Solventogenic
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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
Physiological changes in populations of Clostridium beijerinckii and Clostridium tetanomorphum were monitored by fluorescence staining and flow cytometry. To estimate the number of metabolically active cells in exponential growth, a combination of the dyes propidium iodide and carboxy fluorescein diacetate appeared to be a good choice for both species. During stationary phase, these stains did not reflect physiological changes sufficiently and therefore additional labeling with bis-(1,3-dibutylbarbituric acid) trimethineoxonol was applied. Results of fluorescence staining in solventogenic batch fermentations were compared with substrate-use data, the concentration of key metabolites and growth curves. We demonstrate that measurements by all methods were mutually compatible.
- MeSH
- barbituráty MeSH
- barvení a značení metody MeSH
- bioreaktory MeSH
- Clostridium fyziologie ultrastruktura MeSH
- fermentace * MeSH
- fluoresceiny MeSH
- fluorescenční barviva MeSH
- isoxazoly MeSH
- propidium MeSH
- průtoková cytometrie MeSH
- techniky vsádkové kultivace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
V posledních desetiletích je snahou Evropského společenství snížit závislost svých členských států na ropných zdrojích, což vede k zájmu O procesy, které využívají obnovitelné zdroje energií. Jedním z nich je proces výroby 1-butanolu, který je realizován pomocí různých solventogenních druhů klostridií. Butanol se fermentačním způsobem produkuje při aceton-butanol-ethanolové fermentaci, kdy v první fázi procesu dochází k tvorbě organických kyselin a v druhé fázi procesu, často spojené se sporulací produkčního kmene, se tvoří rozpouštědla. Dvoufázový charakter fermentace výrazně komplikuje regulaci procesu a toxicita produktů brání dosažení jejich vysoké koncentrace. Aby byla zvýšena efektivita tohoto procesu, je prováděn intenzivní výzkum, zaměřený hlavně na zlepšení produkčních vlastností kmenů, zvýšení jejich odolnosti k 1-butanolu, využití levných surovin a zvýšení celkové produktivity procesu. Pokud se podaří výrobní cenu biobutanolu snížit, mohl by díky svým výhodným vlastnostem sloužit jako kosolvent do motorového paliva, do kterého se dnes již přidává ethanol a oddálit tak nevyhnutelné vyčerpání fosilních paliv.
An effort of European Community to reduce its dependence on fossil fuels provokes increased interest on processes using renewable energy resources. One of them is the fermentation process of 1-butanol production using various types of solventogenic clostridia. Biobutanol is produced by aceton-butanol-ethanol fermentation process consisting of two phases - acidogenic and solventogenic, which is associated with sporulation. Biphasic nature of fermentation significantly complicates the process control and product toxicity prevents the achievement of high concentration of solvents. To increase the efficiency of this process, an inventive research focused on improving the production characteristics of strains, increasing their resistance to 1-butanol, the use of cheap raw materials and amelioration of overall productivity of the process is carried out. If the production cost of biobutanol would be reduced, it could be advantageously used as an additive to petrol which could postpone the depletion of fossil fuels.
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
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
Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels.
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
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
- bakteriální proteiny genetika metabolismus MeSH
- Clostridium beijerinckii cytologie genetika MeSH
- fermentace genetika MeSH
- fyziologický stres * genetika MeSH
- glukosa metabolismus MeSH
- kyseliny metabolismus MeSH
- mastné kyseliny metabolismus MeSH
- proteiny teplotního šoku genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- rozpouštědla metabolismus MeSH
- spory bakteriální metabolismus MeSH
- transkriptom genetika MeSH
- vodík metabolismus 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
