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Dr. Brid Quilty

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Dr. Brid Quilty Identification and characterisation o f Y arrow ia lip o lytica R P2 grow ing o n t a l l o w A thesis submitted to Dublin City University in fulfilment of the requirements for the award of the degree of Doctor of Philosophy by Sharon Davin, B.Sc. School of Biotechnology, Dublin City University, Dublin 9, Ireland Research Supervisor: Dr. Brid Quilty J u l y 2 0 0 3 I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D., is entirely my own work and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: I.D. No.: ^ < Q o c\Ol Date: 'No one will ever know or understand the fun there was; for there was fun and there was laughing- foolish, silly fun and foolish, silly laughing; but what it was all about you can 7 remember, can you? Just the memory o f it- that's all you have now- just the memory; and even now, even so soon, it is being distilled o f all its coarseness; and what's left is going to be p re c io u s g o ld ... ’ [Philadelphia Here 1 Come, Brian Fricl] ACKNOWLEDEMENTS I would like to express my deepest thanks to my supervisor, Dr. Brid Quilty, for her interest, understanding, help and encouragement throughout the duration of this project. Thanks to all the staff in the department for your help and advice throughout the years. Thanks to all the members of the faculty staff and to the technical department, especially for all the juicy gossip! and for looking the other way when I had to ‘relocate’ a piece of equipment. Big thanks to Daithi for all the CAD drawings and last, but not least, to Ben for all his help with the fermenters and the lend of a screwdriver (without asking too many questions!). I would like to thank Dublin Products Ltd., Dunlavin, Co. Wicklow for their kind donation of tallow. My sincerest gratitude to Michael, Irene, Therese, Bernie and all the staff of the EPA Regional Water Lab. Kilkenny for their kind use of the TN analyser and support. Many thanks to Kilkenny County Council and Enterprise Ireland for their support during my studies. To all my fellow colleagues in the lab, the oldies and the new gang, Al, John, Henry, Niamh, Mary and Fakhruddin, thanks for all the memories (anyone for cricket?) and pretending to be concerned when I got in a strop about cleaning the place! I wish all the new gang every success (even if you do make me feel like I’m 102!). To the original spicy girls, Auds, Viv, Deirdre and Mir, so many nights, too many funny stories! Big thanks to all the coffee room girlies, especially Jane F., for the million and one reasons to eat chocolate bikkies! Not forgetting the new kids(ish) Connor and Isobelle- hope the legacy of the coffee room laughter lives on in you. To all the girls at No. 32- Susan, P, Jane and Gillian, not forgetting Brian- thanks for all the little things, all the hugs, all the laughter and the important things in life like a good bottle of vino and SITC nights! A special word of thanks to Jane- thank for all the million things that you’ve helped me with and for all the laughter and memories, especially for Friday pints where the problems of the world were solved over a pint of plain! Big thanks to the rest of the dwindling original friday night gang- Al, Therese, Henry and Kieran. To my Mam and Dad, what can I say? Your unwavering support and belief in me throughout my time in DCU has been my shelter in the storms. To Ned, Irene, Catherine and Dave, thanks heaps for the freebies and SOS packs and your constant help, advice and love. Not forgetting my three monsters, to Philip, Rachel and Claire, thanks for not letting me take myself too seriously and remembering my inner child! Table of Contents Abstract List of Tables List of Figures 1.0 Introduction 1.1 Yeast 1.1.1 Yeast classification 1.1.2 Yeast identification 1.1.3 Medical, industrial and environmental importance 1.2 Yarrowia lipolytica 1.2.1 Nomenclature 1.2.2 Dimorphism 1.2.3 Biotechnological importance 1.3 Lipids 1.4 Waste lipids 1.5 Lipid removal from waste streams 1.5.1 Physical and chemical methods of lipid removal 1.5.2 Biological removal of lipids 1.5.2.1 Anaerobic biological removal 1.5.2.2 Aerobic biological removal 