Metabolomics and Dynamic Metabolic Flux Analysis in ABE (Acetone-Butanol-Ethanol) Fermentation

De nos jours, les hydrolysats d'hémicelluloses, des matières premières riches en glucides issues des procédés papetier et forestier, sont largement étudiés pour le développement de procédés de bioraffinerie forestière pour la production de biobutanol, un biodiésel de remplacement du pétrole. Une des avenues biotechnologiques les plus étudiées à ce jour est la fermentation ABE (acétone-butanol-éthanol) par la bactérie Clostridium acetobutylicum. Cependant, certains inhibiteurs de la fermentation ABE sont générés lors du procédé de prétraitement et d’hydrolyse, ce qui limite grandement l'utilisation de ces biomasses riches en substrats peu coûteux. D’autre part, les pratiques actuelles de culture mènent à des rendements en butanol qui ne sont pas suffisants pour assurer la mise en place d’unités industrielles de production qui soient économiquement profitables. L'objectif de ce projet de recherche visait donc à améliorer la productivité du bioprocédé de fermentation ABE, dans un contexte de bioraffinage forestier, par l’amélioration des connaissances et le développement de stratégies de culture. Un milieu synthétique contenant du xylose comme source principale de carbone a été utilisé pour simuler un hydrolysat d’hémicelluloses d’épinette noire pour un bioprocédé de fermentation avec la bactérie Clostridium acetobutylicum. Tout d'abord, nous avons étudié l’effet d’une concentration élevée en chlorure de sodium sur le comportement du bioprocédé. En effet, une quantité élevée en hydroxyde de sodium est ajouté lors du procédé de délignification des copeaux de bois, au préalable de l’étape d’hydrolyse, cet ajout tient au fait du contrôle pH ainsi que pour son pouvoir caustique comme agent de nettoyage. Ainsi, compte tenu de l’intérêt à utiliser cette solution d’hydrolysats d’hémicellulose, il est considéré comme crucial d’évaluer l’effet de cette haute concentration en sodium sur la fermentation ABE. Une concentration seuil de sodium de 200 mM, soit ce qui est normalement mesuré en industrie, a été utilisée et comparée à une culture témoin avec la souche Clostridium acétobutylicum ATCC 824. Les résultats ont révélé que la biomasse et l'ABE étaient sérieusement inhibés par une concentration élevée en sodium, avec une diminution respectivement de 19.50 ± 0.85 % (biomasse), 35.14 ± 3.50 % (acétone), 33.37 ± 0.74 % (butanol) et 22.95 ± 1.81 % (éthanol). De manière intéressante, les productivités spécifiques cellulaires en solvants ont été maintenues comparativement à la culture témoin. Une étude approfondie du métabolisme intracellulaire a permis d’identifier que l'effet principal d’une concentration élevée en sodium se concentre principalement sur la phase d’acidogénèse, phase préalable et requise pour procéder en phase solvantogénèse lors de laquelle les solvants sont produits. Les intermédiaires métaboliques associés à la voie des pentoses phosphates et à la glycolyse ont alors été respectivement inhibés de 80.73 ± 1.47 % et de 68.84 ± 3.42 % face à la culture contrôle sans ajout de sodium. Cependant, l'ATP et le NADH ont été accumulés pendant cette période alors que le ratio entre le NADP+ et le NADPH est demeuré constant pour toute la durée de la culture; un phénomène pouvant être relié à la forte hausse de productivité spécifique en solvants. Dans un deuxième temps, l’effet de la présence d’acétate sur la fermentation ABE a été étudié, compte tenu que ce composé est généré en quantité non négligeable lors de l’étape d’hydrolyse des hémicelluloses. Or, par un heureux hasard, les cultures implémentées initialement avec une concentration en acétate de sodium de 60 mM ont mené à la production d’une quantité élevée de riboflavine, atteignant un maximum ~ 0,2 g L-1 (0,53 mM) contre 0,057 mM dans la culture témoin, soit une augmentation d’un facteur 10x. Parallèlement à une augmentation marquée de production de riboflavine, la production de solvants et le rendement en biomasse ont même été simultanément favorisés. De façon intéressante, l'addition d'acétate a également stimulé l'accumulation intracellulaire de NADH, ce qui a pu contribuer, finalement, à affecter d’autres voies métaboliques par régulation redox. L'analyse métabolique intracellulaire a également permis de spéculer sur les flux stimulés ou inhibés en présence d’acétate et qui les métabolites accumulés lors de l’étape d’acidogénèse vers la phase de solvantogénèse pour la production de solvants. Finalement, un modèle métabolique cinétique a été développé pour simuler ce système de production ABE coproducteur de riboflavine, et utilisé pour l'analyse de la dynamique des flux métaboliques. La cinétique de chaque flux métabolique ainsi que de la croissance de la biomasse sont décrites selon une cinétique de type Michaelis-Menten. Le mécanisme d'activation de la formation de riboflavine et de butanol par l'acétate, ainsi que les mécanismes d'inhibition de la croissance de la biomasse et l'absorption du xylose par le butanol ont été décrits. Le modèle comprend 24 réactions, 23 métabolites et 72 paramètres. La structure du modèle ainsi que la valeur de ses paramètres biocinétiques ont été déterminées en confrontant les simulations du modèle à des données expérimentales en bioréacteur de 3,5 L, en concentrant l’étude des paramètres sensibles identifiés par une étude de sensibilité. Ainsi, le modèle a montré être en mesure de simuler divers phénomènes métaboliques reliés à la transition de la phase acidogène à la phase solvantogène, soit une étape cruciale à l’induction de la production en solvants. Parallèlement, l'analyse dynamique des flux métaboliques, via les simulations du modèle, a permis de révéler que les taux de formation de riboflavine (ribA) et de guanosine triphosphate (GTP, précurseur de la riboflavine) (PurM), étaient tous deux fortement stimulés par l'ajout d’acétate, avec une activité de 9,4 fois et 9,7 fois au moment initial, respectivement. Cette étude supporte donc notre hypothèse que l’ajout d’acétate favorise une stimulation de flux les métabolites accumulés lors de l'acidogénèse vers la production de solvants dans la phase de solvantogénèse. Enfin, une simulation différente de la concentration initiale en acétate a montré que ce modèle était