Ecophysiology of novel intestinal butyrate-producing bacteria
Bui, Thi Phuong Nam - \ 2016
Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462577015 - 202
butyrates - butyric acid bacteria - intestines - microbial interactions - faecal examination - mice - man - infants - genomics - intestinal physiology - microbial physiology - biochemical pathways - lysine - sugar - butyraten - boterzuurbacteriën - darmen - microbiële interacties - fecesonderzoek - muizen - mens - zuigelingen - genomica - darmfysiologie - microbiële fysiologie - biochemische omzettingen - lysine - suiker
The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth.
Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.
Metabolic engineering of acid formation in Clostridium acetobutylicum
Kuit, W. - \ 2013
Wageningen University. Promotor(en): Gerrit Eggink, co-promotor(en): Ana Lopez Contreras. - S.l. : s.n. - ISBN 9789461734464 - 181
clostridium acetobutylicum - butanol - biologische productie - bioengineering - butyraten - acetaten - clostridium acetobutylicum - butanol - biological production - bioengineering - butyrates - acetates
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.
Kinetics of syntrophic cultures: A theoretical treatise on butyrate fermentation
Kleerebezem, R. ; Stams, A.J.M. - \ 2000
Biotechnology and Bioengineering 67 (2000)5. - ISSN 0006-3592 - p. 529 - 543.
afvalwaterbehandeling - kinetica - thermodynamica - anaërobe behandeling - butyraten - fermentatie - waste water treatment - kinetics - thermodynamics - anaerobic treatment - butyrates - fermentation
Numerous microbial conversions in methanogenic environments proceed at (Gibbs) free energy changes close to thermodynamic equilibrium. In this paper we attempt to describe the consequences of this thermodynamic boundary condition on the kinetics of anaerobic conversions in methanogenic environments. The anaerobic fermentation of butyrate is used as an example. Based on a simple metabolic network stoichiometry, the free energy change based balances in the cell, and the flux of substrates and products in the catabolic and anabolic reactions are coupled. In butyrate oxidation, a mechanism of ATP-dependent reversed electron transfer has been proposed to drive the unfavorable oxidation of butyryl-CoA to crotonyl-CoA. A major assumption in our model is that ATP-consumption and electron translocation across the cytoplasmic membrane do not proceed according to a fixed stoichiometry, but depend on the cellular concentration ratio of ATP and ADP. The energetic and kinetic impact of product inhibition by acetate and hydrogen are described. A major consequence of the derived model is that Monod-based kinetic description of this type of conversions is not feasible, because substrate conversion and biomass growth are proposed to be uncoupled. It furthermore suggests that the specific substrate conversion rate cannot be described as a single function of the driving force of the catabolic reaction but depends on the actual substrate and product concentrations. By using nonfixed stoichiometries for the membrane associated processes, the required flexibility of anaerobic bacteria to adapt to varying environmental conditions can be described.