Homeoviscous response of Clostridium pasteurianum to butanol Homeoviscous response of Clostridium pasteurianum to butanol toxicity during glycerol fermentation toxicity during glycerol fermentation

: 17 Clostridium pasteurianum has been shown to ferment glycerol into butanol at higher 18 yields than when sugars are used as the carbon source. C. pasteurianum’ s potential to use 19 biodiesel-derived crude glycerol as the carbon source has been gaining importance in the recent 20 past. This study investigated the homeoviscous response of C. pasteurianum during butanol 21 stress. C. pasteurianum ’s lipid composition of the plasma membrane during butanol challenge 22

Butanol is toxic to cells, as it partitions into the cell membrane and affects both the structural and functional integrity of the cell.The extent of solvent toxicity correlates to the log P value.Solvents with a log P value less than 4 partitions into the lipid membrane bilayer and are considered extremely toxic.Butanol has a log P value of 0.8 and is considered to be one of the most toxic solvents (5).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).The fluidity of the lipid membrane is directly proportional to the amount of saturated fatty acids in the tail of the lipid bilayer (11).Hence, the bacteria that tolerate more butanol have a much higher SFA/UFA ratio in the lipid bilayer (11).This has been observed to be an essential biophysical process of tolerating butanol in various butanol producing organisms from the genus Clostridium (6-8, 10, 12, 13).It has been established that lipid composition and distribution on cell membrane play an essential role not only in maintaining membrane stability, curvature and membrane fluidity but also 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) Membrane lipid composition changes in response to alcohol toxicity by increasing the distribution of unsaturated fatty acids observed in Lactobacillus heterohiochii, Lactobacillus homohiochii, Escherichia coli, and Saccharomyces cereeisiae.(21)(22)(23) However, an opposite effect (decreasing in the amount of saturated fatty acids) in response to alcohol toxicity is also observed in several other microorganisms; Clostridium acetobutylicum (10) and Bacillus subtilis.Timmons et al has studied a comparison of wild-type and ethanol-adapted (EA) Clostridium thermocellum.(24) EA cells preserve the optimum level of fluidity in response to ethanol toxicity with increasing the fatty acids chain lengths of lipid tails and decreasing the unsaturation index in the cell membrane; resulting in higher membrane rigidity (10).
Butanol has been shown to affect the membrane by increasing fluidity and hence reducing lipid ordering (10,(25)(26)(27).Also, butanol's toxic effects lead to the formation of interdigitated phases and phase separation (25,28).Overall, butanol can compromise cellular function of the membrane by altering cell fission, fusion, budding, vesicle formation and cell signaling (28,29).The passive and active transport of substrates and products is also affected, along with the structure and function of integral membrane proteins.This can hinder the ability of the cell to maintain an internal pH and inhibits membrane-bound ATPases and the uptake of glucose (if present), which subsequently inhibits energy generation (9).Membrane bound ATPases are one such examples, which maintain a transmembrane pH for ATP generation.
Butanol inhibits the ATPases and reduces the transmembrane pH resulting in lower ATP formation (30).
To our knowledge, the surface mechanical properties of butanol-tolerant cell membrane producing solvents have not yet been examined.In this study, the membrane extracts of Clostridia Pasteurianum exposed to exogenous addition of different butanol concentrations were used to investigate membrane composition, membrane phase behavior, and membrane fluidization.Materials and Methods

Bacterial Strain
The bacterial strain C. pasteurianum ATCC 6013 was purchased from American Type Culture Collection and the glycerol stock was maintained as described earlier [8].

Effect of n-Butanol
The effect of n-butanol (n-butanol was used throughout all experiments) on the bacterial growth and the stability and change in composition of the membrane were studied by adding butanol to the media containing either glucose or glycerol as the sole carbon source.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 cells were allowed to grow in the presence of butanol for 24 hours after which the membrane was extracted.All experiments were conducted with 10% (v/v) inoculum, pre-grown in RCM.