robuste pour prédire l'ABE et la coproduction de riboflavine dans un milieu de culture contrôle sans ajout d’acétate. En conclusion, cette thèse portant sur l’étude du comportement métabolique d’un bioprocédé de fermentation ABE à l’aide de Clostridium acetobutylicum ATCC 824, apporte des idées, des résultats et des outils qui contribueront à l’établissement de bioprocédés de production de biobutanol, valorisant des résidus d’hydrolysats d’hémicellulose, qui soient économiquement viables. ---------- Nowadays, hemicellulose hydrolysates - sugar-rich feedstocks issued from the pulp-and-paper and forestry industries - are being greatly investigated in butanol biorefinery. Among various biotechnological platforms under study to produce butanol, the acetone-butanol-ethanol (ABE) fermentation process with Clostridium acetobutylicum seems to be the most studied avenue. However, some inhibitors are generated in the pre-treatment and hemicellulose hydrolysis processes, with compounds which are inhibitors of ABE fermentation. Moreover, the productivity yields of the ABE bioprocess are still low and barely enable the economic feasibility of such a bioprocess at an industrial scale, despite the low cost of these feedstocks. Therefore, the main objective of this thesis is focused on ameliorating our fundamental knowledge of ABE fermentation in order to enable the identification of potential bioprocess improvement strategies. The aim of this research is thus to contribute to the development of biobutanol industrialization. A synthetic medium with xylose as the main carbon source was used to simulate hemicellulose hydrolysates of black spruce, used in the pulp-and-paper and forestry industries, and Clostridium acetobutylicum was the culture used to perform ABE fermentation. In the first part, we evaluated the effect of a high sodium chloride concentration in ABE fermentation, since large amounts of sodium hydroxide are applied to wood chips during the hydrolysis process such as in delignification, pH control, and as caustic cleaning agents. These processes then artificially increase the sodium concentration of the resulting solution, and since this solution is to be used as a culture medium for ABE fermentation, it is crucial to characterize the effects of such a high sodium content. A sodium concentration of 200 mM, a level normally observed in industry, was thus assessed and compared to a control culture. The Clostridium acetobutylicum ATCC 824 strain was studied, and a high sodium condition was shown to affect biomass growth and ABE yield, but not the cell-specific productivity in ABE. A further metabolomics study showed that a high sodium concentration mainly influenced the acidogenic phase and biomass synthesis. The ABE fermentation process normally requires an acidogenic phase first, in order to proceed to the solventogenic phase during which solvents are produced, so during acidogenesis, high sodium conditions were shown to inhibit the intermediate metabolites concentration of the pentose phosphate pathway and glycolysis pathways of up to 80.73 1.47 % and 68.84 3.42 %, respectively. However, ATP and NADH were stimulated at high sodium, while the NADP+-to-NADPH ratio was constant for the entire culture duration, a phenomenon which may explain the robustness of solvents’ specific productivities even under a sodium stress. In the second part, we investigated the effect of supplementing acetate on ABE fermentation, since this compound is generated in non-negligible amounts during the hemicellulose hydrolysis step. Indeed, supplementing the culture medium at 60 mM sodium acetate led to the production of a yellow sediment clearly identified as riboflavin. We thus observed that a 60 mM acetate supplementation leads to a 10-fold increase of riboflavin, reaching up to ~ 0.2 g L-1 (0.53 mM) compared to 0.057 mM in the control culture. A metabolomic study showed that acetate supplementation resulted in a higher consumption of GTP, which is the precursor of riboflavin. Moreover, solvents production and biomass yield were also promoted when adding acetate. Interestingly, acetate addition clearly stimulated the accumulation of the reduced form of nicotinamide-adenine dinucleotide (i.e. NADH), which could have affected other metabolic pathways though redox regulation mechanisms. Our metabolomic study also suggests that a high acetate condition stimulates the mobilization of various metabolic intermediates accumulated in acidogenesis towards solvents production in solventogenesis. In the third part, a kinetic metabolic model was developed in order to better understand the effect of adding acetate by simulating the ABE-coproducing riboflavin process and performing a dynamic metabolic flux analysis. Each step in flux kinetics, as well as the biomass specific growth rate, was described using the Michaelis-Menten type approach. The activation mechanism of riboflavin and butanol formation by acetate, as well as the inhibition mechanisms of biomass growth and xylose uptake by butanol, were described. The model includes 24 reactions, 23 metabolites, and 72 parameters. Model structure as well as kinetic parameter value were determined by minimizing simulation errors of experimental data for 3.5-L bioreactor cultures at 60 mM acetate condition. Indeed, the model was shown to be capable of adequately simulating experimental data and predicting culture behavior without acetate addition, as well as the transition from acetogenesis to solventogenesis - a crucial step in the induction of solvents production. Moreover, a dynamic metabolic flux analysis suggests that the riboflavin (ribA) and guanosine triphosphate (GTP, precursor of riboflavin) formation rates (PurM) were strongly stimulated by high acetate with 9.4-fold and 9.7-fold activity early following inoculation, respectively. This in silico study further suggests that a high acetate condition stimulates fluxes which dredged accumulated metabolites in acidogenesis for solvents production. In conclusion, this work on the investigation of the metabolic behavior of ABE fermentation with Clostridium acetobutylicum ATCC 824 has brought thoughts, results, and tools which may contribute to enabling the economic feasibility of producing butanol valorizing hemicellulose hydrolysates wastes

