The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol.

• MeSH
• biologický transport genetika   MeSH
• bioreaktory mikrobiologie   MeSH
• buněčná membrána metabolismus   MeSH
• butanoly toxicita   MeSH
• Clostridium beijerinckii účinky léků   genetika   metabolismus   MeSH
• fyziologický stres genetika   MeSH
• glukosa metabolismus   MeSH
• glykolýza genetika   fyziologie   MeSH
• mastné kyseliny metabolismus   MeSH
• plasmalogeny biosyntéza   MeSH
• proteiny teplotního šoku metabolismus   MeSH
• quorum sensing genetika   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• aceton MeSH
• butanoly MeSH
• Clostridium beijerinckii * MeSH
• Clostridium MeSH
• ethanol MeSH
• fermentace MeSH
• Publikační typ
• časopisecké články MeSH

N-butanol, a valued solvent and potential fuel extender, could possibly be produced by fermentation using either native producers, i.e. solventogenic Clostridia, or engineered platform organisms such as Escherichia coli or Pseudomonas species, if the main process obstacle, a low final butanol concentration, could be overcome. A low final concentration of butanol is the result of its high toxicity to production cells. Nevertheless, bacteria have developed several mechanisms to cope with this toxicity and one of them is active butanol efflux. This review presents information about a few well characterized butanol efflux pumps from Gram-negative bacteria (P. putida and E. coli) and summarizes knowledge about putative butanol efflux systems in Gram-positive bacteria.

• MeSH
• bakteriální proteiny MeSH
• biologický transport MeSH
• Escherichia coli * MeSH
• membránové transportní proteiny MeSH
• metabolické inženýrství MeSH
• mikrobiální viabilita MeSH
• n-butanol * analýza   metabolismus   toxicita   MeSH
• proteiny z Escherichia coli MeSH
• Pseudomonas putida * MeSH
• rozpouštědla MeSH
• transportní proteiny MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy 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
• aminokyseliny genetika   metabolismus   MeSH
• butanoly metabolismus   MeSH
• Clostridium beijerinckii genetika   metabolismus   MeSH
• fermentace MeSH
• genetická transkripce * MeSH
• kovy metabolismus   MeSH
• metabolismus sacharidů genetika   MeSH
• regulace genové exprese u bakterií genetika   MeSH
• sacharidy genetika   MeSH
• vitaminy genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• 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

Biodiesel is a mixture of esters of fatty acids (most often palmitic, stearic and oleic) and lower alcohols (in our work butanol) produced by transesterification. It is a renewable source of energy, prepared from triacylglycerides, which are contained in vegetable oils and animal fats. This work focuses on alkaline catalyzed transesterification of rapeseed oil with butanol and comparison of two catalysts (potassium hydroxide and potassium tert-butoxide). In industry is usually transesterification of rapeseed oil carried out like reaction catalyzed by potassium hydroxide. Potassium hydroxide have high content of K2CO3, KHCO3 and water. Moreover water is formed by neutralization of potassium hydroxide with free fatty acids contained in oil. In cause of tert-butoxide catalyzed reaction, it is not possible because tert-butoxide have not a OH- aniont, which is needed for water forming. The influence of various conditions (addition of water, temperature of separation, intensity of stirring and type of catalyst) on butanolysis process was studied for both catalysts. For both catalysts dependence of conversions on time were plotted. When tert-butoxide was used, satisfactory phase separation was not achieved. The only way was separation of hot crude reaction mixture without adding water. Ester formed by this method had high content of free glycerol and soaps, but reached higher conversion. The best results were obtained with KOH and subsequent separation of cold crude reaction mixture with the addition of water and slow stirring. The difference between reactions catalyzed by potassium hydroxide and potassium tert-butoxide was described.

• MeSH
• biopaliva * MeSH
• butanoly * MeSH
• hydroxidy * MeSH
• katalýza MeSH
• kyseliny mastné mononenasycené MeSH
• olej z řepky MeSH
• oleje rostlin MeSH
• sloučeniny draslíku * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Modern vehicles equipped with Gasoline Direct Injection (GDI) engine have emerged as an important source of particulate emissions potentially harmful to human health. We collected and characterized gasoline exhaust particles (GEPs) produced by neat gasoline fuel (E0) and its blends with 15% ethanol (E15), 25% n-butanol (n-But25) and 25% isobutanol (i-But25). To study the toxic effects of organic compounds extracted from GEPs, we analyzed gene expression profiles in human lung BEAS-2B cells. Despite the lowest GEP mass, n-But25 extract contained the highest concentration of polycyclic aromatic hydrocarbons (PAHs), while i-But25 extract the lowest. Gene expression analysis identified activation of the DNA damage response and other subsequent events (cell cycle arrest, modulation of extracellular matrix, cell adhesion, inhibition of cholesterol biosynthesis) following 4 h exposure to all GEP extracts. The i-But25 extract induced the most distinctive gene expression pattern particularly after 24 h exposure. Whereas E0, E15 and n-But25 extract treatments resulted in persistent stress signaling including DNA damage response, MAPK signaling, oxidative stress, metabolism of PAHs or pro-inflammatory response, i-But25 induced changes related to the metabolism of the cellular nutrients required for cell recovery. Our results indicate that i-But25 extract possessed the weakest genotoxic potency possibly due to the low PAH content.

