Pumping toxic substances through a cytoplasmic membrane by protein transporters known as efflux pumps represents one bacterial mechanism involved in the stress response to the presence of toxic compounds. The active efflux might also take part in exporting low-molecular-weight alcohols produced by intrinsic cell metabolism; in the case of solventogenic clostridia, predominantly acetone, butanol and ethanol (ABE). However, little is known about this active efflux, even though some evidence exists that membrane pumps might be involved in solvent tolerance. In this study, we investigated changes in overall active efflux during ABE fermentation, employing a flow cytometric protocol adjusted for Clostridia and using ethidium bromide (EB) as a fluorescence marker for quantification of direct efflux. A fluctuation in efflux during the course of standard ABE fermentation was observed, with a maximum reached during late acidogenesis, a high efflux rate during early and mid-solventogenesis and an apparent decrease in EB efflux rate in late solventogenesis. The fluctuation in efflux activity was in accordance with transcriptomic data obtained for various membrane exporters in a former study. Surprisingly, under altered cultivation conditions, when solvent production was attenuated, and extended acidogenesis was promoted, stable low efflux activity was reached after an initial peak that appeared in the stage comparable to standard ABE fermentation. This study confirmed that efflux pump activity is not constant during ABE fermentation and suggests that undisturbed solvent production might be a trigger for activation of pumps involved in solvent efflux. KEY POINTS: • Flow cytometric assay for efflux quantification in Clostridia was established. • Efflux rate peaked in late acidogenesis and in early solventogenesis. • Impaired solventogenesis led to an overall decrease in efflux.

• Klíčová slova
• ABE fermentation, Clostridium, Efflux pump, Ethidium bromide, Flow cytometry,
• MeSH
• aceton MeSH
• butanoly MeSH
• Clostridium beijerinckii * MeSH
• Clostridium MeSH
• ethanol MeSH
• fermentace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aceton MeSH
• butanoly MeSH
• ethanol MeSH

BACKGROUND: Thinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data. RESULTS: In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation. CONCLUSIONS: We provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.

• Klíčová slova
• ABE fermentation, Clostridium beijerinckii NRRL B-598, RNA-Seq transcriptome,
• MeSH
• bakteriofágy genetika   MeSH
• butanoly metabolismus   MeSH
• Clostridium beijerinckii genetika   metabolismus   virologie   MeSH
• DNA virů genetika   MeSH
• fermentace MeSH
• genetická transkripce MeSH
• kinetika MeSH
• pseudogeny genetika   MeSH
• sekvenční analýza RNA * MeSH
• stanovení celkové genové exprese * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• butanoly MeSH
• DNA virů MeSH

BACKGROUND: Biobutanol 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. RESULTS: Dual 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. CONCLUSION: We 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.

• Klíčová slova
• ABE fermentation, Butanol, Clostridium, Cytometry, Fluorescence staining, Sporulation, Stress, Viability,
• Publikační typ
• časopisecké články MeSH

We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone-butanol-ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.

• MeSH
• aceton metabolismus   MeSH
• barvení a značení MeSH
• butanoly metabolismus   MeSH
• Clostridium chemie   růst a vývoj   metabolismus   MeSH
• ethanol metabolismus   MeSH
• fermentace MeSH
• fluorescenční barviva chemie   metabolismus   MeSH
• mikrobiální viabilita * MeSH
• průtoková cytometrie metody   MeSH
• rozpouštědla metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aceton MeSH
• butanoly MeSH
• ethanol MeSH
• fluorescenční barviva MeSH
• rozpouštědla MeSH