Development and evaluation of an alkane bioconversion process using genetically modified Escherichia coli

Roux, Philipp Francois (2014-04)

Thesis (MScEng)--Stellenbosch University, 2014.

Thesis

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.

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