Extraction of the Cell Membrane
Cell membranes were extracted using the modified protocol of Bligh and Dyer using dichloromethane/methanol mixtures [29].The cells were harvested (0.5 mL cell suspension) by centrifugation at 13000 rpm for 15 minutes and the pellets were resuspended in 0.5 mL of sterile 1.0% (w/v) NaCl in a 10 mL glass sample tube with PTFE lined caps.To the resuspended pellets, 2 mL of dichloromethane/methanol mixture (1:2 v/v) was added and shaken vigorously for 15 minutes.It was followed by a 2 hour incubation at RT, followed by centrifugation at 2500 rpm and the supernatant (S1) was collected in a fresh tube.The pellet was again resuspended in 0.5 mL 1.0% NaCl and 2 mL of a dichloromethane/methanol mixture (2:1 v/v) was added to the resuspended pellet and shaken vigorously for 15 minutes.Following a 2 hour incubation, the samples were centrifuged at 2500 rpm and the supernatant (S2) was collected.Supernatants S1 and S2 were combined and 1 mL of dichloromethane and 1 mL of sterile 1.0% NaCl were added.
The top phase (aqueous) was removed and the bottom (organic) phase was retained.The solvent was evaporated under a gentle nitrogen stream.Once a dry film was obtained, the headspace was flushed with nitrogen, capped tightly, and stored for further analysis.

NMR Analysis
The dry film of the membrane was dissolved in CDCl3 for 1 H-NMR analysis.Synthetic lipids, DPPC and DOPC were used as standards for NMR analysis.Various ratios of DPPC and DOPC (1:0, 3:1, 1:1, 1:3 and 0:1) in a total lipid concentration of 10 mM were used for calibration (Supplementary Material and Figure S1).The synthetic lipids were dried and dissolved in CDCl3.All 1 H-NMR spectra were recorded on a Varian TM Unity Inova 500 (500 MHz) spectrometer equipped with a 5 mm triple resonance inverse detectable probe.The percentage of unsaturation in the membrane lipid samples was calculated by multiplying the ratio of the intensity of the peak corresponding to the olefinic hydrogen (at 5.31 ppm) to total intensity of all the peaks from the lipid sample by 100 (Eq 1) [30].LS 55) and melting temperature were measured using the L-format configuration with DPH as the hydrophobic bilayer probe from 25 to 50°C at a rate of 1°C/min under continuous mixing as described previously (25).

GC/MS Analysis
The lipid samples were methylated for GC/MS analysis.The dried lipid samples were saponified using 1 mL of 3N sodium hydroxide at 90ºC for an hour and then cooled to RT.The excess sodium hydroxide was neutralized with 1.8 mL of 3.6 N hydrochloric acid at 90 ºC for 10 minutes and cooled to RT.The free fatty acids were extracted using 1 mL of hexane and diethyl ether (1:1 v/v).The organic phase was then separated into a clean and dry round bottom flask.
The fatty acid hydrolyzates were dried using a rotoevaporator and stored under Argon in desiccators.
The dried fatty acid hydrolyzates were derivatized using a 5 mL borontrifluoridemethanol complex at 60 ºC for 5 minutes and then cooled to RT.To the cooled solution, 1 mL water and 1 mL hexane were added and the container shaken multiple times to ensure the transfer of esters into the non-polar solvent.The upper organic layer was removed and transferred into an Erlenmeyer flask conataining 5 g of anhydrous sodium sulfate.The flask was incubated at RT to dry overnight.The sodium sulfate was filtered and the hexane solution was transferred into a clean round bottom flask and dried in a rotoevaporator and the samples were stored under Argon.For GC-MS analysis, samples were dissolved in 250 µL of dichloromethane.
Fatty acid methyl esters (FAME) derivatives were analyzed by a Shimadzu GC-MS QP 2010 system using a SHR5xLB silica capillary column (30m× 0.25mm ID, composed of 100% dimethyl polysiloxane).Manufacturers protocol were followed for FAME analysis.TheThe chloroform was allowed to evaporate and the lipid films to spread at air/water for 15 min.
The system was the equilibrated for 15 min before being compressed at a speed of 10 cm 2 /min.

Elastic modulus / area compressibility modulus
Mechanical properties of the monolayer films were determined by the compressibility modulus.The elastic modulus ( / area compressibility modulus is the reciprocal of the compressibility ( .(31) The elastic modulus corresponds to the elasticity of the Langmuir films under the compression force.values were defined as: where, A is the area per molecule at the particular surface pressure and Π is the corresponding surface pressure.The maximum compressibility or elasticity ( ) provides information on the onset of the plastic region, and the maximum packing condition of monolayer.

Results and Discussion
Clostridial species produce various metabolites in the form of acids and solvents, and have to modify the composition of their lipid membranes to tolerate the toxic effects of the produced metabolites.The fundamental studies to identify the effect of the toxic metabolites on the lipid composition of during the homeoviscous response can be investigated using the tools of chemical biology.This study is focused on investigating the effect of butanol in C. pasteurianum leading to the tolerance response involving changes in the lipid membrane composition using tools to analyze the physical and structural compositions of the membranes along with the membrane phase behavior.

