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Thesis Submitted for the Degree of Doctor of Philosophy University of Bath PHD Microalgae for Wastewater Treatment and Biomass Production from Bioprospecting to Biotechnology Sweiss, Mais Award date: 2017 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Microalgae for Wastewater Treatment and Biomass Production from Bioprospecting to Biotechnology Mais Ahed Sweiss A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry September 2017 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that they must not copy it or use material from it expect as permitted by law or with the consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation with effect from ………………. (date). Signed on behalf of the Faculty of Science ……………………………. Acknowledgements First of all, I would like to express my gratitude to God who guided me throughout my PhD journey. My sincere appreciation goes to my supervisor Prof Rod Scott for giving me the opportunity to learn, for his support and suggestions. I would like to extend my sincere thanks to my co-supervisor Dr Tom Arnot for helping me with my samples collection and for his advice. I would like to express my respect and appreciation to my examiners Prof Alison Smith from Cambridge and Dr Daniel Henk from University of Bath, for their valuable suggestions. I am grateful to Prof David Leak from University of Bath for helping me in designing the equation for assessing the nutrients removal and Prof Ed. Feil from University of Bath for his help in phylogenetic analysis. I would like to thank Prof Saul Purton from the University College London, Dr Kyle Lauersen from Bielefeld University and Katrin Geisler from University of Cambridge for their help and suggestions for microalgae transformation. I would like to thank Dr Baoxiu Qi for her help, support and professional suggestions, Dr Philippe Mozzanega for his day to day supervision, Dr Dimitrios Kaloudis and Dr Holly Smith-Baedorf for their willingness to help. I had the privilege to meet through this journey many truly great people that have been not only lab mates but friends. I would like to thank my colleagues Maha Al Jabri, Julia Tartt, Jane Li, Yaoxio Li, Lidia Al-Halaseh, Kate Petty, Sarah Chordekar, Catherine-Axa Wilkins for the emotional support every time I felt down. I will not forget Ludi Wang, Lian Fan, Mutian Yang, and Ruth Ilesanmi. Some sincere thanks are going to my friend Ruqaya Al Jabri for her great support and help especially when I was writing up my thesis. I would like to extend my thanks to Aidan Berry for his help in the phenotyping experiment in Chapter 5. I would extend my sincere thanks to my sponsors, as without their funding this research would not have been possible. Many thanks to the Ministry of Higher Education and Scientific Research (Jordan), University of Bath (UK), Al Balqa` Applied University (Jordan) and Wessex Water. I owe a lot to my family, my mother, my brother and my sisters for their endless support, they always stood by me, supported me and never let me down. Special thanks go to my uncle Eid Swais and my Auntie Adrienne Swais for their help and support during my PhD Journey. 1 Dedication I would like to dedicate this thesis to my beloved mother, who is a brave, dedicated women with endless love for her family. To my father’s soul who always encouraged me, filled me with hope, and taught me to follow my ambitions. To my brother and my sisters who supported me and showed me the joy in life. 2 Abstract Improving wastewater (WW) treatment process is a major issue in different parts of the world. For a developed country like the UK where eutrophication is a problem that causes environmental and economical losses, and for a developing country like Jordan that is considered one of the most water scarce countries in the world, it is crucially important to improve the quality of the WW for safe reuse. Applying microalgae for WW treatment and biomass production is an economical and environmentally friendly method. However, this method has some challenges that need to be addressed, such as microalgae species selection, harvesting of the microalgae and the large area footprint. In this research, the overall aim was to bioprospect for microalgae that are adapted to the wastewater treatment plants (WWTPs) and evaluate the obtained microalgae depending on specific criteria for a successful application in high rate algal ponds (HRAPs), then there were attempts to improve the phosphorus removal in microalgae to increase the efficiency of the treatment process and reduce the area footprint. Bioprospecting for indigenous microalgae to the WW took place from January to May 2014. Water samples were collected from wastewater treatment plants (WWTPs) in the UK and Jordan. Eight different microalgae isolates were identified from each country. The results showed the Chlorella, Scenedesmus and Desmodesmus are common genera between the two countries and dominated the obtained isolates from the UK and Jordan. The isolates were identified using 18S rDNA and ITS1-5.8S-ITS2 DNA barcoding markers. It was difficult to identify some of the isolates at the species level, as the 18S rDNA is too conserved to differentiate between the closely related species and due to the relatively poor representation of algae in GenBank. Then the obtained microalgae isolates were evaluated by their growth, efficiency in removing nutrients (i.e. nitrogen and phosphorus) and the settleability of the microalgae by gravity. Depending on the results the microalgae species were ranked to come up with some promising candidates to be applied on large scale. From the UK, Avonmouth_12 (Av_12) and Avonmouth_10 (Av_10) and from Jordan, Jordan_18 (Jo_18) and Jordan_29 (Jo_29) were distinguished in their performance in the WW. Since phosphorus is a major cause of eutrophication in the fresh water and it is important to reduce the level of phosphorus in the released WW to the legally permitted limits, this 3 research aimed to study the possibility of improving phosphorus removal by microalgae. Using Chlamydomonas reinhardtii as a model to optimise the protocol to be applied in parallel with Av_12, which is a promising microalga isolate that has been applied on large scale in HRAPs in Beckington WWTP, the strategy was to overexpress a Phosphorus Starvation Response (PSR1) gene. The transformation process was successful in C. reinhardtii but not in Av_12. There was an enhancement of the specific phosphate removal rate in the transformed microalgae isolate CC-1010_B2 and CC-1010_A6 in comparison to the wild type strain CC-1010. 4 Abbreviations and acronyms ANOVA Analysis of variance aph Aminoglycoside 3`-phosphotransferase gene atpB ATPase beta-subunit BBM Bold Basal Medium bp Base pair BPA Bisphenol A ca. Circa (approximately, around) CBCs Compensatory base changes CO2 Carbon dioxide cox1 Cytochrome oxidase 1 cox2-3 spacer Cytochrome oxidase 2-cytochrome oxidase 3 intergenic spacer cox3 Cytochrome oxidase 3 DHA Docosahexaenoic acid DIH2O Deionized water DMSO Dimethyl sulfoxide DW Dry weight EBPR Enhanced biological phosphorus removal EDTA Ethylenediamine-tetraacetic acid EPA Eicosapentaenoic acid EPS Extracellular polymeric substances ETS External transcribed spacer FTIR Fourier Transform Infrared GOI Gene of interest h Hour HRAP High Rate Algal Ponds HSP70A Heat shock protein 70A HTL Hydrothermal liquefaction i1 Intron 1 IGR Intergenic spacers Inhab Inhabitant 5 ITS Internal transcribed spacer kb Kilo base pair kt Kilo ton LSU Large ribosomal subunit MCM Million cubic meters MIC Minimum inhibitory concentration min Minute MPa Megapascal mt Mutant Nucleotide BLAST Nucleotide Basic Local Alignment Search Tool OD Optical density P Phosphorus PE Population equivalent PG phosphatidylglycerol Pi Inorganic phosphorus psaA Code for Photosystem I P700 chlorophyll a apoprotein A1 psbA Code for Photosystem II protein D1 PSR1 Phosphorus Starvation Response 1 qRT-PCR Quantitative reverse transcription polymerase chain reaction rbcL Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit RBCS2 RuBisCO small subunit 2 rDNA Ribosomal deoxyribonucleic acid rRNA Ribosomal RNA RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase SQD1 UDP- sulfoquinovose synthase SQD1 SQD3 UDP- sulfoquinovose synthase SQD3 SQDG Sulfolipids
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