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.
Highlights
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
ABE fermentation with a sporulating phenotype Clostridium beijerinckii grown on tryptone-yeast extract-acetate medium (TYA) medium showed a typical bi-phasic ABE fermentation, starting with the formation of organic acids and subsequent production of solvents accompanied by glucose consumption and partial reutilization of the organic acids (Fig. 1) took place
C. beijerinckii NRRL B-598 used in this study reduced growth rate shortly before the pH breakpoint but continued in growth after commencement of solventogenesis
Summary
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. The main bottlenecks in the process are low butanol yields and final concentration, which increase the final product cost. Other approaches include removal of butanol in situ from the medium [8, 16,17,18], adaptation [19, 20] or engineering of strains [21,22,23,24,25] for higher tolerance, production and improved yield by altering metabolite flux (e.g. by reduction of by-product formation via disruption of respective genes [26]), enhancement of expression of genes responsible for solvent formation [27] or by totally suppressing acid formation and directly stimulating butanol formation from saccharides [28]. Many experiments have revealed that an improved ability of clostridia to tolerate/ survive higher concentrations of butanol might not necessarily be commensurate with increased production [29, 30]
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