Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A
sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high
levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used
chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely
Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and
acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a
viable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate,
inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered by
chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2
tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2
accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate
if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1.
In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using
5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm
whether these strains belonged to the Dekkera or Brettanomyces genus. A method to
accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was
tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2
and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest
tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew
well with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B.
bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed the
same trends as previously found in literature whereas B. bruxellensis strains showed similar
trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two
different media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid,
acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularly
monitored over the time course of each experiment. SO2 had the greatest impact on
B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as
increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent
restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde
was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32
of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2
levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism
to adapt to ensure survival.
In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and
metabolism and that SO2 tolerance is highly strain dependent and a far more complicated
characteristic than currently understood. / AFRIKAANSE OPSOMMING: Die gis Brettanomyces is in 1904 uit bier geïsoleer en daarna met wyn geassosieer. 'n
sporulerende vorm, Dekkera, is later ontdek. Brettanomyces bruxellensis produseer hoë vlakke
van vlugtige fenol afgeure in wyn. Swaweldioksied (SO2) is die mees gebruikte chemiese
preserveermiddel in wyn. Giste het verskeie meganismes om SO2 te hanteer, naamlik Ssu1p, 'n
membraan-gebonde SO2 transporter, sulfietvermindering, sulfiet-oksidasie en
asetaldehiedproduksie. In ongunstige omgewingstoestande kan sekere giste 'n lewensvatbare,
maar nie-kultiveerbare (LMNK)-toestand aanneem wat gekenmerk word deur verlaagde
metaboliese tempo, onvermoë om voort te plant op soliede media en 'n vermindering van die
selgrootte. LMNK kan veroorsaak word deur chemiese stres, soos hoë SO2-vlak. Die doelwitte
van hierdie studie was om die SO2 -bestandheid van B. bruxellensis en Saccharomyces
cerevisiae te ondersoek, hul spoed van SO2 -opneming/akkumulasie en -uitskeiding te
kwantifiseer, die invloed van SO2 op energiemetabolisme te bepaal en te ondersoek of B.
bruxellensis oor ‘n soortgelyke geen as die S. cerevisiae SSU1 beskik.
In hierdie studie is die identiteit van 'n aantal Brettanomyces/Dekkera-stamme bevestig deur
5.8S rDNA-ITS RFLP-analise en DNA-opeenvolging te gebruik. Sporulasietoetse is gebruik om
te bevestig of hierdie stamme aan die genus Dekkera of Brettanomyces behoort. 'n Metode om
SO2 onder laboratoriumtoestande akkuraat te kwantifiseer, is geoptimiseer. Molekulêre SO2-
bestandheid is getoets deur vars giskulture op media met SO2 en/of etanol te groei.
Bestandheid teen SO2 en/of etanol het stam-afhanklike resultate getoon, S. cerevisiae wat die
hoogste toleransievlakke getoon het, terwyl B. bruxellensis SO2 en etanol swak tolereer, maar
sekere stamme het goed gegroei met slegs SO2. Die SO2-akkumulasie en -uitskeidingtempo
van 3 S. cerevisiae-rasse en 3 B. bruxellensis-stamme is bepaal. Daar is gevind dat die S.
cerevisiae-rasse dieselfde tendens soos voorheen in die literatuur beskryf, gevolg het, terwyl B.
bruxellensis-stamme soortgelyke tendense getoon het,maar hoogs veranderlike stamafhanklike
resultate vertoon. B. bruxellensis CB63 en S. cerevisiae VIN13 is ondersoek vir hul reaksie tot SO2 in twee
verskillende media, TA en SWM, oor 'n tydperk van 48-uur en 32-dae onderskeidelik. Asynsuur,
asetaldehied, D-glukose, D-fruktose (slegs in SWM) en etanol (slegs in TA) is gereeld
gemoniteer oor die verloop van elke eksperiment. SO2 het die grootste impak op B. bruxellensis
met ‘n verlaagde tempo van glukoseverbruik en etanolproduksie, sowel as verhoogde asynsuur.
‘n Asetaldehiedhoogtepunt is bereik kort na die SO2-byvoeging met die daaropvolgende
hervatting van suiker wat vir sekere monsters gebruik is. Dit dui daarop dat voldoende
asetaldehied geproduseer is om vry SO2 te bind om SO2-stres te verminder. Vlugtige fenole is
op dag 32 van die SWM-eksperiment gekwantifiseer. 'n Toename van 4-etiel-guajakol korreleer
met hoër molekulêre SO2-vlakke. SO2 het beide giste se energiemetabolisme negatief
beïnvloed, wat die gis dwing om sy metabolisme aan te pas om oorlewing te verseker. Oor die algemeen het SO2 'n negatiewe impak op alle aspekte van giste se groei en
metabolisme, en SO2-bestandheid is hoogs stam–afhanklik. Dit is ook 'n baie meer
ingewikkelde kenmerk as wat tans verstaan word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20104 |
| Date | 03 1900 |
| Creators | Duckitt, Edward |
| Contributors | Divol, Benoit, Du Toit, Maret, Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 89 p. : ill. |
| Rights | Stellenbosch University |
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