ﻟﺘﻘﺪﻳﺮ ﻣﺤﺘﻮﻳﺎﺕ ﺍﻟﺪﻫﻦ ﻭﺍﻟﺒﺮﻭﺗﻴﻦ ﻋﻠﻰ ﻣﺴﺘﻮﻯ ﺧﻠﻴﺔ ﻭﺍﺣﺪﺓ. ﻭﻳﺴﺘﻨﺪ ﻫﺬﺍ ﺍﻷﺳﻠﻮﺏ ﺍﻟﺤﺎﻟﻲ ﻋﻠﻰ ﺗﻠﻮﻳﻦ ﺧﻼﻳﺎ ﺍﻟﻄﺤﻠﺒﻴﺔ ﺑﻤﻮﺍﺩ ﻣﺸﻌﺔ ﺧﺎﺻﺔ ﻟﺘﻘﺪﻳﺮﻧﺴﺒﺔ ﺍﻟﺪﻫﻮﻥ ﻭﺍﻟﺒﺮﻭﺗﻴﻦ. ﺛﻢ ﻳﺘﻢ ﺍﻟﻘﻴﺎﺱ ﺍﻟﻜﻤﻲ ﻭ ﺍﻟﻨﺴﺒﻲ ﻣﻦ ﺧﻼﻝ ﻃﺮﻕ ﻣﻌﺎﻟﺠﺔ ﺍﻟﺼﻮﺭ ﺍﻟﻤﻠﺘﻘﻄﺔ ﻣﻦ ﻣﺮﺑﻊ ﺍﻟﻤﺰﺭﻋﺔ ﻓﻲ ﺃﻳﺎﻡ ﻣﺨﺘﻠﻔﺔ ﻣﻦ ﺗﺤﺖ ﺣﺎﻟﺔ ﺍﻹﺟﻬﺎﺩ ﺍﻟﻨﻴﺘﺮﻭﺟﻴﻦ. ﺣﺎﻟﺔ ﺍﻹﺟﻬﺎﺩ ﻫﺪﻩ ﺗﻠﻌﺐ ﺩﻭﺭ ﻛﺒﻴﺮ ﻓﻲ ﺗﻐﻴﻴﺮ ﻣﺤﺘﻮﻯ ﺍﻟﺪﻫﻮﻥ ﻭﺍﻟﺒﺮﻭﺗﻴﻦ ﺩﺍﺧﻞ ﺍﻟﺨﻼﻳﺎ. ﺍﻥ ﺇﻣﻜﺎﻧﺎﺕ ﺍﻟﻨﻬﺞ ﺍﻟﺘﺼﻮﺭ ﺍﻟﺤﺎﻟﻲ ﻳﺴﻤﺢ ﺗﻘﺪﻳﺮ ﻓﻲ ﺧﻠﻴﺔ ﺣﻴﺔ ﻭﻫﺬﺍ ﺳﻮﻑ ﻳﺆﺛﺮ ﺑﺸﻜﻞ ﻛﺒﻴﺮ ﻋﻠﻰ ﺗﺤﺴﻴﻦ ﻇﺮﻭﻑ ﺍﻟﻨﻤﻮ ﺍﻟﺠﺰﺋﻲ ﻟﺘﺤﺴﻴﻦ ﺇﻧﺘﺎﺟﻴﺔ ﺍﻟﺪﻫﻮﻥ ﻛﻤﺼﺪﺭ ﺑﺪﻳﻞ ﻟﻠﻄﺎﻗﺔ. ﻣﻔﺎﻫﻴﻢ ﺍﻟﺒﺤﺚ ﺍﻟﺮﺋﻴﺴﻴﺔ: ﻃﺤﺎﻟﺐ.ﺍﻟﺪﻫﻮﻥ، ﺍﻟﺨﻠﻴﺔﺍﻟﻄﺤﻠﺒﻴﺔ، ﺍﻟﺒﺮﻭﺗﻴﻦ، ﺍﻟﺘﺤﺪﻳﺪ ﺍﻟﻜﻤﻲ، ﺗﻘﻨﻴﺔ ﺍﻟﺘﺼﻮﻳﺮ. ix Acknowledgements First and foremost, I would like to thank my thesis advisor and supervisor Dr. Mahmoud F. Al Ahmad of the Electrical Engineering Department at United Arab Emirates University. Without his consistent support and help, I most certainly could not have successfully conducted my thesis. I am very grateful for his endless patience towards me! I would also like to thank Mrs. Shaima Raji and Engineer Nisreen Al Naji for their enthusiastic input and help throughout the researching process. Finally, I must express my gratitude to my family (Dr. Mohammad Shakeel Laghari, Anila Shakeel, Anum Laghari, Saad Laghari), including my cats (Coco Pixie, Nala, Luna, Gracie, Chottu) and especially to Pippin for providing me with constant support and encouragement throughout my years of studying engineering and throughout the process of researching and writing this thesis. This accomplishment would not have been possible without them and my supervisor Dr. Mahmoud F. Al Ahmad. Thank you. x Dedication To my treasured family and cats xi Table of Contents Title ............................................................................................................................... i Declaration of Original Work ...................................................................................... ii Copyright ....................................................................................................................iii Approval of the Master Thesis .................................................................................... iv Abstract ....................................................................................................................... vi Title and Abstract (in Arabic) ...................................................................................viii Acknowledgements ..................................................................................................... ix Dedication .................................................................................................................... x Table of Contents ........................................................................................................ xi List of Tables.............................................................................................................xiii List of Figures ........................................................................................................... xiv List of Abbreviations.................................................................................................. xv Chapter 1: Introduction ................................................................................................ 1 1.1 Aim………………………………………………………………………..1 1.2 Motivation .................................................................................................. 1 1.3 Research Objectives ................................................................................... 2 1.4 Thesis Significance .................................................................................... 3 1.5 Thesis Organization ................................................................................... 3 Chapter 2: Literature Review ....................................................................................... 5 2.1 Microalgae Biomass as an Energy Source ................................................. 5 2.2 Conventional Methods for Extraction of Biomolecules............................. 9 2.3 Image Processing by Fluorescent Microscopy ......................................... 13 Chapter 3: Materials and Methods ............................................................................. 18 3.1 Microalgae Cultivation............................................................................. 18 3.1.1 Growth under Controlled Conditions ............................................... 19 3.1.2 Determination of the Cell Concentration ......................................... 19 3.2 Microalgae Fluorescent Staining.............................................................. 20 3.2.1 Protein Visualization by Fluorescent Microscopy ........................... 20 3.2.2 Lipid Visualization by Fluorescent Microscopy .............................. 21 3.3 Image Processing Tools ........................................................................... 22 Chapter 4: Results and Discussion ............................................................................. 24 4.1 Visualization of Lipids, Protein Islands in Microalgal Cells ................... 24 4.1.1 Lipid, Protein Determination Using Biochemistry Method .............. 25 4.1.2 Statistical Analysis ............................................................................ 27 xii 4.2 Lipid, Protein Area Determination from Image Processing..................... 29 4.3 Discussion ................................................................................................ 31 Chapter 5: Conclusions and Future Work .................................................................. 36 5.1 Conclusions .............................................................................................. 36 5.2 Future Work ............................................................................................. 37 References .................................................................................................................. 40 xiii List of Tables Table