ABE fermentace je proces, kterým lze vyrábět organická rozpouštědla. Nejvíce ceněným produktem s největším rozsahem uplatnění je butanol, který může být použit, jako alternativní palivo či příměs pohonných hmot, ale také je to důležitá základní surovina pro výrobu chemikálií. V současnosti je veškerý butanol vyráběn z ropy, kvůli výrazně nižší ceně výroby. V budoucnu by se však ABE fermentace mohla stát opět důležitou technologií k výrobě organických rozpouštědel. Výrazné úspory by mohlo být docíleno využitím alternativních substrátů, kterými mohou být např. surový glycerol, lignocelulózové hydrolyzáty, plynné substráty, řasová biomasa a další.
ABE fermentation is process which could be used for organic solvents production. Butanol is the most valuable product of this process. It can be use as alternative biofuel, its additive and also as raw material for chemical industry. Nowadays, whole butanol production is achieved through the refining of oil because of its lower price. In the future ABE could be important for organic solvents production again. Using of alternative substrates could lead to considerable savings in ABE fermentation. These substrates are for example: crude glycerol, lignocellulosic substrates, algae biomass etc.
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
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
This review emphasises the fact that studies of acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia cannot be limited to research on the strain Clostridium acetobutylicum ATCC 824. Various 1-butanol producing species of the genus Clostridium, which differ in their patterns of product formation and abilities to ferment particular carbohydrates or glycerol, are described. Special attention is devoted to species and strains that do not produce acetone naturally and to the utilisation of lactose, inulin, glycerol and mixtures of pentose and hexose carbohydrates. Furthermore, process-mapping tools based on different principles, including flow cytometry, DNA microarray analysis, mass spectrometry, Raman microscopy, FT-IR spectroscopy and anisotropy of electrical polarisability, which might facilitate fermentation control and a deeper understanding of ABE fermentation, are introduced. At present, the methods with the greatest potential are flow cytometry and transcriptome analysis. Flow cytometry can be used to visualise and capture cells within clostridial populations as they progress through the normal cell cycle, in which symmetric and asymmetric cell division phases alternate. Cell viability of a population of Clostridium pasteurianum NRRL B-598 was determined by flow cytometry. Transcriptome analysis has been used in various studies including the detection of genes expressed in solventogenic phase, at sporulation, in the stress response, to compare expression patterns of different strains or parent and mutant strains, for studies of catabolite repression, and for the detection of genes involved in the transport and metabolism of 11 different carbohydrates. Interestingly, the results of transcriptome analysis also challenge our earlier understanding of the role of the Spo0A regulator in initiation of solventogenesis in C. acetobutylicum ATCC 824. Lastly, the review describes other significant recent discoveries, including the deleterious effects of intracellular formic acid accumulation in C. acetobutylicum DSM 1731 cells on the metabolic switch from acidogenesis to solventogenesis and the development of a high-cell density continuous system using Clostridium saccharoperbutylacetonicum N1-4, in which 1-butanol productivity of 7.99 g/L/h was reached.
- MeSH
- aceton metabolismus MeSH
- butanoly metabolismus MeSH
- Clostridium cytologie genetika metabolismus MeSH
- ethanol metabolismus MeSH
- fermentace * MeSH
- glycerol metabolismus MeSH
- hexosy metabolismus MeSH
- inulin metabolismus MeSH
- laktosa metabolismus MeSH
- pentosy metabolismus MeSH
- průtoková cytometrie MeSH
- Ramanova spektroskopie MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
