Non-inhibitory levels of oxygen during cultivation increase freeze-drying stress tolerance in Limosilactobacillus reuteri DSM 17938

The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500mL/min, with a growth rate of 0.74 +/- 0.13h(-1) which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8%+/- 2.4% vs. 11.5%+/- 4.3%), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs. 3.59 for N-2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5 ' nucleotidase activity (370U/L vs. 240U/L in N-2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimization of L. reuteri DSM 17938

Characterization and adaptation of Caldicellulosiruptor strains to higher sugar concentrations, targeting enhanced hydrogen production from lignocellulosic hydrolysates

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 ( $$Q_{{{\text{H}}_{2} }}$$ Q H 2 ) 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 $$Q_{{{\text{H}}_{2} }}$$ Q H 2 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

Non-inhibitory levels of oxygen during cultivation increase freeze-drying stress tolerance in Limosilactobacillus reuteri DSM 17938

The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500 ml/min, with a growth rate of 0.74 ± 0.13 h-1 which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8 ± 2.4 % vs 11.5 ± 4.3 %), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs 3.59 for N2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5’nucleotidase activity (370 U/L vs 240 U/L in N2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90 min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimisation of L. reuteri DSM 17938

Characterization of carotenoids in Rhodothermus marinus

Rhodothermus marinus, a marine aerobic thermophile, was first isolated from an intertidal hot spring in Iceland. In recent years, the R. marinus strain PRI 493 has been genetically modified, which opens up possibilities for targeted metabolic engineering of the species, such as of the carotenoid biosynthetic pathway. In this study, the carotenoids of the R. marinus type‐strain DSM 4252T, strain DSM 4253, and strain PRI 493 were characterized. Bioreactor cultivations were used for pressurized liquid extraction and analyzed by ultra‐high performance supercritical fluid chromatography with diode array and quadropole time‐of‐flight mass spectrometry detection (UHPSFC‐DAD‐QTOF/MS). Salinixanthin, a carotenoid originally found in Salinibacter ruber and previously detected in strain DSM 4253, was identified in all three R. marinus strains, both in the hydroxylated and nonhydroxylated form. Furthermore, an additional and structurally distinct carotenoid was detected in the three strains. MS/MS fragmentation implied that the mass difference between salinixanthin and the novel carotenoid structure corresponded to the absence of a 4‐keto group on the ß‐ionone ring. The study confirmed the lack of carotenoids for the strain SB‐71 (ΔtrpBΔpurAcrtBI’::trpB) in which genes encoding two enzymes of the proposed pathway are partially deleted. Moreover, antioxidant capacity was detected in extracts of all the examined R. marinus strains and found to be 2–4 times lower for the knock‐out strain SB‐71. A gene cluster with 11 genes in two operons in the R. marinusDSM 4252T genome was identified and analyzed, in which several genes were matched with carotenoid biosynthetic pathway genes in other organisms.Vetenskapsrådet, Grant/Award Number: 622-2010-333; Svenska Forskningsrådet Formas, Grant/Award Number: 239-2013-971 and 942-2015-1952; Seventh Framework Programme, Grant/Award Number: 311932Peer Reviewe

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

Formation and Conversion of Oxygen Metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under Different Growth Conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h(−1)). Accumulation of H(2)O(2) in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H(2)O(2) accumulated only temporarily during the lag phase. H(2)O(2) is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min(−1) mg of protein(−1)), which reduced oxygen to water, whereby superoxide was formed as a by-product. H(2)O(2) originated from the dismutation of superoxide by superoxide dismutase. Although H(2)O(2) was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H(2)O(2)-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H(2)O(2) and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H(2)O(2)