Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions
Abstract 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 an...
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| Format: | Article |
| Language: | English |
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BMC
2018-04-01
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| Series: | Biotechnology for Biofuels |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13068-018-1096-x |
| _version_ | 1811336946325127168 |
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| author | Barbora Branska Zora Pechacova Jan Kolek Maryna Vasylkivska Petra Patakova |
| author_facet | Barbora Branska Zora Pechacova Jan Kolek Maryna Vasylkivska Petra Patakova |
| author_sort | Barbora Branska |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-13T17:46:56Z |
| format | Article |
| id | doaj.art-6e60dd561b07480b9da15a339e363c7b |
| institution | Directory Open Access Journal |
| issn | 1754-6834 |
| language | English |
| last_indexed | 2024-04-13T17:46:56Z |
| publishDate | 2018-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels |
| spelling | doaj.art-6e60dd561b07480b9da15a339e363c7b2022-12-22T02:36:55ZengBMCBiotechnology for Biofuels1754-68342018-04-0111111610.1186/s13068-018-1096-xFlow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditionsBarbora Branska0Zora Pechacova1Jan Kolek2Maryna Vasylkivska3Petra Patakova4Department of Biotechnology, University of Chemistry and Technology PragueDepartment of Biotechnology, University of Chemistry and Technology PragueDepartment of Biotechnology, University of Chemistry and Technology PragueDepartment of Biotechnology, University of Chemistry and Technology PragueDepartment of Biotechnology, University of Chemistry and Technology PragueAbstract 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.http://link.springer.com/article/10.1186/s13068-018-1096-xCytometryClostridiumFluorescence stainingViabilityABE fermentationButanol |
| spellingShingle | Barbora Branska Zora Pechacova Jan Kolek Maryna Vasylkivska Petra Patakova Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions Biotechnology for Biofuels Cytometry Clostridium Fluorescence staining Viability ABE fermentation Butanol |
| title | Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions |
| title_full | Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions |
| title_fullStr | Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions |
| title_full_unstemmed | Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions |
| title_short | Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions |
| title_sort | flow cytometry analysis of clostridium beijerinckii nrrl b 598 populations exhibiting different phenotypes induced by changes in cultivation conditions |
| topic | Cytometry Clostridium Fluorescence staining Viability ABE fermentation Butanol |
| url | http://link.springer.com/article/10.1186/s13068-018-1096-x |
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