Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Reliable energy resources could be considered as one of the cornerstones of the prosperity of the
human race. The growing human population is constantly exerting more pressure on the world’s
natural resources, which include natural fossil fuels that are non‐renewable. There are concerns
regarding the use of fossil fuels due to its growing scarcity and its negative impact on the
environment. There is thus a growing need in the world for energy sources that are renewable, more
or less carbon neutral and therefore with a minimum environmental impact. Renewable energy is
currently being harnessed from the wind, water and sun, but to a limited extent. These forms of
natural resources are very attractive for the production of renewable energy, but these technologies
are difficult to apply in the current transportation sector. Biofuels provide an alternative to the
current use of liquid fossil fuels and it could be able to sustain the current fleet of automobiles
worldwide in the intermediate to long term with minimal adjustment to the engines of these
vehicles.
Extensive research has been done on the production processes for biofuels. Previous processes
included the use of high temperatures and acids that further increased the total production cost and
thus making biofuels less attractive as an alternative energy source. Recent research has suggested a
wide range of organic materials as substrate for the production of biofuels, which include lignin,
hemi‐cellulose, cellulose and starch. Processes based on hemi‐cellulose, cellulose and lignin as
substrate are still in its early research stages and commercial application of these processes will only
occur over the medium‐ to long‐term.
Starch is a very good alternative source for the production of biofuels, but there is a need for a
microbial system for the conversion of starch to bio‐ethanol in a single step, referred to as
Consolidated Bioprocessing (CBP). This would reduce the overall production cost of bio‐ethanol and
thus making starch‐based substrates more attractive as an alternative energy source. The cost saving
will be mainly due to the elimination of the pre‐treatment of raw starch at high temperatures and
the addition of enzymes for the liquefaction and saccharification of starch to simple sugars.
However, as there is no currently no known microbial organism known that can produce the
required enzymes (i.e. amylases) as well as ferment the resulting sugars to ethanol, heterologous
expression of these enzymes in a host strain able to ferment sugars could provide the best
alternative system. In the first part of this study, 36 fungal strains known for the production of amylases were screened
and compared for the highest extracellular enzyme activity on raw corn starch. The best two
candidates, i.e. Aspergillus tubingensis (T8.4) and Mucor cincinelloides (1180), were then further
evaluated to determine which organism has the highest efficiency when combined with a
Saccharomyces cerevisiae laboratory strain. In fermentation experiments, A. tubingensis (T8.4) in
combination with S. cerevisiae Y102 yeast strain resulted in the highest yield of ethanol.
Literature on A. tubingensis is limited compared with other Aspergillii and it was previously accepted
that A. tubingensis has the highest homology with Aspergillus niger. However, other reports –
including the present study ‐ found that A. tubingensis is closer related to other Aspergillus spp. with
regard to its amylolytic enzymes. The α‐amylase gene of A. tubingenis has a homology of 99.00%
with that of Aspergillus kawachii whereas the glucoamylase gene has a homology of 99.26% with
that of Aspergillus shirousami.
In the second part of this study, two recombinant S. cerevisiae strains were constructed to express
the wild type A. tubingensis α‐amylase (Atamy) and glucoamylase (Atglu), respectively. The
combination of the two recombinant yeast strains was able to completely hydrolyse and also utilize
raw corn starch for the production of bio‐ethanol, with a yield of 11.04 g/l of ethanol, which
translates to 98% of the theoretical yield from starch with a 52% conversion of the total raw starch.
This rate of conversion is lower than other reports which indicated up to 82% and 96% of the
theoretical yield of ethanol from raw and soluble starch, respectively, by α‐ and glucoamylase.
Furthermore, the combined expressed of the two genes was much more effective than when only
one of the two genes were expressed, with a yield of 0.32 g/l ethanol for only Atamy and 2.52 g/l
ethanol for Atglu. This proved that the combination of the A. tubingensis genes were best suited for
the production of biofuels from raw starch. This also proved that the concept of constructing an
amylolytic yeast strain capable of raw starch hydrolysis and fermentation was indeed feasible. / AFRIKAANSE OPSOMMING: Betroubare energiebronne kan as een van die boublokke vir die vooruitgang van die mensdom
beskou word. Die groeiende menslike populasie is gedurig besig om meer druk op die wêreld se
natuurlike hulpbronne te plaas, insluitende nie‐hernubare fossielbrandstowwe. Daar is kommer
rakende die gebruik van fossielbrandstowwe weens ‘n afname in die beskikbaarheid en die
negatiewe impak wat dit op die omgewing het. Daar is dus ‘n groeiende behoefte in die wêreld vir ‘n
hernubare, min of meer koolstof‐neutrale energiebron wat ‘n minimale omgewingsimpak sal hê.
