The role of lipids in the flocculation of Saccharomyces cerevisiae

Strauss, Catharina J

The role of lipids in the flocculation of Saccharomyces cerevisiae

dc.contributor.advisor	Kock, J. L. F.
dc.contributor.advisor	Van Wyk, P. W. J.
dc.contributor.advisor	Lodolo, E. J.
dc.contributor.author	Strauss, Catharina J
dc.date.accessioned	2015-11-23T14:03:26Z
dc.date.available	2015-11-23T14:03:26Z
dc.date.copyright	2005-11
dc.date.issued	2005-11
dc.date.submitted	2005-11
dc.description.abstract	English: Although beer production is one of the oldest biotechnologies in the world, a major constraint in brewing remains controlling flocculation. Evidence points towards a possible role of lipids, associated with the cell surfaces, as a major factor responsible for flocculation. Therefore, the aim in this study became to evaluate the contribution of lipids, especially oxylipins, in the flocculation of Saccharomyces cerevisiae UOFS Y-2330. Saccharomyces cerevisiae UOFS Y-2330 was selected as a model, since it was found to demonstrate both Flo1 and NewFlo phenotype flocculation behaviour, when cultivated in different media. In a defined medium with glucose as a sole carbon source, this strain immediately flocculated strongly and lost this ability before stationary phase was reached. In a complex medium containing glucose, this yeast strongly flocculated towards the stationary growth phase without losing this ability during this phase. This inverse pattern may be ascribed to a switch in sensitivity of the yeast to flocculate in the presence of glucose as well as pH level, which may, in turn, influence the availability of calcium ions. In both media, matured cells produced protuberances upon flocculation as observed by electron and immunofluorescence microscopy, which may be involved in cell adhesion. This was followed by further investigations into the role of lipids over the growth cycle of this yeast. Here, it was uncovered that Sacch. cerevisiae UOFS Y-2330 does not only demonstrate inverse flocculation, but is also characterised by two different lipid turnover patterns. During Flo1 phenotype flocculation, this yeast showed two neutral lipid accumulating stages (i.e. at 8 h and from 12 h). This is probably triggered by flocculation, which may be regarded as a survival mechanism where cells accumulate especially neutral lipids as reserve energy source - a similar mechanism is probably operative when cells enter stationary growth. Contrary to Flo1 behaviour, this strain in NewFlo phenotype mode demonstrates only a single lipid accumulation phase i.e. when cells enter stationary growth, which coincides with the increase in flocculation. In addition, an increase in phospholipids was experienced during active growth in both flocculation behaviours, probably as a result of active membrane production. These results prompted us to investigate the possible role of oxylipins present on the cell surfaces during the flocculation process. It was found that some strains of Sacch. cerevisiae (include strains used in fermentation processes) produce short chain (mainly 8 carbon) oxylipins and not potent inflammatory long chain (20 carbon) oxylipins such as prostaglandins. When aspirin was added to cultures of Sacch. cerevisiae UOFS Y-2330, flocculation was significantly inhibited as well as the production of 3-hydroxy (OH) 8:0 thereby linking flocculation and this oxylipin. Furthermore, no traces of 3-OH 8:0 could be detected before flocculation onset in this yeast. Next, the involvement of these oxylipins in co-flocculation was assessed. According to the lectin-theory, the yeast Schizosaccharomyces pombe lacks the specific receptors necessary to facilitate co-flocculation with Sacch. cerevisiae species. In this study we demonstrate oxylipin associated co-flocculation between Sacch. cerevisiae UOFS Y-2330 and S. pombe strains using differential cell staining, immunofluorescence and ultrastructural studies. Using a 3-OH oxylipin specific antibody coupled to a fluorescing compound, 3-OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3-OH oxylipins was confirmed using gas chromatography-mass spectrometry. Whether these 3- OH oxylipins play a role in affecting co-flocculation of Sacch. cerevisiae with S. pombe cells through possibly entropic-based hydrophobic interactions and/or hydrogen bonds still needs to be verified. Studies on the physiological, genetic as well as colloidal aspects of flocculation using this model strain may lead to important new insights in this fascinating phenomenon as well as applications in industry.	en_ZA
