Metabolic engineering of acid formation in Clostridium acetobutylicum

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.</p

Vortex trapping and expulsion in thin-film YBCO strips

A scanning SQUID microscope was used to image vortex trapping as a function of the magnetic induction during cooling in thin-film YBCO strips for strip widths W from 2 to 50 um. We found that vortices were excluded from the strips when the induction Ba was below a critical induction Bc. We present a simple model for the vortex exclusion process which takes into account the vortex - antivortex pair production energy as well as the vortex Meissner and self-energies. This model predicts that the real density n of trapped vortices is given by n=(Ba-BK)/Phi0 with BK = 1.65Phi0/W^2 and Phi0 = h/2e the superconducting flux quantum. This prediction is in good agreement with our experiments on YBCO, as well as with previous experiments on thin-film strips of niobium. We also report on the positions of the trapped vortices. We found that at low densities the vortices were trapped in a single row near the centers of the strips, with the relative intervortex spacing distribution width decreasing as the vortex density increased, a sign of longitudinal ordering. The critical induction for two rows forming in the 35 um wide strip was (2.89 + 1.91-0.93)Bc, consistent with a numerical prediction

Surgical site infection after wound closure with staples versus sutures in elective knee and hip arthroplasty:a systematic review and meta-analysis

PURPOSE: This systematic review and meta-analysis aimed to study surgical site infection of wound closure using staples versus sutures in elective knee and hip arthroplasties. METHODS: A systematic literature review was performed to search for randomized controlled trials that compared surgical site infection after wound closure using staples versus sutures in elective knee and hip arthroplasties. The primary outcome was surgical site infection. The risk of bias was assessed with the Cochrane risk of bias assessment tool. The relative risk and 95% confidence interval with a random-effects model were assessed. RESULTS: Eight studies were included in this study, including 2 studies with a low risk of bias, 4 studies having ‘some concerns’, and 2 studies with high risk of bias. Significant difference was not found in the risk of SSI for patients with staples (n = 557) versus sutures (n = 573) (RR: 1.70, 95% CI: 0.94–3.08, I(2) = 16%). The results were similar after excluding the studies with a high risk of bias (RR: 1.67, 95% CI: 0.91–3.07, I(2) = 32%). Analysis of studies with low risk of bias revealed a significantly higher risk of surgical site infection in patients with staples (n = 331) compared to sutures (n = 331) (RR: 2.56, 95% CI: 1.20–5.44, I(2) = 0%). There was no difference between continuous and interrupted sutures (P > 0.05). In hip arthroplasty, stapling carried a significantly higher risk of surgical site infection than suturing (RR: 2.51, 95% CI: 1.15–5.50, I(2) = 0%), but there was no significant difference in knee arthroplasty (RR: 0.87, 95% CI: 0.33–2.25, I(2) = 22%; P > 0.05). CONCLUSIONS: Stapling might carry a higher risk of surgical site infection than suturing in elective knee and hip arthroplasties, especially in hip arthroplasty. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s42836-021-00110-7

Measurement of the tt̄W and tt̄Z production cross sections in pp collisions at √s = 8 TeV with the ATLAS detector

The production cross sections of top-quark pairs in association with massive vector bosons have been measured using data from pp collisions at s√ = 8 TeV. The dataset corresponds to an integrated luminosity of 20.3 fb−¹ collected by the ATLAS detector in 2012 at the LHC. Final states with two, three or four leptons are considered. A fit to the data considering the tt̄W and tt̄Z processes simultaneously yields a significance of 5.0σ (4.2σ) over the background-only hypothesis for tt¯Wtt¯W (tt̄Z) production. The measured cross sections are σtt̄W = 369 + 100−91 fb and σtt̄Z = 176 + 58−52 fb. The background-only hypothesis with neither tt̄W nor tt̄Z production is excluded at 7.1σ. All measurements are consistent with next-to-leading-order calculations for the tt̄W and tt̄Z processes