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Microalgal Co-Cultures for Biomanufacturing Applications

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Microalgal Co-Cultures for Biomanufacturing Applications Microalgal co-cultures for biomanufacturing applications. Gloria Padmaperuma Department of Chemical and Biological Engineering Thesis submitted for the degree of Doctor of Philosophy To the University of Sheffield, Sheffield, UK 20th December 2017 This copy of the thesis has been submitted with the condition that anyone who consults it is understood to recognize that the copyright rests with its author. No quotation and information derived from this thesis may be published without prior written consent from the author or the University (as may be appropriate). Declaration This is a declaration to state that this thesis is an account of the author’s work, which was conducted at The University of Sheffield, United Kingdom. This work has not been submitted for any other degree of qualification. I II Acknowledgments “If life's journey be endless, where is its goal? The answer is, it is everywhere.” - R. Tagore The PhD, in itself, has been a roller coaster of self-discovery and dwelling deeper into the unknown… or at least that is how it felt like. The main pillars of my life, Ammi and Thathi; I have, and always will dedicate all my achievements. My two supervisors, Raman and Jim, have been sources of guidance and reference. I would like to thank you both, for your time, valuable contributions and support. Your experience and inputs have indeed shaped my research and taken me into discovering knowledge pools to me previously hidden. I am grateful to the EPSRC for funding me. Thank you to Rahul, Esther, Joy, Tamas, James and the Workshop guys, for taking time out their busy working hours to help me and encourage. My PhD has not just been an academic journey. My friends have been with me through good and bad times. In the time of need, I did not feel alone. I thank you all for seeing me through this and for never doubting that I could make it. To my Sister and Efi… I am very lucky to know that you will always have my back. III IV Publications Microbial consortia: a critical look at microalgae co-cultures for enhanced biomanufacturing Gloria Padmaperumaa, Rahul Vijay Kapoorea, Daniel James Gilmourb and Seetharaman Vaidyanathana aChELSI Institute, Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, The University of Sheffield, Sheffield, UK; bDepartment of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK CRITICAL REVIEWS IN BIOTECHNOLOGY, 2017 https://doi.org/10.1080/07388551.2017.1390728 V VI List of Abbreviations AHLs N-acylhomoserine lactones BBM Bold’s Basal Medium BBM+ Bold’s Basal Modified Medium BSA Bovine Serum Albumin Ca, Chl a Chlorophyll a Cb, Chl b Chlorophyll b CBE Chemical and Biological Engineering CCAP Culture Collection of Algae and Protozoa CFU Colony formation unit CHO Chinese Hamster Ovary CTC Copper Tartarate Cx+c Carotenoids D. salina Dunaliella Salina DCW Dry Cell Weight DHAP Dihydroxyacetone phosphate DIC Dissolved Inorganic Carbon DIN Dissolved Inorganic Nitrogen EPS Extracellular Polymeric Substances ETC Electron Transport Chain FACS Fluorescence-Activated Cell Sorting FAME Fatty Acid Methyl Esthers GDPH Glyceraldehyde 3-phosphate dehydrogenase GHGs Greenhouse Gases H. salinarum Halobacterium salinarum HEPES Hepes Medium HEPES+ Hepes Modified medium MBB Molecular Biology and Biotechnology MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide NCIMB National Collection of Industrial, Food and Marine Bacteria VII NCYC National Collection of Yeast Cultures OD Optical Density PC1 Principle Component 1 PCA Principle Component Analysis QSI Quorum Sensing Inhibiting R. toruloides Rhodosporidium toruloides RIs Retention Indices RuBisCO ribulose-1, 5-busphate carboxylase/oxygenase S. obliquus Scenedesmus obliquus TAGs Triglycerides THF Tetrahydrofuran UNFCCC United Nations Framework Convention on Climate Change VOSCs Volatile Organic Sulphur Compounds YM Yeast Mold Media VIII Abstract High demands in consumer goods and pressures from governments to meet environmental regulations have pushed industries to find innovative, carbon-neutral solutions. Sustainable methods in biotechnology are sought to increase productivity whilst keeping at bay one of the major problems in monoculture production routes: contamination. The use of engineered consortia is seen as a viable option. In nature, microorganisms exist as part of complicated networks known as consortia. Within the consortia, each member plays a role in facilitating communication, tasks distribution, nutrients acquisition and protection. This emerging field uses the conundrums that govern natural microbial assemblages to create artificial co-culture within the laboratory. Purpose fit, co-cultures have been created, to enhance productivity yields of desired products, for bioremediation and to circumvent contamination. The use of microalgae in co-cultures is the focus of this study. Microalgae have application in many fields and are ideal candidates for bioproduction and carbon sequestration. The results of two different systems are presented, which aim to increase the productivity of microalgae biomass and of β-carotene or lipids. The natural consortium of Dunaliella salina, Halomonas and Halobacterium salinarum showed both an increase in microalgae cell concentration by 79% and higher β-carotene productivity compared to the monoculture. This association also showed that Halomonas is able to aid D. salina when subjected to abiotic stress. The artificial co-culture of Scenedesmus obliquus and Rhodosporidium toruloides showed an increase in microalgae biomass by 20%; however, the FAME levels of 26% dw were not a significant increase, compared to monocultures. Both systems demonstrated that if one member of the assemblage is in dire stress, this stress will translate to the entire community. Characterisation of exopolymeric substances and metabolites provided a fuller picture on how these microorganisms co-exist. Additionally, a novel method, duo-plates, was developed and successfully tested to trap metabolites within co-cultures. IX X Table of Contents Introduction ............................................................................................................................ 1 Chapter 1: Literature Review .................................................................................................. 3 1.1. Introduction ................................................................................................................. 3 1.2. Microbial Consortia ..................................................................................................... 5 1.2.1. Consortia in Nature .............................................................................................. 5 1.3. Artificial Co-cultures: learning from Nature ................................................................ 7 1.4. Co-culture design ......................................................................................................... 9 1.4.1. Shortlisting suitable candidates ......................................................................... 10 1.5. Selecting co-culture partners .................................................................................... 10 1.5.1. Co-culture media ............................................................................................... 11 1.5.2. Inoculation: ratio and timing ............................................................................. 11 1.6. Reactor design and available technologies for co-culture ........................................ 12 1.7. Critical considerations ............................................................................................... 12 1.8. Case study: microalgae co-cultures for biotechnological application ....................... 13 1.9. Microalgae co-cultures: current status ..................................................................... 14 1.9.1. Factors Affecting Microalgae Co-Cultures ......................................................... 16 1.9.2. Microalgae Co-Culture: Future Potential ........................................................... 16 1.10. Work presented in the thesis ................................................................................ 17 1.11. Co-cultures and consortia: challenges and future possibilities ............................. 18 1.11.1. Co-culture database ....................................................................................... 19 1.12. Conclusions ............................................................................................................ 20 1.13. References ............................................................................................................. 20 Chapter 2: Materials and Methods…………………………………………………………………………………..32 2.1.Introduction .................................................................................................................... 32 2.2. Microorganisms studied ........................................................................................... 32 2.2.1. Dunaliella salina CCAP 19/18 ............................................................................. 32 2.2.2. Halomonas (isolated from D. salina) ................................................................. 33 2.2.3. Halobacterium salinarum NCIMB 764 ............................................................... 33 2.2.4. Scenedesmus obliquus ....................................................................................... 33 2.2.5. Rhodosporidium toruloides NCYC 912 ..............................................................
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