Dissertation (PhD(Agric))--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Considerable efforts have been made to improve strains of the wine yeast
Saccharomyces cerevisiae through the use of genetic engineering. Although the
process is well defined, globally there is much resistance towards the use of
genetically modified organisms (GMOs), primarily because little is known about their
environmental fate and their potential effect on naturally occurring organisms. The
public concern is mainly focused on the uncertainty associated with the impact of the
deliberate or accidental release of a GMO into the environment. As a consequence,
thére is an urgent need to assess the potential risks involved with the use of this new
technology. For the eventual global acceptance of any GMO, it is imperative that the
consumer must be convinced that it is ultimately safe for human consumption and the
environment. In order to achieve this, certain risk assessment procedures must be
performed on each and every GMO that is planned to be released into the
environment. Although some of the genetically modified (GM) yeasts that have been
developed comply with the strict legislation of most countries and have been cleared
by regulatory authorities for commercial use, GM yeasts have not, as yet, been used
for the commercial production of GM bread, beer or wine. Nevertheless, the use of
GM yeasts in the market appears imminent and there is an urgent need to assess
and address the perceived health and environmental risks associated with GM foods.
The overall objective of this research was to evaluate key environmental issues
concerning the use of GM yeasts. The focus was on comparing the behaviour of
specific parental and GM yeast strains in model systems in order to determine
whether the GM strains may have any selective advantage, which could lead to their
spreading. Specifically, it involved monitoring of the growth behaviour of selected
GM yeasts within a vineyard microbial community and in fermentations, as well as
the interaction of these yeasts with sand and glass surfaces in an aqueous
environment. The GM yeasts under investigation were recombinant strains of a
well-known, industrial strain of S. cerevisiae VIN13 expressing an a-amylase
(designated GMY1); an endo-p-1,4-glucanase and endo-p-xylanase (designated
GMY2); and a pectate lyase and polygalacturonase (designated GMY3).
The GM yeasts were mist-inoculated onto individually-contained blocks
consisting of one-year old grapevines in a secluded glasshouse environment.
Specifically, the numbers and dynamics of GM yeast survival, as well as the effect of
an introduced GM yeast on the yeast community dynamics and numbers, were
investigated. Overall, it was found that the most prevalent wild yeasts isolated from
the grapevines were Rhodo torula, Yarrowia lipolytica, Pichia and Candida spp.
VIN13 and the GM yeasts did not affect the overall ecological balance of the
microflora on the grapevines. Wild strains of S. cerevisiae were seldom isolated from
the grapevines. With a few exceptions, the overall detection of GM yeasts was
numerically limited. Co-inoculation of (VIN13+GMY1) and (GMY1+GMY2) revealed detection approximately in the same ratio at which they were inoculated, with small
differences in the order of GMY2>GMY1 >GMY3. GM yeasts were rarely isolated
from bark and soil samples. Spontaneous fermentation of the grapes harvested from
the different treated blocks indicated that the GM yeasts survived on the berries, that
the natural fermenting ability of VIN13 was conserved in the recombinant strains, and
that the GM yeasts did not have any competitive advantage.
The soil environment forms an important part of the biosphere and the transport
and attenuation of a GM yeast in this matrix will to a large extent affect their ultimate
fate in the environment. In soil, microorganisms either occur as suspended cells in
pore water or as biofilms on soil surfaces. Although less extensive than a typical soil
yeast, Cryptococcus, epifluorescent staining of biofilms confirmed that VIN13 and
GMY1 were capable of existing in a biofilm mode on sand granules and glass. Data
on effluent numbers detected in flow cells indicated that GMY1 had no advantage
due to the genetic modification and had the same reproductive success as VIN13.
These strains either had no difference in biofilm density or GMY1 was less dense
than VIN13. When co-inoculated, GMY1 had no negative influence on the mobility of
Cryptococcus through a sand column, as well as the ability of Cryptococcus to form
biofilms. Furthermore, it was found that GMY1 did not incorporate well into a stable
biofilm community on glass, but did not disrupt the biofilm community either.
