Use of fluorescent staining and flow cytometry for monitoring physiological changes in solventogenic clostridia

Patáková, Petra; Linhová, Michaela; Vykydalova, Pavla; Branská, Barbora; Rychtera, M.; Melzoch, Karel

doi:10.1016/j.anaerobe.2013.10.006

Cited by 16 publications

(11 citation statements)

References 17 publications

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“…C. beijerinckii was not an exception and four clearly recognisable populations were apparent on FC dot-plot diagrams. The development of a staining pattern clearly indicated the beginning of a decline in viability together with the pH breakpoint and onset of solventogenesis, which was consistent with previous observations for C. beijerinckii CCM 6218 [ 59 ] and C. beijerinckii NRRL B-598 [ 35 ].…”

Section: Discussionsupporting

confidence: 90%

“…This applies doubly for solventogenic clostridia, which have long resisted efforts to identify and implement a protocol to determine their viability and physiological state based on fluorescence staining [ 36 – 38 , 58 ]. Nevertheless, these studies provided a robust basis for the design of quick and reliable methodologies that were applied for C. beijerinckii [ 35 ], Clostridium pasteurianum [ 11 ] and Clostridium tetanomorphum [ 59 ]. From our results, as well as those published, it is clear that viability was seldom close to 100%, as can be observed for a wide range of microorganisms in exponential phase [ 60 – 62 ].…”

Section: Discussionmentioning

confidence: 99%

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Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions

Branská

Pechacova

Kolek

et al. 2018

Biotechnol Biofuels

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BackgroundBiobutanol production by clostridia via the acetone–butanol–ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol.ResultsDual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained—viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation.ConclusionWe have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions

Branská

Pechacova

Kolek

et al. 2018

Biotechnol Biofuels

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“…The method proposed showed 100% spore staining efficiency, since spores were always stained by HO or PI. Viability tests have been previously proposed for either lactic acid bacteria by immunodetection of intracellular proteins with species-specific antibodies or solventogenic clostridia by flow cytometry (Hannon et al, 2006;Patakova et al, 2014) but to the best of our knowledge there has been no prior application of these coupled stains to Clostridium species.…”

Section: Staining Ofmentioning

confidence: 99%

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy

D’Incecco

Ong

Gras

et al. 2018

Micron

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Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments.

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“…The characterization of metabolic phases in the ABE fermentation based on batch processes is linked to sophisticated prearrangements and poor reproducibility [16]. In the future, experimental characterization of individual cell populations in the different metabolic phases using flow cytometry [34][35][36][37], electrooptical measurements [38] or single cell RNA sequencing [39,40] can be applied to steady state samples of the CCSTR. Already, the here-presented model, showing good agreement with the experimental data, delivers prospects of the composition and the characteristics of the individual cell populations.…”

Section: Discussionmentioning

confidence: 99%

Process Engineering of the Acetone-Ethanol-Butanol (ABE) Fermentation in a Linear and Feedback Loop Cascade of Continuous Stirred Tank Reactors: Experiments, Modeling and Optimization

Karstens

Trippel

Götz

2021

Fuels

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The production of butanol, acetone and ethanol by Clostridium acetobutylicum is a biphasic fermentation process. In the first phase the carbohydrate substrate is metabolized to acetic and butyric acid, in the following second phase the product spectrum is shifted towards the economically interesting solvents. Here we present a cascade of six continuous stirred tank reactors (CCSTR), which allows performing the time dependent metabolic phases of an acetone-butanol-ethanol (ABE) batch fermentation in a spatial domain. Experimental data of steady states under four operating conditions—with variations of the pH in the first bioreactor between 4.3 and 5.6 as well as the total dilution rate between 0.042 h−1 and 0.092 h−1—were used to optimize and validate a corresponding mathematical model. Beyond a residence time distribution representation and substrate, biomass and product kinetics this model also includes the differentiation of cells between the metabolic states. Model simulations predict a final product concentration of 8.2 g butanol L−1 and a productivity of 0.75 g butanol L−1 h−1 in the CCSTR operated at pHbr1 of 4.3 and D = 0.092 h−1, while 31% of the cells are differentiated to the solventogenic state. Aiming at an enrichment of solvent-producing cells, a feedback loop was introduced into the cascade, sending cells from a later state of the process (bioreactor 4) back to an early stage of the process (bioreactor 2). In agreement with the experimental observations, the model accurately predicted an increase in butanol formation rate in bioreactor stages 2 and 3, resulting in an overall butanol productivity of 0.76 g L−1 h−1 for the feedback loop cascade. The here presented CCSTR and the validated model will serve to investigate further ABE fermentation strategies for a controlled metabolic switch.

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Use of fluorescent staining and flow cytometry for monitoring physiological changes in solventogenic clostridia

Cited by 16 publications

References 17 publications

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy

Process Engineering of the Acetone-Ethanol-Butanol (ABE) Fermentation in a Linear and Feedback Loop Cascade of Continuous Stirred Tank Reactors: Experiments, Modeling and Optimization

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