Thesis (MScEng)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Alkanes can be used as an inexpensive feedstock to produce more valuable alcohols. The
biotransformation of alkanes to alcohols provides an alternative to conventional chemical
procedures.
The scope of this research was to develop a process utilising a biocatalyst to catalyse the
oxidation of an alkane to its corresponding alcohol on a larger scale than had been reported on
in previous research. The research utilised a recombinant E. coli BL21(DE3) cell, containing the
CYP153A6 operon in pET 28 vector, as the biocatalyst. The CYP153A6 enzyme catalyses the
oxidation of octane to 1-octanol. The principle objective of the research was to determine the
amount of 1-octanol that can be produced by a system utilising this strain of recombinant E.
coli as a biocatalyst on a three orders of magnitude larger scale than what had previously been
reported on for this reaction system. An additional objective was to model the 1-octanol
production performance in the bioreactor.
Bioconversion batch reactions, with excess octane used as a substrate, were conducted in 30ml
McCartney bottles and in a 7.5L BioFlo 110 Modular Benchtop Fermentor (New Brunswick).
The McCartney bottles were not equipped to actively control process conditions.The bioreactor
was equipped to control process conditions such as temperature, pH and dissolved oxygen
concentration. Experiments in the bioreactor were therefore described as being performed
under controlled conditions. The procedures used to grow, maintain and harvest the biocatalyst
cells were based on those developed by the Department of Microbial, Biochemical and Food
Biotechnology at the University of the Free State. The product and substrate concentrations
were determined through gas chromatography (GC) analysis. The McCartney bottle bioconversion reactions, with a 1.33ml reaction volume, produced 1.88
mg 1-octanol per gram of dry cell weight per hour. The bioreactor under controlled conditions,
with a 2L reaction volume, produced 14.89 mg 1-octanol per gram of dry cell weight per hour.
The formation of a secondary product, octanoic acid, was observed for the bioreactor under
controlled conditions experiment at a production of 1.12 mg per gram of dry cell weight per
hour. The McCartney bottle experiments did not produce any by-products.
The 1-octanol production performance in the bioreactor experiments was empirically modelled.
The empirical rate law was based on the form of the Monod equation, with the addition of a
product inhibition term. The model achieved an average Root Mean Square Error of less than 5% when compared to experimental data, and was therefore concluded to be accurate within
the range of experimental data and conditions tested for.
The principal finding of the research is that the cells produced an order of magnitude more
product in the bioreactor than in the McCartney bottles. The literature on this reaction system,
however, reports only on smaller scale research than that performed in the bioreactor. The
improved production results in the bioreactor therefore give the first insight into the potential
that this technology has for being scaled up.
Of equal significance is the finding that a secondary product developed during the
biotransformations performed in the bioreactor. This refutes the assumption that the
biocatalyst cells are unable to catalyse any secondary reactions. This aspect of the cells’
performance must be addressed before the biocatalyst cell strain can be considered to be a
viable option for utilisation in large-scale processes. / AFRIKAANSE OPSOMMING: Alkane kan gebruik word as ‘n bekostigbare bron om meer waardevolle alkohol te produseer.
Die biotransformasie van alkane na alkohol bied dus ‘n alternatief vir konvensionele chemiese
prosedure.
Die oogmerk en omvang van hierdie navorsing was om ‘n proses te ontwikkel waarin ‘n
biokatalisator gebruik word om die oksidasie van ‘n alkaan tot sy ooreenstemmende alkohol te
kataliseer, en om vas te stel hoeveel 1-oktanol vervaardig kan word deur ‘n herverenigde E. coli
as katalisator gebruik. ‘n Rekombinante E. coli BL21(DE3) sel, wat die CYP153A6 operon in pET
28 vector bevat, is as biokatalisator gebruik. Die CYP153A6 ensiem kataliseer die oksidasie van
oktaan na 1-oktanol.
Biokonversie lot-reaksies, met oormatige oktaan wat as substraat gebruik word, is in 30ml
McCartney bottels en in 7.5L BioFlo 110 Modular Benchtop Fermentor (New Brunswick)
uitgevoer. Die bioreaktor was toegerus om kondisies van die proses soos temperatuur, pH and
opgeloste suurstof-konsentrasie te kontroleer. Die prosedures wat gebruik is om die groei,
onderhoud en oes van die biokatalisator selle te bewerkstellig, is gebaseer op prosedures wat
ontwikkel is deur the Department van Microbiese, Biochemiese and Voedsel Biotegnologie van
die Universiteit van die Vrystaat. Die produk- en substraat-konsentrasies is vasgestel deur gaschromatografie
(GC) ontleding.
Die McCartney bottel biokonversie-reaksie met ‘n 1.33ml reaksie-volume het 1.88 mg 1-oktanol
per gram droeë-sel gewig opgelewer. Die bioreaktor, wat onder beheerde toestande ‘n 2L
reaksie-volume het, het 14.89 mg 1-octanol per gram droeë-sel gewig gelewer. Onder beheerde
eksperimentele kondisies is die vorming van ‘n sekondere produk, oktanol-suur, by die
bioreaktor waargeneem teen 1.23 mg per gram droeë-sel gewig per uur. Die McCartney bottel
eksperimente egter het geen newe-produkte opgelewer nie. Die ontwikkeling van die 1-oktanol in die bioreaktor-ekperimente is empiries gemodelleer. Die
empiriese ‘rate law’ is gebaseer op ‘n vorm van die Monod- vergelyking, met byvoeging van ‘n
produk-inhiberingsterm. Die model het ‘n gemiddelde vierkantswortel foutvariansie van minder
as 5% opgelewer, vergeleke met die eksperimentele data, en word dus binne die rykwydte van
die eksperimentele data, en die kondisies waarvoor getoets is, as akkuraat beskou.
Die belangrikste bevinding is dat die selle in die bioreaktor ‘n orde van grootte meer produk
gelewer het as die selle in die McCartney bottels. Die literatuur oor hierdie reaksie-sisteem berig egter slegs oor kleiner skaalse navorsing as wat in die bioreaktor gedoen is. Die verbeterde
opbrengsresultate van die bioreaktor dui daarop dat laasgenoemde tegnologie die potensiaal
inhou om opgegradeer te word.
Die bevinding dat ‘n sekondere produk in die biotransformasie in die bioreaktor gevorm het, is
beduidend. Dit weerspreek die aanname dat die biokatalisator-selle nie sekondere reaksies
kataliseer nie. Hierdie aspek moet aangespreek word alvorens die biokataliseer-selle oorweeg
kan word as ‘n lewensvatbare alternatief vir gebruik in grootskaalse prosesse.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/86271 |
| Date | 04 1900 |
| Creators | Roux, Philipp Francois |
| Contributors | Clarke, K. G., Callanan, L. H., Smit, M. S., Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 141 p. : ill. |
| Rights | Stellenbosch University |
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