This is the first report of the assessment of the fate of GM strains of VIN13 that
are suitable for the wine and baking industry. The investigation of the GM yeasts in
this study under different scenarios is a good start to an extensive and necessary risk
assessment procedure for the possible use of these GM yeasts in the industry. This
study could lead to the provision of much-needed scientific and technical information
to both industry and regulating bodies. The outcome of this research is also intended
to serve as a basis for information sharing with public interest groups. / AFRIKAANSE OPSOMMING: Aansienlike pogings is reeds aangewend om rasse van die wyngis, Saccharomyces
cerevisiae, deur middel van genetiese manipulering te verbeter. Alhoewel hierdie
proses goed gedefinieerd is, is daar wêreldwyd heelwat teenkanting teen die gebruik
van geneties gemanipuleerde organismes (GMO's). Dit is hoofsaaklik te wyte
daaraan dat so min bekend is oor hul lot in die omgewing en hul potensiële effek op
die organismes wat natuurlik voorkom. Die publiek is veral besorg oor die
onsekerheid verbonde aan die bestemde of toevallige vrylating van 'n GMO in die
omgewing. Gevolglik is daar 'n dringende behoefte om die potensiële risiko's in die
gebruik van hierdie nuwe tegnologie te bepaal. Dit is van uiterste belang dat die
verbruiker oortuig sal word van die veiligheid vir menslike gebruik en die omgewing
voordat enige GMO uiteindelik wêreldwyd aanvaarbaar sal word. Om dit te kan
bereik sal sekere risiko-bepalende prosedures toegepas moet word op ieder en elke
GMO wat beplan word om vry gelaat te word in die omgewing. Alhoewel sommige
van die geneties gemanipuleerde (GM) giste aan die streng wetgewing van die
meeste lande voldoen en deur die owerhede vir kommersiële gebruik goedgekeur is,
word GM-giste nog steeds nie vir die produksie van GM brood, bier of wyn gebruik
nie. Ten spyte hiervan, blyk die gebruik van GM-giste onafwendbaar te wees en is
daar dus 'n dringende behoefte om die voorspelde gesondheids- en
omgewingsrisiko's wat met GM voedsel geassosieer word, aan te spreek.
Die oorhoofse doel van hierdie navorsing was om belangrike
omgewingskwessies aangaande die gebruik van GM-giste te evalueer. Die fokus
was op die vergelyking van die gedrag van spesifieke oorspronklike gisrasse en
GM-gisrasse in modelsisteme sodat daar bepaal kon word of die GM-gisrasse enige
selektiewe voordele het wat moontlik tot hulonbeheerde verspreiding in die natuur
sou kon lei. Dit het spesifiek die monitering van die groei van geselekteerde GMgiste
binne 'n mikrobiese gemeenskap op wingerd en in fermentasies behels, asook
die interaksie van hierdie giste met grond en glas oppervlaktes in 'n wateromgewing.
