ULVA GROWTH, DEVELOPMENT AND APPLICATIONS by Fatemeh Ghaderiardakani A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences The University of Birmingham August 2019 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The green marine macroalgae Ulva (Ulvophyceae) are common algae distributed worldwide, which play a key role in aquatic ecosystems. Ulva species are a potentially valuable resource for food, feed, fertiliser and fuel but can also cause massive nuisance blooms if they grow unchecked. For correct growth and development, Ulva requires the presence of a combination of regulatory morphogenetic compounds released by associated epiphytic bacteria in addition to nutritional parameters. The first results chapter examines the extent of specificity or generality of bacteria-induced morphogenesis in Ulva, by cross-testing bacteria isolated from several Ulva species on Ulva mutabilis and Ulva intestinalis. We show that pairs of bacterial strains isolated from Ulva species can fully rescue U. mutabilis or U. intestinalis morphology. In the second results chapter, activity of algal growth- and morphogenesis-promoting factors (AGPFs) derived from bacteria were estimated in a land-based integrated multitrophic aquaculture system (IMTA) of fish and macroalgae (located at the coastal lagoon Ria de Aveiro, Portugal), using a standardised bioassay with axenic cultures of Ulva. Nutrient availability was also assessed in this IMTA system. The study thus informs aspects of the various potential aquaculture-environment interactions. It was observed that both the water from the lagoon (external to the farm system) and the water from the fish pond (input for algae cultures) could completely restore the normal growth and morphology of the macroalga under axenic conditions. The results highlight the presence of a sufficient chemical cocktail of AGPFs in this IMTA system required for growth and morphogenesis of Ulva. In addition, the water of fish farming increases the nutrient availability (nitrate and ammonium) needed for macroalgae production. The conclusion of this chapter is that Ulva´s sustainable growth and development can benefit from multitrophic aquaculture systems and shallow water systems, due to the naturally enriched AGPFs and their in-situ production by bacteria in intensive algal aquacultures. In the final results chapter, the effects of U. intestinalis extracts on germination and root development in the model land plant Arabidopsis thaliana were examined. Ulva extract concentrations above 0.1% inhibited Arabidopsis germination and root growth. Ulva extract <0.1% stimulated root growth. All concentrations of Ulva extract inhibited lateral root formation. An abscisic-acid insensitive mutant showed altered sensitivity to germination- and root growth-inhibition. Ethylene- and cytokinin-insensitive mutants were partly insensitive to germination-inhibition. This suggests that different mechanisms mediate each effect of Ulva extract on early Arabidopsis development and that multiple hormones contribute to germination-inhibition. Taken together, the results of this thesis highlight: (i) Specific Ulva-associated bacterial functions (promoting cell division, or cell differentiation) that cannot be assigned to a specific genus/taxonomic group of bacteria, (ii) an IMTA system ensuring an adequate supply of nutrients and a sufficient chemical mixture of AGPFs for reliable Ulva cultivation and (iii) the first-characterised mechanisms to date by which Ulva extract can impact germination and growth in Arabidopsis. This thesis and all my academic achievements are dedicated to my mother and father. Without their endless support and encouragement, I would never have been able to complete my graduate studies. Acknowledgements I would like to take this opportunity to express my heartfelt gratitude and sincere thanks to all those who helped me to complete my thesis. Dr. Juliet Coates, my fantastic supervisor, I would like to thank you for agreeing to take me as PhD student, for your regular advice, immense support, thoughtful guidance, critical comments, correction of my papers and thesis and eventually for boosting my confidence throughout the entire PhD journey. I feel so lucky to have you as my supervisor. Dr. Thomas Wichard, I thank you from the bottom of my heart for accepting me as visiting researcher in your lab and mentoring my experiments at the Friedrich Schiller University Jena. You have been a constant source of motivation, encouragement and inspirational guidance. You