| 1. |
- Bhattacharya, Abhishek, et al.
(författare)
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Cross-Feeding and Enzymatic Catabolism for Mannan-Oligosaccharide Utilization by the Butyrate-Producing Gut Bacterium Roseburia hominis A2-183
- 2022
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 10:12
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Tidskriftsartikel (refereegranskat)abstract
- β-Mannan is abundant in the human diet and in hemicellulose derived from softwood. Linear or galactose-substituted β-mannan-oligosaccharides (MOS/GMOSs) derived from β-mannan are considered emerging prebiotics that could stimulate health-associated gut microbiota. However, the underlying mechanisms are not yet resolved. Therefore, this study investigated the cross-feeding and metabolic interactions between Bifidobacterium adolescentis ATCC 15703, an acetate producer, and Roseburia hominis A2-183 DSMZ 16839, a butyrate producer, during utilization of MOS/GMOSs. Cocultivation studies suggest that both strains coexist due to differential MOS/GMOS utilization, along with the cross-feeding of acetate from B. adolescentis E194a to R. hominis A2-183. The data suggest that R. hominis A2-183 efficiently utilizes MOS/GMOS in mono- and cocultivation. Notably, we observed the transcriptional upregulation of certain genes within a dedicated MOS/GMOS utilization locus (RhMosUL), and an exo-oligomannosidase (RhMan113A) gene located distally in the R. hominis A2-183 genome. Significantly, biochemical analysis of β-1,4 mannan-oligosaccharide phosphorylase (RhMOP130A), α-galactosidase (RhGal36A), and exo-oligomannosidase (RhMan113A) suggested their potential synergistic role in the initial utilization of MOS/GMOSs. Thus, our results enhance the understanding of MOS/GMOS utilization by potential health-promoting human gut microbiota and highlight the role of cross-feeding and metabolic interactions between two secondary mannan degraders inhabiting the same ecological niche in the gut.
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| 2. |
- Byrne, Eoin, et al.
(författare)
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Characterization and adaptation of Caldicellulosiruptor strains to higher sugar concentrations, targeting enhanced hydrogen production from lignocellulosic hydrolysates
- 2021
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Ingår i: Biotechnology for Biofuels. - : BioMed Central Ltd. - 1754-6834. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H2/mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high sugar concentrations, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C.owensensis, C. kronotskyensis, C.bescii, C.acetigenus and C.kristjanssonii, were developed to tolerate higher sugar concentrations through an adaptive laboratory evolution (ALE) process. The developed mixed population C.owensensis CO80 was further studied and accompanied by the development of a kinetic model based on Monod kinetics to quantitatively compare it with the parental strain. Results: Mixed populations of Caldicellulosiruptor tolerant to higher glucose concentrations were obtained with C.owensensis adapted to grow up to 80 g/L glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. The C.owensensis CO80 mixed population was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/L glucose, but with reduced volumetric hydrogen productivities (QH2) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of the mixed population C.owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical sugar concentration of the cells increased fourfold when cultivated at higher concentrations. When co-cultured with the adapted C.saccharolyticus G5 mixed culture at a hydraulic retention time (HRT) of 20 h, C.owensensis constituted only 0.09–1.58% of the population in suspension. Conclusions: The adaptation of members of the Caldicellulosiruptor genus to higher sugar concentrations established that the ability to develop improved strains via ALE is species dependent, with C.owensensis adapted to grow on 80 g/L, whereas C.kristjanssonii could only be adapted to 30 g/L glucose. Although C.owensensis CO80 was adapted to a higher sugar concentration, this mixed population demonstrated reduced QH2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated sugar concentrations, this approach does not result in improved fermentation performances at these higher sugar concentrations. Moreover, the observation that planktonic mixed culture of CO80 was outcompeted by an adapted C.saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of CO80 mixed culture should be improved for industrial application. © 2021, The Author(s).
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| 3. |
- Sreenivas, Krishnan, et al.
(författare)
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Evaluation of Pyrophosphate‐Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions
- 2024
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Ingår i: Microorganisms. - 2076-2607. ; 12:3
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L−1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.