Effect of Exogenous Butanol
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).C. pasteurianum cultures do not undergo solventogenesis during the fermentation of glucose, as the fermentation is predominantly only in the acidogenic phase, resulting in butyric acid as the major fermentation product with no butanol formation.
Butanol was added at concentrations of 0 g/L (control), 2.5 g/L (0.0335 M), 5 g/L (0.067 M), 7. The data from GC-MS on the fatty acid composition (Figure 2) of the membranes corroborates the presence of the homeoviscous response to butanol.Furthermore, there was an increase in the percentage of higher carbon saturated fatty acids (C19 to C22), which are almost completely absent in the control samples with no external butanol present.A similar trend was also observed in the disappearance or reduction in the shorter chain fatty acids with an increase in the dose of butanol stress (Figure 2).The shorter length fatty acids C10, were completely absent in the cells exposed to higher butanol concentrations, while the other shorter fatty acids from C11 to C16 were found to decrease proportionately with increase in butanol dose.Similarly, the longer fatty acids, C19 to C22, were not observed in the control which was not exposed to butanol and had a proportionate increase in the percentage with corresponding increase in the butanol dosage.The compositional analysis through GC-MS not only supports the results from NMR and fluorescence anisotropy, but also substantiates that C. pasteurianum exerts a homeoviscous response to butanol in EB1 condition by two major changes in the fatty acid composition to counteract the fluidity of the toxic butanol.First, it increases the percentage of longer chain fatty acids at the expense of shorter chain fatty acids and secondly by increasing the ratio of saturated to unsaturated fatty acids.
The effect of butanol on the lipid composition and the fluidity of the lipid membrane has also been reported for C. acetobutylicum (6,7,10,12).C. acetobutylicum produces butanol by fermenting glucose and the effect of butanol challenge was studied during the growth of C. acetobutylicum in glucose (butanol producing media) (6,7,10,12).C. acetobutylicum tolerates butanol through a homeoviscous response that predominantly involves an increase of saturated fatty acids at the expense of unsaturated fatty acids in the lipid membrane (6,7,10,12).Lepage et al reported the composition of the C. acetobutylicum's fatty acid composition in the lipid membrane, which consisted of fatty acids from C12 to C19 (10).The ratio of the unsaturated to saturated fatty acids was found to be close to 1 without any butanol exposure but the ratio was reduced to 0.87 and 0.77 respectively with an exposure to 4 g/L and 8 g/L butanol respectively, due to the reduction in unsaturation and an increase in saturated fatty acids in the membrane lipids, driven by a homeoviscous response.
As a control, the change in the degree of unsaturation and anisotropy of the lipid membrane during the production of endogenous butanol was studied and compared (Supplementary material and Figure S2 and S3).The addition of butanol during glycerol fermentation results in a similar result as observed during the endogenous butanol production.
The only change was observed in the anisotropy data which accounts for the fluidizing effect of the exogenous butanol on the membrane.The difference observed in the anisotropy data is consistent with the previously reported fluidizing effect of butanol observed through fluorescence anisotropy of synthetic lipids and reconstituted membrane lipids (8,25).

The Differential Response to Butanol Toxicity
The effect of butanol during EB1 showed a conventional homeoviscous response by increasing the fatty acid length and the ratio of saturated to unsaturated fatty acids.To further investigate the sole effect of butanol toxicity the cells grown in glucose were exposed to butanol (EB2).Intially, the EB2 experiment was conducted to match the same butanol stress dosage from 0 g/L to 10 g/L but was also performed at higher concentrations of butanol of up to 20 g/L.
As explained earlier, the extracted membranes were studied using 1 H-NMR and fluorescent anisotropy.Figure 3 summarizes the change in the fluidity of the membrane in terms of anisotropy and the degree of unsaturation in the membranes of C. pasteurianum grown on glucose.The 1 H-NMR results indicated an increase in the percentage of unsaturation in the fatty acid tails of the lipid membranes extracted from the cells stressed with exogenous butanol.This result is completely contrasting to the results obtained earlier during butanol stress on glycerol fermentation.The degree of unsaturation in the lipid membranes increased proportionately with increasing concentration of butanol in the media.For an exogenous butanol concentration of 5 g/L, a small drop in the percentage of unsaturation in the fatty acid tail was observed when compared to the control with no external butanol.The data obtained from fluorescent anisotropy of the reconstituted membrane, using DPH as the probe, supported the data obtained from 1 H-NMR (Figure 3).The anisotropy, <r>, measured at 37°C decreased gradually with an increase in the concentration of butanol in the media.
The lipid membranes extracted from the cells were also analyzed by GC-MS to determine the constituent fatty acids.Figure 3 summarizes the results from GC-MS analysis of the cells stressed with exogenous butanol.Thirteen different fatty acids were identified using GC-MS, of which 11 were saturated.The data on the composition of the fatty acids from Figure 4 shows not only an increase in the degree of unsaturation in the fatty acids, but also an increase in the percentage of fatty acid length (≥ C16) with an increase in the concentration of exogenous butanol.
The degree of unsaturation was found to increase (Figure 3 and 4) in the presence of exogenous butanol, but without endogenous butanol production (cells gown on glucose).An increase in unsaturation coincided with an increase in membrane fluidity (Figure 4).This is in contrast with the response observed for EB1(Figure 2).
It has been shown that an increase in the percentage of saturated lipids in the model liposomes (model membranes from DPPC and DOPC) results in an increase in the anisotropy of the liposomes (25).Furthermore, the addition of butanol to model membranes comprised of DPPC, DOPC or mixture of the two have shown a decrease in anisotropy due to the fluidizing effects of butanol on the lipid membrane (25,29).Butanol fluidizes membranes by reducing inter lipid interactions and the surface tension within the membrane.Increase in the degree of unsaturation in lipid membranes has been shown to augment the fluidizing effects of butanol (25).Hence, the results obtained from the addition of exogenous butanol to the cells of C. pasteurianum in EB2 contradict the results obtained during EB1.This led to the question of whether other non-lipid entities could be involved in the homeoviscous adaptation of the membrane in EB2.
The fatty acid composition of the cells also varied considerably when the cells are grown on glycerol and glucose .The cells grown on glucose and exposed to exogenous butanol show neither a decrease in shorter (C6 to C10) fatty acids nor an increase in longer (C19 and greater) fatty acids (Figure 4).The fatty acid composition of the cells are also different under the two condition, glucose and glycerol fermentation.The fatty acid chain length is as low as 6 carbons during glucose fermentation, while the the lowest fatty acid in glycerol fermentation is 10.
Similar distinction is also observed in the maximum chain length as well for the two carbon sources.Moreover, the percentage of unsaturated fatty acids increased with increasing concentrations of exogenous butanol during EB2 (Figure 4).
The reduction in shorter chain fatty acids and an increase in longer chain fatty acids in C.
acetobutylicum resulted in a decrease of membrane fluidity (6,10).It was also observed in C.
acetobutylicum that challenging the cells with butanol resulted in the formation of longer chain fatty acids at the expense of shorter chain fatty acids (6,10).
The increase in unsaturation (exogenous butanol on glucose) observed with all three different analytical methods cannot be explained by a homeoviscous adaptation, as it led to an increase in the fluidity of the membrane consistent with the toxic effect of butanol (Figure 3 and    4).However, an increase in unsaturation in the lipid membrane of cells exposed to solvent stress has been reported for E. coli during ethanol stress and for C. butyricum during 1,3-PDO stress (14,32).Dombek and Ingram reported that the plasma membrane became more rigid during ethanol challenge experiments, but the extracted lipid membrane exhibited higher fluidity in comparison to the control cells that were unexposed to ethanol.This shows that the rigidity of the membrane is not only dependent on the ratio of saturated to unsaturated fatty acids, or the presence of shorter or longer fatty acids, but is also dependent on the lipid to protein ratio in the membrane (34).Membrane proteins can rigidify the membrane despite irrespective of the lipid composition.Hence, an increase in protein:lipid ratio can account for the net increase in the rigidity of the membrane and compensates for the increase in fluidity as a result of the increased unsaturation of the lipid tails (Figure 3).
During the formation of butanol (EB1), the bacteria must be synthesizing butanol efflux pumps in the membrane that serve as butanol transporters to the extracellular environment (33).
Dunlop et al. have shown that cloning and expressing of efflux pumps from different microorganisms for various solvents resulted in an increase of solvent tolerance.But, overexpression of butanol and iso-butanol efflux pumps did not improve butanol tolerance leading to a conclusion that the toleration of butanol is a much more complex phenomenon (33).
There should also be a correlation between the transcriptional regulation of the genes involved in butanol production and the genes responsible for butanol tolerance through homeoviscous adaptation.If there was no correlation, the homeoviscous adaptation of C. pasteurianum must be similar during both, endogenous production (from glycerol) and exogenous addition (growth on glucose).