introduced me a sense of discipline and attention to details in my work. Thanks for the time you spent to answer my questions and all discussions. Xulyu Cao, Clare Clayton and Alexandros Phokas, I would like to thank you as my fantastic friends for sharing your knowledge and all cheerful time we spent together. I would also like to thank my lab mates in Jena, Ralf Kessler, Anne Weiss, Jan Frieder Mohr and Gianmaria Califano for your help and advice. Dr. Daniel Gibbs, I am grateful to you and your wonderful group for supporting me. Thanks to Dr. Helena Abreu and all amazing members of ALGAplus company for your assistance, support and advices to perform my investigation in Portugal and for that particular dream you helped to come true. Finally, a big thank you to my sister and brothers, for those lots of support you all gave me during my long educational journey, to my mother-in-law and father-in-law for your understanding and sacrifices, to my sisters-in-law and brothers-in-low, you have been a source of strength and motivation during moments of distress and discouragement and to Reza, for putting up with me at home throughout my PhD and for all seaweed collecting journeys. I would like to acknowledge the Islamic Development Bank for funding my PhD studentship, networking support by the COST Action ‘Phycomorph’ FA1406 through Short Term Scientific Missions, the British Phycological Society, International Phycological Society, Estuarine and Coastal Sciences Association and Society for Experimental Biology for travel grants that enabled me to present my research in the UK, Denmark, Poland and Italy. Contents CHAPTER 1: INTRODUCTION 1 1.1 Phylogeny of macroalgae 2 1.2 The functions and benefits of macroalgae 4 1.2.1 The ecological importance of macroalgae 4 1.2.2 Nutritional and biomedical values of macroalgae 6 1.2.3 Seaweed aquaculture 11 1.3 The genus Ulva 14 1.4 Identification of Ulva 15 1.5 Ulva’s life cycle 17 1.6 Features of the Ulva mutabilis life cycle 20 1.7 The ecological and economic importance of Ulva species 22 1.7.1 Ecological and economic benefits 22 1.7.1.1 Ulvan, a unique polysaccharide found in Ulva genus with potential values in biotechnology and industry 22 1.7.1.1.1 Elicitor activity of ulvan 23 1.7.1.2 Ulva as a co-culture species in integrated multi-trophic aquaculture (IMTA) 25 1.7.2 Ecological and economic disadvantages of Ulva 29 1.7.2.1 Ulva settlement and its regulators 30 1.8 Model organisms 31 1.8.1 Arabidopsis thaliana a well-defined model organism 31 1.8.2 Algal model systems 32 1.9 Aims of this research 35 1.10 Concluding remarks 37 CHAPTER 2: MORPHOGENESIS AND DEVELOPMENT INDUCED BY BACTERIA IN ULVA SPECIES 38 2.1 The symbiosis between marine macroalgae and bacterial communities 39 2.1.1 Microbial abundance and diversity 39 2.1.2 Importance and function of algal-bacteria interaction 40 2.2 Uniqueness and variability of the epiphytic bacterial community on macroalgae 42 2.2.1 Is the epibacterial community on macroalgae host-specific? 42 2.2.2 The lottery hypothesis 43 2.2.3 The sea lettuce Ulva only adopts a typical morphotype with the right bacteria 43 2.3 A symbiotic tripartite community as a bioassay-driven approach 44 2.3.1 Cross-testing experiment 45 2.3.2 A new tripartite system more suited to the UK 46 2.4 Chapter aims 49 2.5 Materials and methods 49 2.5.1 Algal samples 49 2.5.2 Preparation of Ulva culture medium (UCM) 52 2.5.3 Cultivation conditions 52 2.5.4 Artificial induction of gametogenesis and preparation of axenic cultures 53 2.5.4.1 Purification of gametes 54 2.5.5 Phylogenetic characterisation of Ulva 55 2.5.5.1 Genomic DNA extraction 55 2.5.5.2 Primer sequences and PCR conditions 56 2.5.5.3 Cloning the tufA sequences 57 2.5.5.4 Agarose gel electrophoresis 57 2.5.5.5 Sequence analysis of rbcL and tufA genes 57 2.5.5.6 Phylogenetic analysis 58 2.5.6 Bacterial strain selection 58 2.5.6.1 Phylogenetic characterisation of bacteria 61 2.5.6.1.1 Preparation of bacterial isolates 61 2.5.6.1.2 DNA extraction, primer sequences and PCR conditions 61 2.5.6.1.3 Taxonomic classification 62 2.5.7 The Ulva bioassay array 62 2.5.7.1 Gamete distribution 62 2.5.7.2 Bacteria inoculation 63 2.5.7.3 Microscopy and quantification of parameters 64 2.6 Results 65 2.6.1 Identification of Ulva samples 65 2.6.1.1 Tubular-shape samples 65 2.6.1.2 Flat-shape samples 68 2.6.2 Identification of bacteria 71 2.6.2.1 Initial screening of morphologically active Ulva spp. surface-associated bacterial strains 71 2.6.2.2 Assay of nine further U.