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| 4. |
- Sreenivas, Krishnan
(författare)
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Explorations of interlinked energy and redox metabolism in two industrially applied microorganisms
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anaerobic fermentation, compared to aerobic processes, remains the most scalable method for bioproduction of compounds. This is due to that aerobic microbial processes require large amounts of energy and are unable to satisfy the oxygen demand of a high-density microbial biomass. Thus, anaerobic microbes are readily scalable to large volumes. It was found that most anaerobic microbes have evolved with the Embden-Meyerhof-Parnas pathway for efficient and luxurious growth under these conditions due to adequate energy production provided by the pathway. It is also found that most of these microbes are able to establish a balanced redox and energy metabolism either by the use of anaerobic respiration or other forms of ATP synthesis. Some microbes, however, are of industrial relevance but are not capable of luxurious growth without the use of external electron acceptors or other external conditions. Among these microbes, the suspected issues are usually attributed to redox or energy imbalances. Thus, the thesis expands on our understanding of the regulatory mechanisms and explores possible engineering methods to improve their growth rates by focusing on two specific microbes, namely the bacterium Limosilactobacillus reuteri and the yeast Saccharomyces cerevisiae.Oxygen tolerance of Lb. reuteri is important as it is one of the mechanisms to alleviate the redox imbalance. This study expands on the variations of oxygen tolerance between strains and shows that Lb. reuteri DSM 17938 does not necessarily produce more peroxide per biomass but has greater resistance than its counterparts. In parallel the various lactate dehydrogenases present in Lb. reuteri DSM 17938 were enzymatically characterised to explore the presence of alternative control mechanisms that may be present due to the simultaneous utilisation of two different central carbon pathways. The impact of overexpression of the native phosphofructokinase candidates which are predicted to be from a minor family revealed issues related to protein burden in lean media.The introduction of a proton pumping pyrophosphatase (H+-PPase) to supplement the proton pumping ATPase (H+-ATPases) in S. cerevisiae was also explored. Under stressful conditions, the study revealed that the H+-PPase could improve the growth rate and successfully act in restoring pH homeostasis. The H+-PPase improved growth of S. cerevisiae in high acetic acid concentrations and showed that there may be more limiting factors in xylose engineered S. cerevisiae. The study also revealed new avenues for improving productivity for ethanol production using lignocellulosic biomass as well as possible alternative methods that could be implemented to increase production of existing compounds that are currently ATP limited.
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| 5. |
- Vongkampang, Thitiwut, et al.
(författare)
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Characterization of simultaneous uptake of xylose and glucose in Caldicellulosiruptor kronotskyensis for optimal hydrogen production
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: Caldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profles of C. kronotskyensis in the presence of mixtures of glucose–xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study, we aimed to investigate further whether C. kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose).Results: Interestingly, growth of C. kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus, C. kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C. kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μmax=0.57 h−1) as on glucose (μmax=0.28 h−1). A study of the sugar uptake was made with diferent glucose–xylose ratios to fnd a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be KI,glu=0.01 cmol L−1 and KI,xyl=0.001 cmol L−1, hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatics analysis could not exclude that C. kronotskyensis possesses more than one transporter for glucose. As a next step it was investigated whether glucose uptake by C. kronotskyensis improved in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose; nevertheless, the growth rate on each sugar remained similar.Conclusions: C. kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C. kronotskyensis has adapted more strongly to pentose rather than hexose, thereby having obtained a specifc survival edge in thermophilic lignocellulosic degradation communities.
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| 6. |
- Vongkampang, Thitiwut, et al.
(författare)
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Immobilization techniques improve volumetric hydrogen productivity of Caldicellulosiruptor species in a modified continuous stirred tank reactor
- 2023
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Ingår i: Biotechnology for Biofuels and Bioproducts. - : Springer Science and Business Media LLC. - 2731-3654. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Co-cultures and cell immobilization have been used for retaining biomass in a bioreactor, with the aim to improve the volumetric hydrogen productivity (QH2). Caldicellulosiruptor kronotskyensis is a strong cellulolytic species that possesses tāpirin proteins for attaching on lignocellulosic materials. C. owensensis has its reputation as a biofilm former. It was investigated whether continuous co-cultures of these two species with different types of carriers can improve the QH2. Results: QH2 up to 30 ± 0.2 mmol L−1 h−1 was obtained during pure culture of C. kronotskyensis with combined acrylic fibres and chitosan. In addition, the yield of hydrogen was 2.95 ± 0.1 mol H2 mol−1 sugars at a dilution rate (D) of 0.3 h−1. However, the second-best QH2 26.4 ± 1.9 mmol L−1 h−1 and 25.4 ± 0.6 mmol L−1 h−1 were obtained with a co-culture of C. kronotskyensis and C. owensensis with acrylic fibres only and a pure culture of C. kronotskyensis with acrylic fibres, respectively. Interestingly, the population dynamics revealed that C. kronotskyensis was the dominant species in the biofilm fraction, whereas C. owensensis was the dominant species in the planktonic phase. The highest amount of c-di-GMP (260 ± 27.3 µM at a D of 0.2 h−1) were found with the co-culture of C. kronotskyensis and C. owensensis without a carrier. This could be due to Caldicellulosiruptor producing c-di-GMP as a second messenger for regulation of the biofilms under the high dilution rate (D) to prevent washout. Conclusions: The cell immobilization strategy using a combination of carriers exhibited a promising approach to enhance the QH2. The QH2 obtained during the continuous culture of C. kronotskyensis with combined acrylic fibres and chitosan gave the highest QH2 among the pure culture and mixed cultures of Caldicellulosiruptor in the current study. Moreover, it was the highest QH2 among all cultures of Caldicellulosiruptor species studied so far.
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