• MeSH
• benzin analýza   toxicita   MeSH
• biopaliva analýza   toxicita   MeSH
• buněčné linie MeSH
• butanoly analýza   toxicita   MeSH
• ethanol chemie   MeSH
• genetická transkripce účinky léků   MeSH
• látky znečišťující vzduch analýza   toxicita   MeSH
• lidé MeSH
• MAP kinasový signální systém účinky léků   MeSH
• organické látky chemie   toxicita   MeSH
• oxidační stres účinky léků   MeSH
• pevné částice toxicita   MeSH
• plíce účinky léků   patologie   MeSH
• polycyklické aromatické uhlovodíky analýza   toxicita   MeSH
• poškození DNA MeSH
• stanovení celkové genové exprese MeSH
• výfukové emise vozidel analýza   toxicita   MeSH
• zánět chemicky indukované   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biofilmy růst a vývoj   MeSH
• buněčná membrána účinky léků   MeSH
• buněčná stěna účinky léků   MeSH
• butanoly metabolismus   toxicita   MeSH
• Clostridium účinky léků   genetika   metabolismus   MeSH
• fylogeneze MeSH
• genom bakteriální MeSH
• mutageneze MeSH
• průmyslová mikrobiologie metody   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The production of acetone, butanol and ethanol by fermentation of renewable biomass has potential to become a valuable industrial process. Mechanisms of solvent production and sporulation involve some common regulators in some ABE-producing clostridia, although details of the links between the pathways are not clear. In this study, we compare a wild-type (WT) Clostridium beijerinckii NRRL B-598 with its mutant strain OESpo0A, in which the gene encoding Spo0A, an important regulator of both sporulation and solventogenesis, is overexpressed in terms of solvent and acid production. We also compare morphologies during growth on two different media: TYA broth, where the WT culture sporulates, and RCM, where the WT culture does not. In addition, RT-qPCR-based analysis of expression profiles of spo0A, spoIIE, sigG, spoVD, ald and buk1 genes involved in sporulation or solvent production in these strains, were compared. The OESpo0A mutant did not produce spores and butanol titre was lower compared to the WT, but increased amounts of butyric acid and ethanol were produced. The gene spo0A had high levels of expression in the WT under non-sporulating culture conditions while other selected genes for sporulation factors were downregulated significantly. Similar observations were obtained for OESpo0A where spo0A overexpression and downregulation of other sporulation genes were demonstrated. Higher expression of spo0A led to higher expression of buk1 and ald, which could confirm the role of spo0A in activation of the solventogenic pathway, although solvent production was not affected significantly in the WT and was weakened in the OESpo0A mutant.

• MeSH
• aceton metabolismus   MeSH
• bakteriální proteiny genetika   MeSH
• butanoly metabolismus   MeSH
• Clostridium beijerinckii genetika   metabolismus   MeSH
• ethanol metabolismus   MeSH
• fermentace MeSH
• mutace MeSH
• polymerázová řetězová reakce MeSH
• regulace genové exprese u bakterií MeSH
• rozpouštědla metabolismus   MeSH
• spory bakteriální růst a vývoj   MeSH
• transkripční faktory genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Flow cytometry, in combination with fluorescent staining, was used to evaluate population heterogeneity in acetone-butanol-ethanol fermentation that was carried out with type strain Clostridium beijerinckii NCIMB 8052 and non-type C. pasteurianum NRRL B-598. A combination of propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), PI plus Syto-9 and bis-oxonol (BOX) alone were employed to distinguish between active and damaged cells together with simultaneous detection of spores. These strategies provided valuable information on the physiological state of clostridia. CFDA and PI staining gave the best separation of four distinct subpopulations of enzymatically active cells, doubly stained cells, damaged cells and spores. Proportional representation of cells in particular sub-regions correlated with growth characteristics, fermentation parameters such as substrate consumption and product formation in both species under different cultivation conditions.

• MeSH
• aceton metabolismus   MeSH
• biomasa MeSH
• bioreaktory MeSH
• butanoly metabolismus   MeSH
• Clostridium fyziologie   MeSH
• energetický metabolismus * MeSH
• ethanol metabolismus   MeSH
• fermentace * MeSH
• glukosa metabolismus   MeSH
• mikrobiální viabilita MeSH
• průtoková cytometrie MeSH
• spory bakteriální * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH