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Abstract
Clostridium pasteurianum ATCC 6013 achieves high n-butanol production when glycerol is used as the sole carbon source. In this study, the homeoviscous membrane response of C. pasteurianum ATCC 6013 has been examined through n-butanol challenge experiments. Homeoviscous response is a critical aspect of n-butanol tolerance and has not been examined in detail for C. pasteurianum. Lipid membrane compositions were examined for glycerol fermentations with n-butanol production, and during cell growth in the absence of n-butanol production, using gas chromatography–mass spectrometry (GC–MS) and proton nuclear magnetic resonance (1H-NMR). Membrane stabilization due to homeoviscous response was further examined by surface pressure–area (π–A) analysis of membrane extract monolayers. C. pasteurianum was found to exert a homeoviscous response that was comprised of an increase lipid tail length and a decrease in the percentage of unsaturated fatty acids with increasing n-butanol challenge. This led to a more rigid or stable membrane that counteracted n-butanol fluidization. This is the first report on the changes in the membrane lipid composition during n-butanol production by C. pasteurianum ATCC 6013, which is a versatile microorganism that has the potential to be engineered as an industrial n-butanol producer using crude glycerol.
Highlights
Clostridium pasteurianum, an anaerobic spore-forming firmicute, ferments glycerol as the sole substrate, resulting in a mixture of butanol, ethanol, and 1,3-propanediol (PDO) along with acetate and butyrate [1,2,3,4]
For investigating the effect of butanol, exogenous butanol was added to the media varied from 0 to 1% (w/v) (0 gL-1 to 10 gL-1) containing either glycerol (25 gL-1) or glucose (50 gL-1) in Biebl media [6] were used to study the effect of exogenous butanol during an active butanol biosynthesis on glycerol and its absence when grown in glucose, as C. pasteurianum does not produce butanol when growing on glucose
The homeoviscous response of C. pasteurianum to the addition of exogenous butanol was studied under two conditions, where the first condition involved the addition of exogenous butanol during the production of butanol by C. pasteurianum during glycerol fermentation (EB1), while the second condition involved the addition of exogenous butanol while no butanol was produced, during the fermentation of glucose (EB2)
Summary
Clostridium pasteurianum, an anaerobic spore-forming firmicute, ferments glycerol as the sole substrate, resulting in a mixture of butanol, ethanol, and 1,3-propanediol (PDO) along with acetate and butyrate [1,2,3,4]. Solvents with a log P value less than 4 partitions into the lipid membrane bilayer and are considered extremely toxic. When Clostridia are exposed to solvents, the solvents exhibit a fluidizing effect on the phospholipid bilayer, which causes the organism to alter the lipid composition of the bilayer This response of bacteria to tolerate toxic solvents by altering the composition of the lipid bilayer is known as homeoviscous response (membrane viscosity is proportional fluidity). To compensate for the fluidizing effects of butanol, Clostridia increase the ratio of saturated to unsaturated fatty acids (SFA/UFA) in the lipid membrane, thereby reducing the fluidity of the membrane and increasing butanol tolerance [6,7,8,9,10,11,12,13,14,15,16]. It has been established that lipid composition and distribution on cell membrane play an essential role in maintaining membrane stability, curvature and membrane fluidity and in modulating protein function and insertion on the membranes.[17,18,19] Membrane fluidity is essential for maintaining the proper distribution and diffusion of embedded proteins in membrane.[20]
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