Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions

Abstract

BackgroundBiobutanol 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.ResultsDual 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.ConclusionWe 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.

Metabolic Engineering of Histidine Kinases in Clostridium beijerinckii for Enhanced Butanol Production

“…In addition, overexpression of spo0A in C. beijerinckii NRRL B-598, sharing high genome homology with C. beijerinckii NCIMB 8052, led to cessation of production at a low ABE concentration, indicating that Spo0A is a master regulator of solventogenesis in C. beijerinckii (Kolek et al, 2017). Furthermore, without spore formation, C. beijerinckii NRRL B-598 could produce more butanol in RCM medium, compared with the sporulating phenotype (Branska et al, 2018). Therefore, we speculated that Cbei2073 and Cbei4484 played a role on phosphorylation of Spo0A due to their high similarity in sequence with Cac3319, Cac0903, and Cac0323 and further regulated both butanol synthesis and sporulation frequency.…”

Matrices (re)loaded: Durability, viability, and fermentative capacity of yeast encapsulated in beads of different composition during long‐term fed‐batch culture

“…42 The lower viability reported here may stem from cells being subjected to repeated cycles of feast and famine, each of which 83,84 Since the S. cerevisiae strain used here is purported to be tolerant of ethanol up to 18%, 59 it is unlikely that a maximum theoretical concen- 100 as well as an association between lower viability and pH changes. 101 3.7 | Encapsulated cells are more heat-shock resistant than planktonic cells, but no one matrix confers greater heat-shock resistance than another Simultaneous saccharification and fermentation has been widely implemented in the bioethanol industry due to its higher efficiency, 102,103 even though most enzymes operate optimally well above yeasts' preferred growing temperatures. 104,105 Thus, a more thermotolerant yeast could improve the efficiency of this step.…”

Experimental Design to Improve Cell Growth and Ethanol Production in Syngas Fermentation by Clostridium carboxidivorans

“…However, not all species and strains of solventogenic clostridia have the same behavior. Lipovsky et al [31] and Branska et al [32] verified butanol production associated with cell growth by clostridia via the acetone-butanol-ethanol (ABE) pathway.…”