dc.description.abstract	Afrikaans: Alhoewel bierproduksie een van die oudste biotegnologiese prosesse ter wêreld is, bly die beheer oor flokkulasie ‘n groot probleem. Data dui daarop dat lipiede wat met die seloppervlaktes gedurende flokkulasie geassosieer word, moontlik ‘n groot bydraende faktor tot flokkulasie kan wees. Die doelstellings van hierdie studie is dus om die bydrae van lipiede, veral oksielipiene, tot die flokkulasie proses in Saccharomyces cerevisiae UOFS Y-2330 te evalueer. Saccharomyces cerevisiae UOFS Y-2330 is as model geselekteer, aangesien daar bevind is dat dit inverse flokkulasie patrone tydens kweking in verskillende media vertoon. In ‘n gedefinieerde medium met glukose as enigste koolstofbron, het hierdie stam dadelik sterk begin flokkuleer, maar het egter hierdie vermoë verloor voordat die stasionêre fase bereik is. In ‘n komplekse medium wat glukose bevat, het hierdie gis sterk begin flokkuleer aan begin van die stasionêre fase, sonder om hierdie vermoë gedurende hierdie fase te verloor. Hierdie inverse patroon kan moontlik toegeskryf word aan ‘n omskakeling in die sensitiwiteit van hierdie gis om by verskillende glukose-konsentrasies te flokkuleer asook pH-vlakke, wat gevolglik die beskikbaarheid van kalsium moontlik kan beïnvloed. In beide media, soos waargeneem deur elektron- en immunofluoresensiemikroskopie, het volwasse selle uitstulpings tydens flokkulasie getoon, wat moontlik betrokke kan wees in seladhesie. Dit is gevolg deur verdere ondersoeke na die rol van lipiede oor die groeisiklus van hierdie gis (op komplekse – en sintetiese media). Dit is ontdek dat Sacch. cerevisiae nie slegs inverse flokkulasie-patrone demonstreer nie, maar ook deur twee verskillende omskakelingspatrone van lipiede gekenmerk word. Gedurende Flo1- fenotipe flokkulasie, het hierdie gis twee neutrale akkumuleringstadiums in lipiede getoon (naamlik by 8 h en vanaf 12 h). Dit word waarskynlik deur flokkulasie ontlok, wat as ‘n oorlewingsmeganisme beskou word, waar selle – veral neutral lipidiede – as ‘n reserwe energiebron akkumuleer. ‘n Soortgelyke meganisme is waarskynlik teenwoordig wanneer selle die stasionêre groeifase binnegaan. In teenstelling met die Flo1-gedrag, demonstreer hierdie stam tydens NewFlo fenotipe gedrag slegs ‘n enkele akkumuleringsfase van lipiede, naamlik wanneer hierdie selle stasionêre groei binnegaan, wat met die toename in flokkulasie ooreenstem. Daar is ook ‘n toename in fosfolipiede ondervind gedurende aktiewe groei in beide flokkulasie fenotipes, waarskynlik as gevolg van aktiewe membraanproduksie. Hierdie resultate het ons geïnspireer om die moontlike rol van oksielipiene te ondersoek, wat op die seloppervlaktes gedurende flokkulasie teenwoordig is. Daar is bevind dat sommige stamme van Sacch. Cerevisiae (insluitend stamme wat in fermentasieprosesse gebruik word) kortkettingoksielipiene (hoofsaaklik 8 koolstof lank) en nie potente inflammatoriese langketting-oksielipiene (20 koolstof lank), soos prostaglandiene produseer nie. Nadat aspirien by kulture van Sacch. cerevisiae UOFS Y-2330 gevoeg is, is daar bevind dat flokkulasie aansienlik geïnhibeer word, asook die produksie van 3-hidroksie (OH) 8:0 - en dus word flokkulasie aan hierdie oksilipien gekoppel. Boonop kon geen bewyse van hierdie oksilipien opgespoor word net voor die begin van flokkulasie in hierdie gis nie. Vogende is die betrokkenhied van hierdie oksielipiene in ko-flokkulasie ondersoek. Na aanleiding van die lektienteorie, het die gis Schizosaccharomyces pombe, ’n gebrek aan spesifieke reseptore wat noodsaaklik is om ko-flokkulasie met Sacch. cerevisiae-spesies te fasiliteer. In hierdie studie demonsteer ons oksilpien-geassosieerde ko-flokkulasie tussen Sacch. cerevisiae UOFS Y-2330 en S. pombe -stamme deur gebruik te maak van differensiële selkleuring, immunofluoresensie en ultrastrukturele studies. Deur ‘n 3-OH oksilipien spesifieke teenliggaam gekoppel aan ‘n fluoresserende verbinding te gebruik, is bevind dat 3-OH oksielipiene op die selwande van Sacch. cerevisiae en S. pombe teenwoordig is. Dit is bevestig deur gaskromatografie – massa spektrometrie. Of hierdie 3-OH oksielipiene ‘n rol speel in ko-flokkulasie van Sacch. cerevisiae met S. pombe selle deur moontlike entropies-gebaseerde hidrofobiese interaksies en/of waterstofbindings, moet nog bevestig word. Studies op die fisiologiese, genetiese asook kolloïdale aspekte van flokkulasie op hierdie modelstam, kan moontlik tot nuwe insigte in hierdie fassinerende verskynsel lei, asook toepassings in die bedryf.	af
dc.description.sponsorship	National Research Foundation (NRF)	en_ZA
dc.identifier.uri	http://hdl.handle.net/11660/1744
dc.language.iso	en	en_ZA
dc.publisher	University of the Free State	en_ZA
dc.rights.holder	University of the Free State	en_ZA
dc.subject	Industry	en_ZA
dc.subject	Inverse flocculation	en_ZA
dc.subject	Lipids	en_US
dc.subject	Saccharomyces cerevisiae	en_ZA
dc.subject	Schizosaccharomyces pombe	en_ZA
dc.subject	3-Hydroxy oxylipins	en_ZA
dc.subject	Yeast	en_ZA
dc.subject	Fatty acids	en_ZA
dc.subject	Co-floccculation	en_ZA
dc.subject	Yeast fungi	en_ZA
dc.subject	Thesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2005	en_ZA
dc.subject	Flocculation	en_ZA
dc.title	The role of lipids in the flocculation of Saccharomyces cerevisiae	en_ZA
dc.type	Thesis	en_ZA