Developing systems for the commercial culture of Ulva species in the UK Guang Gao A thesis submitted to Newcastle University in candidature for the Degree of Doctor of Philosophy School of Marine Science and Technology May 2016 Abstract The green seaweed, Ulva, is highly valued in terms of animal feed, food and biofuel, as well in the delivery of crucial remediation services including wastewater treatment and CO2 removal. Accordingly, Ulva cultivation has gained significant research interest worldwide. Notwithstanding these research efforts, Ulva cultivation is still in its infancy and knowledge to underpin such developments remains limited. A common challenge in Ulva cultivation is the fluctuating productivity with time due to vegetative fragmentation and/or periodic reproduction. In this study, three methods were employed to address this challenge. Firstly, culture conditions were optimised to establish a balance between growth and reproduction. Secondly, a refined culture method was developed, which more than tripled growth of Ulva over an 18-day cultivation as compared to a standard method. Thirdly, a sterile strain was obtained by mutating a wild strain with ultraviolet radiation. This new strain grew five times faster over an 18-day cultivation and absorbed nitrate and phosphate 40.0% and 30.9% quicker compared to the wild strain respectively. The chemical composition of the sterile strain showed a lipid content of more than double that of the wild strain, while the protein content was 26.3% lower than the wild strain. Several tissue preservation techniques were developed to enable settlement and growth trials to be conducted on demand. The merits or otherwise of the preservation techniques were determined for gametes, germlings and thalli. In addition to cultivation-related techniques, the co-effects of climate change factors (global warming and ocean acidification) and eutrophication on Ulva cultivation were investigated. These three variables interacted in a complex pattern to differentially affect life history stages, as well as altering the chemical composition and functional properties of Ulva. These findings make tangible contributions to the ability to successfully and commercially cultivate Ulva in terms of culture conditions, tissue preservation and the development of mutant strains. Further, by placing Ulva culture in a climate change context, this work provides valuable insight into the limits to resilience of Ulva to a changing climate. This will inform the future development of the Ulva culture industry over the coming decades. i ii Acknowledgements I am very happy that I was able to complete this project within three years. But it would have been impossible to manage it without help from many people. Therefore, I would like to express my sincere gratitude to them. The first people I would like to acknowledge are my supervisors, Dr Gary Caldwell, Prof. Tony Clare, and Dr Craig Rose. It is you that brought me to Newcastle from China. It is you that inspired me when I got stuck in the experiments. It is you that improved my writing with perpetual patience. Your expertise and ethic in research have impressed me and would have a profound and lasting influence on my future career. Massive thanks should also be given to staff and colleagues in the School of Marine Science and Technology (MaST). I would like to say thank you to the technicians David Whitaker and Pete McParlin. Your excellent work tangibly facilitated the process of my research. I also want to express my appreciation of Dr Nicholas Aldred and Dr John Finlay. I hope that you were not annoyed by some stupid questions from me. Dr Sofia Cota Franco and Carol Barnett your enthusiastic help and sweet smiles often make me think of angels. Amanda Benson, Philippa Richard, Bita Sabbaghzadeh, Wahyu Nugraha, Adibi Md Nor, please forgive me that I cannot mention everyone in MaST here, I actually love you all. Special thanks should go to Dr Qingchun Zhang in the Institute of Oceanology, Chinese Academy of Sciences, who helped me identify the Ulva species, Eleni Chatzidimitriou who assisted me in analysing fatty acids in School of Agriculture, Food and Rural Development, and Georgina Robinson for her support in statistical analysis. Life is always up and down just like the English weather. I am lucky to make some intimate friends in Newcastle. They are Serena Lim, Hongbin Zhang, Bill Gibson, Roger Darsley, HaiboYang, Danqing Feng, etc. You are my fortune because you blowed away cloud and brought over sunshine for me. This research would not have been possible without the financial support from MaST in Newcastle University. Last but not least, I would like to express my debt to my wife. You carry the burden of whole family with your tender shoulders. You take care of parents at both sides and raise our son up alone. Without your self-giving contribution and support, I could never have proceeded to my current position. I also owe a great deal to my Mum and Dad for their long-suffering support and endless encouragement! iii iv Table of Contents Abstract ......................................................................................................................... i Acknowledgements ...................................................................................................... iii List of Tables ............................................................................................................... xi List of Figures ............................................................................................................ xvi Chapter 1. Introduction ................................................................................................. 1 1.1 Physiology of Ulva .............................................................................................. 1 1.1.1 Life history of Ulva ....................................................................................... 1 1.1.2 Settlement of Ulva swarmers ......................................................................... 3 1.1.3 Factors affecting germination ........................................................................ 4 1.1.4 Factors affecting growth and reproduction..................................................... 7 1.2 Cryopreservation of algae .................................................................................... 7 1.2.1 Freezing methodology ................................................................................... 7 1.3 Values of seaweed ............................................................................................. 10 1.3.1 Food ............................................................................................................ 10 1.3.2 Animal feed ................................................................................................ 14 1.3.3 Bioremediation ............................................................................................ 15 1.4 Ulva cultivation: history and methods ................................................................ 16 1.4.1 Open water cultivation ................................................................................ 18 1.4.2 Land based cultivation ................................................................................ 19 1.4.3 Integrated Multi-Trophic Aquaculture ......................................................... 19 1.4.4 Advantages and disadvantages of Ulva cultivation ...................................... 20 1.5 Effects of ocean acidification and warming on seaweeds ................................... 22 1.5.1 Effects of ocean acidification on seaweeds .................................................. 22 1.5.2 Effects of ocean warming on seaweeds ........................................................ 23 1.5.3 Combined effects of ocean acidification and warming ................................. 25 1.6 Research aims and objectives............................................................................. 26 Chapter 2. Growth conditioning of Ulva rigida ........................................................... 27 2.1 Introduction ....................................................................................................... 27 2.1.1 Nitrate ......................................................................................................... 27 2.1.2 Light ........................................................................................................... 27 2.1.3 Temperature ................................................................................................ 28 2.1.4 Aeration ...................................................................................................... 28 2.1.5 Aims and objectives .................................................................................... 29 v 2.2 Materials and methods ....................................................................................... 29 2.2.1 Seaweed collection and identification .......................................................... 29 2.2.2 Seaweed culture under different light and nutrient levels ............................. 30 2.2.3 Seaweed culture under different temperatures ............................................. 31 2.2.4 Seaweed culture under different aeration conditions .................................... 31 2.2.5 Multiple harvests method ............................................................................ 31 2.2.6 Nitrogen and phosphorus uptake determination ..........................................