Metabolic engineering of acid formation in Clostridium acetobutylicum

W. Kuit

Research output: Thesisinternal PhD, WU


During the last few decades, there has been an increasing search for alternative resources for the production of products traditionally derived from oil, such as plastics and transport fuels. This has been prompted by the finite nature of our oil reserves, the desire for energy security, and by concerns about anthropogenic global warming. Petrol and diesel are the two main fuel types for land based transportation and are currently derived from oil. Butanol, a four-carbon alcohol that can be produced by certain bacteria in a renewable way, can be used as a direct petrol replacement. It also has multiple applications as chemical intermediate and as a solvent. Although it is similar to ethanol it has superior properties with regard to energy density, vapour pressure, and water solubility when applied a biofuel.

The acetone-butanol-ethanol (ABE) fermentation of sugars as carried out by various bacteria of the genus Clostridium has been widely applied in the first part of the 1900s as a commercial method to produce butanol and acetone. The two most used species have been Clostridium acetobutylicum and C. beijerinckii. Both produce not only solvents but also the unwanted acids acetate and butyrate. In the second part of the 20th century, the ABE-process became no longer economically competitive with the petrochemical process for the production of these solvents. But today’s high oil prices make the fermentation process interesting again, although there are still challenges that have to be tackled before the process can be re-commercialised. These include finding ways to make it possible to use cheap biomass feedstocks (such as lignocelluloses) as substrate rather than using traditional feedstocks such as starch and molasses, which are relatively expensive. In addition this replacement would avoid the food-versus-fuel dilemma. Another challenge is to improve butanol production, yield, and titre. The work described in this thesis focuses on the enhancing of butanol production and diminishing acid formation by C. acetobutylicum.

A metabolic engineering approach was taken to reduce the number and amount of by-products in C. acetobutylicum fermentations. Production pathways of the acids acetate and butyrate were targeted, as we hypothesised that inhibiting acid formation would also prevent acetone production by C. acetobutylicum, resulting in only alcohols as the liquid fermentation products. To carry out our metabolic engineering work, we first developed an essential tool for gene disruption.

During this work we studied storage conditions for electro-competent C. acetobutylicum cells, allowing for the batch preparation of these cells for later use for up to 54 months (Chapter 2 part 1). The principle on which it is based, exclusion of oxygen, suggests that it might also be applicable to the storage of other obligate anaerobes.

The second part of Chapter 2 describes the adaptation of the TargeTron gene knock-out system for use in C. acetobutylicum. The TargeTron system uses a mobile group II intron that can be ‘retargeted’, i.e. reprogrammed, to insert into a specific site in the genome in a process called retrohoming. We targeted the acetate kinase (ack) gene and successful insertion of the intron was demonstrated using a PCR test. But only after the development of a colony PCR protocol for C. acetobutylicum as described in Chapter 4, we were able to apply our system and quickly detect pure mutants amongst the parental strain.

Another research group also developed a clostridial version of the TargeTron system and called it ClosTron. The advantage of this system over the one we developed is that inserted intron copies carry an activated erythromycin resistance gene and can therefore easily be selected. In Chapter 3 we used this system to obtain an acetate kinase gene knockout, which was extensively characterised in pH‑controlled batch fermentations on two media; CGM and Clostridial Medium 1 (CM1). Enzyme assays showed a 98 % reduction in in vitro acetate kinase activity, however the mutant strain continued to produce wild-type levels of acetate in CGM which does not contain any added acetate. In CM1 that does contain acetate, acetate production could still be seen, but was severely reduced. These results suggest that alternative ways of acetate production may be active in C. acetobutylicum. The solvent production of the ack strain was not significantly affected in CM1. When grown on CGM our wild-type strain produced large amounts of lactate and was therefore not suitable as a production medium. Interestingly our ack mutant strain performed better.

Subsequently we created a strain with an inactivated butyrate kinase gene termed BUK1KO, as described in Chapter 4. The phenotype of this strain was essentially that of an acetate-butanol producer. Analysis of the fermentation behaviour indicated that the strain never seemed to switch from an acidogenic to an solventogenic state, as the wild-type did. Furthermore, the growth on CM1 in batch culture demonstrated a strong influence of the pH on the fermentation behaviour. There was a good correlation between increasing fermentation pH and higher acetate levels within the pH range from 4.5 to 5.5, suggesting that the produced acetate levels might actually be the growth inhibiting compound. In addition, the mutant cells never produced the clostridial cell-types associated with spore formation. This is in line with the absence of a solventogenic switch. Also in parallel with the increasing fermentation pH was an increased acetoin accumulation with a maximum of 49 mM at pH 6.5 compared to 12 mM for the wild type under control conditions. Growth on CM1 without acetate at a pH of 5.5 resulted in a 21 % increase in butanol levels to 195 mM (14.5 g/L) compared to the wild type under its optimal conditions and 127 % under the same conditions. There was also a 60 % reduction in acetone levels and slightly increased ethanol levels.

A subsequent inactivation of the acetate kinase gene in the buk1 negative background using our own TargeTron system (see Chapter 2) resulted in isolation of an ack buk1 double mutant. Despite abolishment of both acetate kinase and butyrate kinase enzyme activity in vitro, the mutant continued to produce both acids. In CM1, acetate levels were severely reduced compared to the parenteral buk1 strain, but when acetate was removed from the medium, large amounts of acetate were produced again. This behaviour is reminiscent of the ack mutant and supports the hypothesis that unknown alternative acid producing pathways or enzymes exist in C. acetobutylicum. Alcohol production was negatively affected as compared to the parental strain and acetone production was not eliminated. Also at certain pH‑levels acetoin production was even further increased to 100 mM, the highest reported value for this organism.

In an alternative take on improving butanol production titre, we envisioned a homo-fermentative 2‑butanol strain. 2‑butanol is less toxic to the cell and should, in the proposed pathway, be produced redox-neutral from glucose. In addition it retains all the beneficial biofuel properties. As a first step towards this goal, we demonstrated in Chapter 5 that an alcohol dehydrogenase from Clostridium beijerinckii, over-expressed in C. acetobutylicum, can accept natively produced d‑ and l‑acetoin as its substrate and reduce it to d‑ and meso‑2,3‑butanediol. In addition we showed that our C. acetobutylicum WUR strain already produces small amounts (approximately 3 mM) of meso‑2,3‑butanediol through an unknown pathway, most likely from d‑acetoin. No production of meso‑2,3‑butanediol was observed for the ATCC 824 strain. Completion of the pathway requires a dehydratase and a secondary-alcoholdehydrogenase to produce methyl-ethyl ketone and 2‑butanol respectively.

In the general discussion (Chapter 6) the results described in this thesis were put into perspective, and the existence of an alternative acid pathway in C. acetobutylicum is suggested. Furthermore the disadvantages and advantages of C. acetobutylicum as a butanol production platform are discussed together with developments of butanol production in heterologous hosts.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Eggink, Gerrit, Promotor
  • Lopez Contreras, Ana, Co-promotor
Award date7 Jan 2013
Place of PublicationS.l.
Print ISBNs9789461734464
Publication statusPublished - 2013


  • clostridium acetobutylicum
  • butanol
  • biological production
  • bioengineering
  • butyrates
  • acetates

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