Die GM-giste wat in hierdie studie gebruik is, was rekombinante rasse van 'n
bekende industriële ras van S. cerevisiae, VIN13, wat geneties gemodifiseerd was
om 'n a-amylase (aangedui as GMG1); 'n endo-p-1,4-glukanase en 'n
endo-B-xilanase (aangedui as GMG2); en 'n pektaatliase en 'n poligalaktorinase
(aangedui as GMG3) uit te druk. Die GM-giste is op afsonderlike blokke van
eenjaaroue wingerdplante binne-in 'n beskutte kweekhuis gesproei-inokuleer. Daar
was spesifiek na die selgetalle en dinamika van die oorlewende GM-giste gelet,
asook wat die invloed was van die inokulasie van 'n GM gis op die selgetalle van die
natuurlike gisgemeenskap. Daar is bevind dat die wildegiste Rhodotorula, Yarrowia
Iipolytica, Pichia en Candida spp die gereeldste van die wingerd geïsoleer is. VIN13
en die GM-giste het nie die ekologiese balans van die natuurlike mikrobiese
populasie op die wingerd versteur nie. Wilde rasse van S. cerevisiae is selde
geïsoleer vanaf die wingerd. In die meeste gevalle is daar bevind dat wanneer GM-giste opgespoor is, hulle in lae selgetalle voorgekom het. Waar giste saam
geïnokuleer was, was die opsporing van (VIN 13+GMY1) en (GMY1 +GMY2)
ongeveer in dieselfde verhouding as waarin hul geïnokuleer was, terwyl klein
verskille in die orde van GMY2>GMY1 >GMY3 opgemerk is. GM-giste is selde vanaf
bas- en grond-monsters geïsoleer. Spontane fermentasies van druiwe wat geoes
vanaf die verskillende behandelde blokke is, het daarop gedui dat die GM-giste wel
op die druiwe oorleef, dat die natuurlike vermoë van VIN13 om te kan fermenteer in
die gemodifiseerde gisrasse behoue gebly het en dat die GM-giste geensins deur die
genetiese modifikasies bevoordeel was nie.
Grond is 'n belangrike deel van die biosfeer en die verspreiding en aanhegting
van 'n GM-gis in hierdie matriks sal sy algehele lot in die omgewing tot 'n groot mate
beïnvloed. In die grond kom mikroorganismes as gesuspendeerde selle in
poriewater of as biofilms op die oppervlaktes van grond voor. Alhoewel
biofilmvorming van VIN13 en GMG1 swakker was as in die geval van 'n tipiese
grondgis, Cryptococcus, het epifluoresserende kleuring van hierdie S. cerevisiaegiste
bevestig dat VIN13 en GMG1 in staat was om as biofilms op sandkorrels en
glas te oorleef. Gebaseer op seltellings in vloeiseluitlaat, kon daar afgelei word dat
GMG1 geen selektiewe voordeel geniet het as gevolg van die genetiese modifikasie
nie en dat die gis net so reproduktief was as VIN13. Hierdie gisrasse het geen
verskil in biofilmdigtheid getoon nie of die biofilmvorming van GMG1 was minder dig
as die van VIN13. Wanneer GMG1 saam met Cryptococcus geïnokuleer was, het
GMG1 geen negatiewe invloed op die beweeglikheid van Cryptococcus deur 'n
sandkolom gehad nie en die vermoë van Cryptococcus om biofilms te vorm is ook nie
beïnvloed nie. Daar is verder ook bevind dat GMG1 nie goed binne-in 'n
gestabiliseerde biofilmgemeenskap op glas geïnkorporeer het nie, maar dat die gis
ook nie die biofilmgemeenskap versteur het nie.
Hierdie studie verteenwoordig die eerste ondersoek ooit oor die lot, oorlewing en
groeigedrag van GM-wyngiste in biologies-afgesonderde wingerd-, fermentasie-,
modelgrond- en modelwater-ekosisteme. Die bestudering van hierdie GM-giste
onder verskillende omgewingstoestande in afgeslote ekosisteme lê 'n stewige basis
vir verdere ondersoeke en die ontwikkeling van omvattende en noodsaaklike
risikobepalingsprosedures betreffende die moontlike toekomstige gebruik van
GM-giste in die industrie. Hierdie studie baan die weg tot die verkryging van
noodsaaklike wetenskaplike en tegniese inligting oor die veiligheidsaspekte rakende
GM-wyngiste en dit kan van groot waarde vir die industrie, owerhede en
verbruikerspubliek wees.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50489 |
| Date | 03 1900 |
| Creators | Schoeman, Heidi |
| Contributors | Wolfaardt, G. M., Van Rensburg, P., Pretorius, I. S., Grossmann, M. K., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 142 p. : ill. |
| Rights | Stellenbosch University |
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