Solvent-based approaches to evaluate the ABE extractive fermentation

The reindustrialization of ABE fermentation is hampered by significant production costs, linked to high product inhibition and limited intrinsic yield. The reduction of these costs depends on the effective application of integrated toxic product removal techniques. The evaluation of ABE extractive fermentation with solvents of different nature in terms of extraction capacity or biocompatibility is the main objective of this thesis. Attention is focused on the assessment of the solvent influence, not only on the physical effects but also on the metabolism and microbial population dynamics evolution. A mathematical model based on the evolution of the heterogeneous culture inside the bioreactor was proposed and validated ABE extractive fermentation is techno and economically evaluated on a solvent-based comparative basis. The integration of this process within a LCB biorefinery using a 2G type substrate is also considered

Metabolic engineering of Clostridium saccharoperbutylacetonicum for improved solvent production

In order to avert irreversible damage to the global climate, the global community has committed to reaching net zero carbon emission in the coming years. To meet this ambitious target, substantial changes will be needed. To minimise the disruption to people’s lives, there is a need for renewable technologies which are compatible with existing infrastructure, such as biofuels and drop in chemical compounds. A compound which can fulfil both roles is n-butanol. Clostridium are natural butanol producers but have fallen out of use as they have been unable to compete with fossil fuel-based production methods. The aim of this thesis was to improve the production of butanol in an asporogenic strain of Clostridium saccharoperbutylacetonicum N1-4. A systems scale approach was taken to improve butanol production. Combined analysis of Clostridium metabolism using flux balance analysis and 13C-metabolic flux analysis was used to guide metabolic engineering strategies, with the aim of increasing butanol production in asporogenic C. saccharoperbutylacetonicum N1-4 spo0AI261T. Flux balance analysis was used to gain an understanding of Clostridium metabolism and to explore manipulations that could lead to increased butanol production in wild type C. saccharoperbutylacetonicum N1-4. Key features of in silico engineered strains were compared to experimental data and identified an increase in NADH generation and key butanol synthesising genes as targets for increasing butanol production. A second round of flux balance analysis identified further manipulations relevant to C. saccharoperbutylacetonicum N1-4 spo0AI261T, mainly that the rate of glucose uptake appeared to be limiting butanol production. Simulations of strains with increased glucose uptake and butanol production suggested that ATP consuming enzymes would have to be engineered into the asporogenic strain to balance ATP. Additional investigation was performed using 13C-metabolic flux analysis, which was able to resolve intracellular fluxes of asporogenic C. saccharoperbutylacetonicum N1-4. It also identified a feature of C. saccharoperbutylacetonicum N1-4 spo0AI261T metabolism that was unexpected, that ATP was in excess to biomass synthesis requirements, resulting in a futile cycle. The results from this flux analysis confirmed the rationale of the flux balance analysis guided strategies. In the final chapter, the developed strategies were incorporated into the asporogenic strain of C. saccharoperbutylacetonicum N1-4. These mutant strains were analysed in fermentations. While none of the strains produced more butanol than the parent strain, this work incorporated several novel approaches to increase butanol production in Clostridium and will serve as a starting point for future metabolic engineering work

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