Succinic acid production using metabolically engineered Escherichia coli
Sammanfattning: The prospects of peak oil, climate change and the dependency of fossil carbon have urged research and development of production methods for the manufacture of fuels and chemicals from renewable resources (biomass). To date, the primary emphasis has been placed on the replacement of oil for transportation fuels. A highly significant subset of petroleum usage is the production of chemicals, which represents 10-15% of the petroleum usage. White biotechnology, also called industrial biotechnology, is a fast evolving technology with a large potential to have a substantial impact on the industrial production of fuels and chemicals from biomass. This work addresses the issue of chemical production by investigating the production of bio-based succinic acid, which can be used in a wide range of applications to replace petroleum based chemicals. Succinic acid can be produced by fermentation of sugar by a number of organisms; one is Escherichia coli (E. coli). It is known that E. coli under anaerobic conditions produces a mixture of organic acids. In order to obtain a cost-effective production it is necessary to metabolically engineer the organism to produce succinic acid in greater yield than the other acids. In the current work, E. coli mutant AFP184 was used. AFP184 originates from a near wild type strain, the C600 (ATCC 23724), which can ferment both five and six carbon sugars and has mutations in the glucose specific phosphotransferase system (ptsG), the pyruvate formate lyase system (pfl) and in the fermentative lactate dehydrogenase system (ldh). The previous studies using different organisms have all used cultivation mediums supplemented to some degree with different nutrients like biotin, thiamine and yeast extract. In order to apply the technology to large scale, production must be cost-effective and it is important to minimise the use of additional supplements. The first part of this work aimed to investigate the fermentation characteristics of AFP184 in a medium consisting of corn steep liquor, inorganic salts and different sugar sources without supplementation of other additional nutrients. It addresses questions regarding the effect of different sugars on succinic acid kinetics and yields in an industrially relevant medium. In order to gain a sustainable production of succinic acid from biomass feedstocks (sugar from biomass) it is important to investigate how well the organism can utilise different sugars in the biomass. The sugars studied were sucrose, glucose, fructose, xylose and equal mixtures of glucose-fructose and glucose-xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilise all sugars and sugar combinations except sucrose for biomass generation and succinate production. Using glucose resulted in the highest yield, 0.83 (g succinic acid per g sugar consumed anaerobically). Fructose resulted in a yield of 0.66 and xylose of 0.5. Using a high initial sugar concentration made it possible to obtain volumetric productivities of almost 3 g L-1h-1, which is above estimated values for feasible economic production. Succinic acid production ceased at final concentrations greater than 40 g L-1. In order to further increase succinic acid concentrations, this inhibitory effect was studied in the second part of the present work. The inhibitory effects can be two-fold including pH-based inhibition and an anion specific effect on metabolism. It has been reported that high concentrations of ammonia inhibit E. coli growth and damage cell membranes. In order to limit toxic and inhibitory effects different neutralising agents were tested. First the use of NH4OH was optimised with respect to fermentation pH and it was found that the best results were obtained at pH 6.5-6.7. Optimal pH was then used with NaOH, KOH, and Na2CO3 as neutralising agents and it was shown that NaOH, KOH, and Na2CO3 neutralised fermentations could reach succinic acid concentrations of 69 and 61 and 78 g L-1 respectively without any significant decrease in succinic acid productivity. It was observed that cells lost viability during the cause anaerobic phase. It resulted in decreasing succinic acid productivities. It is believed that the viability decrease is a combined effect of organic acids concentration and the osmolarity of the medium. The work done in this thesis is aimed towards increasing the economical feasibility of a biochemical succinic acid production.
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