Lignocellulose is a promising renewable resource for producing platform chemicals, such as acetone, butanol, and ethanol, via ABE fermentation by solventogenic clostridia. This study investigates the effects of common lignocellulose derived inhibitory compounds: ferulic acid, coumaric acid, and furfural on Clostridium beijerinckii. Dual-staining with propidium iodide and CFDA, combined with flow cytometry, was employed to assess physiological variability. The results showed that phenolic acid-induced stress helped maintain a higher proportion of viable cells during the production phase, enhancing solvent yields and reducing sporulation. At 0.4 g/L, ferulic and coumaric acids did not reduce cell viability; however, coumaric acid exposure led to an acid-crash profile. Conversely, a more robust inoculum exposed to both phenolic acids simultaneously exhibited effects similar to ferulic acid alone, including slower viability decline, reduced growth and sporulation, and improved solvent production. Furfural exposure at 1.5 g/L resulted in immediate viability loss in 20% of the population, though the overall decline accompanied by the highest sporulation rate occurred later than in the control. Additionally, furfural transformation was slower, suppressing butyrate production and reducing solvent production by 13%. This study suggests that delaying cell death mechanism may explain the stimulatory effects of inhibitors, advancing lignocellulose use in the future.

• Keywords
• Clostridium beijerinckii, ABE, Butanol, Carboxyfluorescein diacetate, Cytometry, Lignocellulose inhibitors, Viability,
• Publication type
• Journal Article MeSH

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl.

• Keywords
• Bacillus subtilis, Clostridium beijerinckii, Monascus, bacterial spores germination, nitrite, red yeast rice,
• Publication type
• Journal Article MeSH

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.

• Keywords
• ABE fermentation, Clostridium, Efflux pump, Ethidium bromide, Flow cytometry,
• MeSH
• Acetone MeSH
• Butanols MeSH
• Clostridium beijerinckii * MeSH
• Clostridium MeSH
• Ethanol MeSH
• Fermentation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetone MeSH
• Butanols MeSH
• Ethanol MeSH

In-depth knowledge of cell metabolism and nutrient uptake mechanisms can lead to the development of a tool for improving acetone-butanol-ethanol (ABE) fermentation performance and help to overcome bottlenecks in the process, such as the high cost of substrates and low production rates. Over 300 genes potentially encoding transport of amino acids, metal ions, vitamins and carbohydrates were identified in the genome of the butanol-producing strain Clostridium beijerinckii NRRL B-598, based on similarity searches in protein function databases. Transcriptomic data of the genes were obtained during ABE fermentation by RNA-Seq experiments and covered acidogenesis, solventogenesis and sporulation. The physiological roles of the selected 81 actively expressed transport genes were established on the basis of their expression profiles at particular stages of ABE fermentation. This article describes how genes encoding the uptake of glucose, iron, riboflavin, glutamine, methionine and other nutrients take part in growth, production and stress responses of C. beijerinckii NRRL B-598. These data increase our knowledge of transport mechanisms in solventogenic Clostridium and may be used in the selection of individual genes for further research.

• MeSH
• Amino Acids genetics   metabolism   MeSH
• Butanols metabolism   MeSH
• Clostridium beijerinckii genetics   metabolism   MeSH
• Fermentation MeSH
• Transcription, Genetic * MeSH
• Metals metabolism   MeSH
• Carbohydrate Metabolism genetics   MeSH
• Gene Expression Regulation, Bacterial genetics   MeSH
• Carbohydrates genetics   MeSH
• Vitamins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Butanols MeSH
• Metals MeSH
• Carbohydrates MeSH
• Vitamins MeSH

Clostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABE) fermentative pathway. The pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. The description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. The study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. Surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Clostridium beijerinckii cytology   genetics   MeSH
• Fermentation genetics   MeSH
• Stress, Physiological * genetics   MeSH
• Glucose metabolism   MeSH
• Acids metabolism   MeSH
• Fatty Acids metabolism   MeSH
• Heat-Shock Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Solvents metabolism   MeSH
• Spores, Bacterial metabolism   MeSH
• Transcriptome genetics   MeSH
• Hydrogen metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Glucose MeSH
• Acids MeSH
• Fatty Acids MeSH
• Heat-Shock Proteins MeSH
• Solvents MeSH
• Hydrogen 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.

• Keywords
• ABE fermentation, Clostridium beijerinckii NRRL B-598, RNA-Seq transcriptome,
• MeSH
• Bacteriophages genetics   MeSH
• Butanols metabolism   MeSH
• Clostridium beijerinckii genetics   metabolism   virology   MeSH
• DNA, Viral genetics   MeSH
• Fermentation MeSH
• Transcription, Genetic MeSH
• Kinetics MeSH
• Pseudogenes genetics   MeSH
• Sequence Analysis, RNA * MeSH
• Gene Expression Profiling * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Butanols MeSH
• DNA, Viral 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.

• Keywords
• ABE fermentation, Butanol, Clostridium, Cytometry, Fluorescence staining, Sporulation, Stress, Viability,
• Publication type
• Journal Article MeSH