Alisa Mihaila
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http://researchcommons.waikato.ac.nz/ Research Commons at the University of Waikato Copyright Statement: The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). The thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: Any use you make of these documents or images must be for research or private study purposes only, and you may not make them available to any other person. Authors control the copyright of their thesis. You will recognise the author’s right to be identified as the author of the thesis, and due acknowledgement will be made to the author where appropriate. You will obtain the author’s permission before publishing any material from the thesis. Investigating the anti-methanogenic properties of select species of seaweed in New Zealand. A thesis submitted in fulfilment of the requirements for the degree of Master of Science (Research) at The University of Waikato by Alisa Mihaila 2020 Abstract Enteric methane emissions from ruminants constitute a large proportion of agricultural greenhouse gas emissions, particularly in New Zealand. The recent increase in enteric methane emissions has driven the development of innovative strategies for mitigating these emissions. Red seaweed from the genus Asparagopsis has demonstrated elimination of enteric methane due to the presence of the active anti-methanogenic component, bromoform. Spatial variation in bromoform content for Asparagopsis armata throughout the North Island, New Zealand, was quantified to determine the region that produces the highest concentration of bromoform. Alongside Asparagopsis, the New Zealand red seaweed species Bonnemaisonia hamifera, Delisea compressa, Plocamium sp., Vidalia colensoi, and identified aquaculture-target seaweed species, Ecklonia radiata, and Ulva sp. B, were investigated as ruminant feed additives to reduce enteric methane emissions. Polyphenol quantification and compositional analyses were carried out for these seaweed species to provide a baseline for interpreting anti-methanogenic effects. Seaweed species were included at 0 %, 2 %, 6 %, and 10 % of feed organic matter (ryegrass hay) during in vitro fermentation assays using rumen inoculant from non-lactating cows. Total gas, methane, hydrogen, volatile fatty acid (VFA), and ammonia production were measured during the incubations. Bromoform concentration was highest in A. armata sampled from Matheson’s Bay at 1 % of the biomass dry weight. Species of red seaweed had a high halogen content, while E. radiata and Ulva. sp. B had a high iodine and crude protein content, respectively. Inclusions of A. armata and B. hamifera demonstrated near elimination of enteric methane production at doses of 2 and 6 % organic matter, respectively, while the remaining species (except for Ulva sp. B) caused moderate reductions at doses of 6 and 10 % organic matter in comparison to these two species. The anti-methanogenic effects of A. armata and B. hamifera resulted in a 22 % reduction in total VFA production, accompanied by changes in the relative i proportions of individual VFAs, and had little or no effect on organic matter degradation. The effectiveness of A. armata and B. hamifera demonstrates the potential of these species for mitigating ruminant methane emissions at low inclusion rates, dependent on the concentration of their active components, while E. radiata and Ulva sp. B could be used as feed additives for nutritional benefit. The undertaking of larger scale sampling of A. armata throughout New Zealand, the identification of the active component(s) in B. hamifera, and the development of methods and infrastructure required for successful large-scale aquaculture and application of these seaweed species to livestock management systems are key areas of future research highlighted by this thesis. ii Acknowledgements Firstly, I want to thank my supervisors Marie Magnusson and Rebecca Lawton for their utmost support and guidance. Marie, you have truly been a superhero to me – even out of the thousands of words that make up this thesis, there are no words that really can express the degree of gratitude I have for your input and ongoing support and encouragement. Rebecca, I greatly appreciate that you were always happy to help me with anything I came to you with; without you, I would have no stats to discuss, so thank you for the extra time you put in. Chris Glasson, thank you for allowing me to pick your knowledgeable brain when it came to all things chemistry, your insight was highly welcomed. Ari Brandenburg, a huge thank you for showing me how to do everything when it came to lab work. Thank you also to Cheryl Ward. Stefan Muetzel, I thank you immensely for the time, patience, and knowledge you provided me with while I was based at AgResearch; I learnt a great deal from you. A similar thanks to German Molano, your humour kept me from running away from the horrible smells in the rumen fermentation lab. This project was funded by the Tertiary Education Commission (TEC) and the University of Waikato via the Macroalgal Biotechnologies Programme funded under the TEC Entrepreneurial Universities scheme. I was awarded a scholarship under this same scheme, which provided me with financial support whilst studying and was largely appreciated, thank you. I thank my partner Andrew, who constantly listened to me talk about cows and seaweed, you were the greatest outside support I could have asked for in every way. A huge thank you to all of my friends who supported, sent love, and checked up on me throughout this thesis. I would like to extend a special thanks to my good friend Jess; not many friends would read an entire thesis by choice, you are indeed a trooper. Thanks to all my fellow masters students. And lastly, to my family, I can’t thank you enough. My sister Sara, you have always been there for me. Mum and Dad, you have always been my biggest supporters and I am forever grateful. Mum, I really couldn’t have done this without you, you deserve a medal, thank you. iii Table of Contents Abstract ................................................................................................................... i Acknowledgements ............................................................................................... iii Table of Contents .................................................................................................. iv List of Figures ........................................................................................................ vii List of Tables .......................................................................................................... ix Chapter 1 – General introduction ........................................................................... 1 1.1 Contribution of methane emissions to climate change .......................... 1 1.2 Livestock production as a source of methane emissions ....................... 5 1.3 Methane mitigation strategies ............................................................... 7 1.3.1 Legislation ........................................................................................... 8 1.3.2 Selective breeding .............................................................................. 9 1.3.3 Nutritional strategies ........................................................................ 11 1.3.4 Feed additives ................................................................................... 13 1.3.5 Antibiotics ......................................................................................... 15 1.3.6 Vaccines ............................................................................................ 17 1.4 Algal bioactive compounds ................................................................... 19 1.5 The use of Asparagopsis spp. ............................................................... 20 1.6 Other potential species of seaweed ..................................................... 21 1.7 Thesis aims ........................................................................................... 22 Chapter 2 – Species biochemistry; Spatial differences in secondary metabolites of selected seaweed species in New Zealand ............................................... 23 2.1 Introduction .......................................................................................... 23 2.2 Aims and objectives .............................................................................. 27 2.3 Materials and methods ......................................................................... 27 2.3.1 Sample collection .............................................................................. 27 2.3.2 Quantification of bromoform ........................................................... 29 2.3.3 Compositional analysis ..................................................................... 30 iv 2.3.4 Quantification of polyphenols .......................................................... 31 2.3.5 Statistical analysis ............................................................................. 32 2.4 Results .................................................................................................. 33 2.4.1 Bromoform quantification ................................................................ 33 2.4.2 Compositional analysis ..................................................................... 34 2.4.3 Polyphenol quantification ................................................................. 36 2.5 Discussion