Bioprospecting for Extremophile Oleaginous Yeasts

Abstract

Palm Oil is the highest produced edible oil globally, with over 66 million tonnes produced annually. It has been estimated that up to 50% of all products sold in the supermarket contain palm oil in some form. point and texture due to Palm oil has attractive properties such as a high melting a balanced ratio of unsaturated and saturated fatty acids. It contains approximately 40% oleic acid (monounsaturated fatty acid (polyunsaturated fatty acid), 45% palm itic acid and 5% stea ), 10% linoleic a cid ric acid (saturated fatty acid), that results in an edible oil that is cosmetics products . suitable for use in a variety of food , detergent and In addition, palm oil is the least expensive oil produced due to its high productivity and extensive production. Due to the high demand for the product, vast amounts of rainforest have been cleared to make way reducing biodiversity and releasing huge levels of carbon dioxide for more plantations, into the atmosphere. There is a clear need for an alternative lipid that can match palm oils properties but can be produced sustainably. Recent work suggests that some yeasts are capable of producing a similar oil to palm oil and can be grown on waste resources. In this thesis a novel bioprospecting protocol was developed to isolate yeasts that can survive the harsh conditions necessary for industrial biotechnology. In this way a vineyard and the local area was sampled for yeasts which were then cultured under extremes of pH, m ultiple sugars and inhibitors caused from the breakdown of lignocellulose. The wild yeast were cultured in four stages: minimal medium with Lysine; minimal medium with inhibitors; minimal medium with xylose as sole carbon only arabinose and cellobiose as carbon-- source; and lastly minimal medium with sources. Only strains that survived each stage were taken forward to the next, to isolate species that were truly suited to these conditions. Out of the estimated 1000s of strains screened this resulted in 12 st yeast, mostly in the Metschnikowia pulcherrima rains of , group being able to cope with the conditions. The 12 strains were further lignocellulosic feedstocks analyzed by culturing them in an array of 4 different namely wheat straw, corn S tover, model sugarcane bagasse, and palm kernel cake hydrolysates. Other conditions incorporated in these analysis were a range of pH from pH 1.5 to pH 7.0; four levels of a mixture of 5 inhibitors; and two different temperatures. All of the 12 strains showed similar behaviour where inhibitor tolerance was only marked at higher pH, and at low pH the strains could not grow at all. Though all strains were able to grow on the hydrolysate models, even those with little glucose and/or xylose content. The lipid profile of t he strains was also assessed and proved to be similar to most terrestrial crops, with suitable lipid profiles for a rapeseed oil, and in some cases palm oil substitute. Lastly, to further ambiguity in the evaluate the accurate identification of the strains as there are so Metschnikowia pulcherrima me group , we applied an approach only widely used for Pathogenic Bacteria/Yeast identification, Multilocus Sequence Typing (MLST). Using 25 strains (7 of this collection), 6 type species and some isolates from the original culture collection in Bath. Sequences of 6 genes was analysed using the Bayesian statistical method. The result showed grouping of different for each gene. M . Corniflorae being the M. pulcherrima into 34 groups 9 outgroup. In all 3 genes success sequenced: M. Fruticola fully ; R6; Mp DAH 3; and ICS48 were consistently shown to be clonal. The work presented here demonstrates a new method for bioprospecting strains capable of isolating strains for industrial biotechnology, and for characterisation of yeast in the Metschnikowia genus. Some of the yeasts identified were oleaginous, and could potentially be used as a novel source of palm oil substitute.

Date of Award	1 Apr 2017
Original language	English
Awarding Institution	University of Bath
Sponsors	MARA, Government of Malaysia
Supervisor	Chris Chuck (Supervisor) & Roderick Scott (Supervisor)