1.6 Biodégradation of lipid 1.6.1 Lipase catalysed hydrolysis 1.6.2 Lipid émulsification 1.6.3 Fatty acid uptake 1.6.4 p- oxidation 1.6.5 Lipid accumulation 38 1.7 The role of environmental conditions 40 1.7.1 Temperature, pH and dissolved oxygen 40 1.7.2 The role of nutrients 41 1.8 Design of lipid biodégradation systems 44 1.9 By-products of lipid biodégradation 47 1.9.1 Bioconversion of fats 47 1.9.2 Single cell oil 47 1.9.3 Single cell protein 48 1.9.4 Biosurfactant production 50 1.10 Aims of the project 51 2.0 Materials and Methods 2.1 Materials 52 2.1.1 Yeast isolates 52 2.1.2 Source of chcmicals and materials 52 2.1.3 Media 53 - Minimal medium 53 - Nutrient broth 53 - Olive oil agar 53 - Olive oil broth 54 2.1.4 Buffers 54 - Citrate phosphate buffer 54 - Potassium phosphate buffer 54 - Sodium phosphate buffer, N a2HP 0 4 -NaH 2PQ 4 55 - Sodium phosphate buffer, NaOH-NaFbPC^ 55 - Sodium phosphate buffer (inoculum preparation) 55 2.1.5 Stains 55 2.2 Methods 56 2.2.1 Identification of yeast isolates 56 2.2.1.1 Tests used in yeast identification - Ascospore test 56 - Carbon assimilation 56 - Cell morphology 57 - Colony morphology 57 - Cycloheximide resistance 58 - Diazonium Blue B (DBB) colour test 58 - Fermentation of carbohydrates 59 - Growth at 37°C 60 - Growth in 10% sodium chloridc-5% glucose medium 60 - Growth in 50% glucose-yeast extract medium 61 - Liquid morphology 61 - Nitrate assimilation 61 - Production of ammonia from urea (urease test) 62 2.2.1.2 Identification scheme for yeast isolates 63 2.2.2 Yeast maintenance 63 2.2.3 Inoculum preparation 64 2.2.4 Measurement of growth 64 2.2.4.1 Cell number and cell viability 64 2.2.4.2 Cell dry weight 64 2.2.5 Measurement of mycelium ratio 65 2.2.6 Growth studies 65 2.2.6.1 Shake flask studies 6 6 2.2.6 .2 2 L bench-top fermenter studies 6 6 - Fermenter configuration 6 6 - Fermenter set-up 67 2.2.6 .3 10 L pilot-scale fermenter studies 6 8 - Fermenter configuration 6 8 - Fermenter set-up 69 2.2.6.4 Scale-up of agitation from 2 L to 10 L fermenter 70 2.2.7 Measurement of glucose 71 2.2.8 Measurement of percentage free fatty acids in tallow 72 2.2.9 Measurement of fat removal 73 2.2.10 Intracellular analysis 73 2.2.10.1 Preparation of freeze-dried cells 73 2.2.10.2 Measurement of intracellular fat 73 2.2.10.3 Cell disruption with glass beads 74 2.2.10.4 Measurement of intracellular protein 74 2.2.10.5 Measurement of cellular total nitrogen 75 2.2.11 Biosurfactant measurement 75 2.2.11.1 Surface tension measurement 75 2.2.11.2 Measurement of relative biosurfactant concentration 76 2.2.11.3 Emulsification measurement 76 2.2.11.4 Validation of biosurfactant measurement 76 2.2.12 Metal analysis 77 2.2.12 Sludge Volum e Index (SVI) 77 2.2.14 Data analysis 78 - Growth rate 78 - Specific rate of substrate removal 79 - Yield coefficient 79 3.0 Results 3.1 Identification and selection of yeasl isolates capable of growth on 80 tallow as the sole carbon source 3.1.1 Identi fication of the isolates 80 3.1.1.1 Macroscopic appearance of the isolates 80 3.1.1.2 Microscopic appearance of the isolates 84 3.1.1.3 Physiological characteristics of the isolates 88 3.1.1.4 Isolate identification schemes 92 3.1.2 Selection of the isolates 104 3.2 Characterisation and optimisation of the environmental conditions 106 for the degradation of tallow (20 g I/1) by Yarrowia lipolytica RP2 in shake flask culture 3.2.1 Characteristics of the growth of Y. lipolytica RP2 on tallow (20 g 106 L'1) in minimal medium 3.2.2 Optimisation of growth temperature 112 3.2.3 Effect of chemical surfactant addition on tallow émulsification 114 and biodégradation 3.2.4 Role of pH control 120 3.2.4.1 Optimisation of medium pH between pH 3.0 - 8.0 with 120 0.1 M citrate phosphate buffer 3.2.4.2 Influence of various buffers (0.1 M) at pH 7.0 on tallow 124 biodégradation 3.2.4.3 Optimisation of the concentration of potassium phosphate 125 buffer, pH 7.0 and growth temperature 3.2.4.4 Characteristics of the growth of Y. lipolytica RP2 on tallow 129 (20 g L'1) at optimised growth temperature, 25°C and pH, 7.0 3.2.5 Effect of agitation on growth and fat removal 133 3.2.6 Influence of the method of inoculum preparation of 7.
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