Hernubare energie word tans tot ‘n beperkte mate uit wind, water en die son verkry. Hierdie vorms
van natuurlike energie hulpbronne is baie aanloklik vir die vervaardiging van hernubare energie,
maar hierdie tegnologië is moeilik toepasbaar in die huidige vervoersektor. Biobrandstowwe
voorsien ‘n alternatief vir die huidige gebruik van fossielbrandstowwe en kan moontlik die huidige
voertuigvloot wêreldwyd oor die medium‐ tot langtermyn onderhou met minimale enjinaanpassings
van hierdie voertuie.
Deeglike navorsing is alreeds op die vervaardigingsprosesse vir biobrandstowwe gedoen. Vorige
prosesse het die gebruik van hoë temperature en sure ingesluit wat produksiekostes verder verhoog
en gevolglik die gebruik van biobrandstowwe as ‘n alternatiewe energiebron minder aantreklik
gemaak het. Onlangse navorsing het die gebruik van organiese materiaal as substraat vir die
produksie van biobrandstowwe voorgestel, wat lignien, hemi‐sellulose, sellulose en stysel insluit.
Prosesse met die gebruik van hemi‐sellulose, sellulose en lignien as substraat is nog in die beginfase
van ontwikkeling en kommersialisering van hierdie prosesse sal eers oor die medium‐ tot langtermyn
plaasvind.
Stysel is ‘n baie goeie alternatiewe bron vir die produksie van biobrandstowwe, maar ‘n mikrobiese
sisteem word vir die omskakeling van stysel in bio‐etanol in ‘n enkele stap benodig, bekend as
gekonsolideerde bioprosessering (GBP). Dit sal die algemene produksiekoste van bio‐etanol verlaag
en dus styselsubstrate as ‘n alternatiewe energiebron meer aantreklik maak. Die kostebesparing sal
hoofsaaklik realiseer omdat die vooraf‐behandeling van rou stysel byhoë temperature en die
toevoeging van ensieme vir die vervloeiing en versuikering van stysel tot eenvoudige suikers,
uitgeskakel word. Aangesien daar tans geen bekende mikrobe organisme is wat die nodige ensieme
(nl. amilases) kan produseer en ook die suikers wat daardeur vrygestel is, na etanol kan fermenteer
nie, kan die heteroloë uitdrukking van hierdie ensieme in ‘n gasheer‐ras wat die suikers kan
fermenteer, moontlik die beste alternatief verskaf. In die eerste deel van hierdie studie is 36 fungi rasse wat bekend is vir hul amilase produksie geevalueer
en met mekaar vergelyk vir die hoogste ekstrasellulêre ensiemaktiwiteit op rou
mieliestysel. Die beste twee kandidate, naamlik Aspergillus tubingensis en Mucor cincinelloides, is
verder ge‐evalueer om te bepaal watter organisme het die hoogste effektiwiteit in kombinasie met
‘n Saccharomyces cerevisiae laboratorium gisras. In fermentasie‐eksperimente het A. tubingensis in
kombinasie met S. cerevisiae Y102 gisras die hoogste etanol opbrengs gelewer.
Inligting rakende A. tubingensis is beperk relatief tot ander Aspergillii en dit was voorheen aanvaar
dat A. tubingensis die hoogste homologie met Aspergillus niger het. Ander verslae – insuitende die
huidige studie ‐ het egter gevind dat A. tubingensis nader verwant aan ander Aspergillus spp. in
terme van amilolitiese ensieme is. Die α‐amilase geen van A. tubingensis het ‘n homologie van
99.00% met dié van Aspergillus kawachii en die glukoamilase ‘n homologie van 99.26% met dié van
Aspergillus shirousami getoon.
In die tweede gedeelte van hierdie studie is twee rekombinante S. cerevisae gisrasse gekonstrueer
om onderskeidelik die α‐amilase (Atamy) en glukoamilase (Atglu) van A. tubingensis uit te druk. Die
kombinasie van die twee rekombinante gisrasse was in staat om die volledige hidrolise en benutting
van rou mieliestysel vir die produksie van bio‐etanol deur te voer met ‘n opbrengs van 11.04 g/l wat
gelykstaande is aan 98% van die teoretiese opbrengs vanaf stysel met ‘n omskakeling van 52% van
die totale rou stysel. Hierdie omskakelingskoers is laer as ander studies wat onderskeidelik 82% en
96% van die teoretiese opbrengs van rou en oplosbare stysel vir α‐ en glukoamilase getoon het.
Verder was die kombinasie van die twee gene meer effektief as wanneer slegs een gebruik is, met ‘n
0.32 g/l opbrengs vir Atamy en 2.52g/l vir Atglu. Hierdie het bewys dat die kombinasie van die
A. tubingensis meergeskik vir die produksie van bio‐etanol was. Dit het ook bewys dat die beginsel
van ‘n amilolitiese gisras wat in staat is om rou stysel te hidroliseer en te fermenteer, inderdaad
moontlik is.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6880 |
| Date | 03 1900 |
| Creators | Viktor, Marko Johann |
| Contributors | Bloom, Marinda, Van Zyl, Willem Herber, 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 | v, 84 p. : ill. |
| Rights | Stellenbosch University |
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