Π-A isotherms of reconstituted cell membrane (RCM) monolayers
To gain more understanding of the interaction within lipid-lipid and lipid-protein, it is essential to first characterize the phase behavior of the RCM monolayers spread at the air/water interface.The Π-A isotherms of RCM is plotted in Figure 5. Three different temperatures (25,37, and 50 °C) and sample extract at different conditions (without butanol and with 10 g/l butanol) have been examined.Without exogenous butanol in media at 25 o C, the experimental isotherms exhibited a liquid expanded (LE) phase and a collapse phase.The trends in Π-A isotherms were slightly different with those of unsaturated phospholipids (34) and to E. coli lipid extract (17).Π of RCM monolayers was much lower than the unsaturated lipids and the E. coli lipid extract and lift off area was not observable on RCM monolayers.The collapse of the RCM monolayers took place at ~25 mN/m indicating that the monolayers had low viscosity due to strong lipid interaction and a plausible displacement of relaxation membrane protein.At 37 o C RCM monolayers isotherms existed in single LE phase and collapse phase disappeared that could be attributed to the increase of disordered lipid tails and disordered organic membrane protein lowering lipid-protein interactions in membrane monolayers.With increasing temperature up to 50 o C, Π-A isotherms occurred at lower pressure.The appearance of the lift-off area was able to observe.A plateau region on Π-A isotherms indicates that the G-LE phase transition occurred.
Increases temperature lowered hydrogen bonding between lipid head groups and hydrophobic interactions between lipid tails and extended the relaxation phenomena in membrane proteins.
In addition, the length of plateau region could be associated with the ratio of unsaturated/saturated lipid (U/S).A short plateau region displayed as a high ratio of saturated lipid.The addition of 10 g/l butanol in media had markedly different effects on the Π-A isotherms (Figure 5) and the maximum elastic modulus, (Figure 6).With temperature changes (25, 37, and 50 °C) Π-A isotherms of RCM monolayer existed in the LE phase and occurred at higher surface pressure than without butanol present in media.This reveals that the content of hydrophobic proteins (Supplementary material and Figure S4) adsorbed at air/water interface had increased creating a higher Π-A and an increased (more rigid).The decreased proportionally with increasing temperature due to disordering structure of lipid and expanded relaxation phenomena.
Based on our interpretation data, the effect of protein can be interpreted as shown in Figure 7.At low surface pressure ( < ) 25 o C, hydrophobic membrane protein from cell grew in media without butanol exhibited relaxation phenomena within lipid membrane monolayers and water molecules were between the hydrophilic head groups of lipids.As a result, repulsive contribution at polar headgroup was high, and attractive van der Waals interactions between tails of lipids and interfacial interaction at head/tail of tails were low promoting a weak molecular packing.At high surface pressure, > external pressure compressed the protein network until it failed and displaced from the interface, forming collapse protein multilayers in the aqueous bulk phase near to the interface; thus, the collapse phase appeared.In the case of the reconstituted cell membranes extracted from cells grew in media with butanol, the content of hydrophobic membrane protein was increased resulted in longer relaxation phenomena.Therefore, the collapse pressure appeared higher.It is clear that homeoviscous C. pasteurianum producing solvents altered the lipid membrane composition to modulate protein function and insertion on the membranes in determining membrane integrity and activity.

Conclusion
This study is the first report of the analysis of the homeoviscous adaptation to butanol by C. pasteurianum and this study is also the first study that quantifies lipid composition in C.
pasteurianum.The analysis of the extracted membranes from cells exposed to various concentration of butanol in two different media, butanol producing medium containing glycerol and non-butanol producing medium containing glucose, resulted in two completely different responses with respect to the changes in the composition of the lipids.During butanol production, when stressed with exogenous butanol, C. pasteurianum responds by increasing the ratio of the saturated to unsaturated fatty acids in the membrane.This is also the response found in other butanol producing species of Clostridia, along with the response exhibited by other microbes towards tolerating toxic organic compounds (14)(15)(16)35).C. pasteurianum exhibits a different response when grown on glucose (no butanol produced), but challenged with exogenous butanol, which can be explained on the hypothesis of altering the ratio of the protein to lipids in the membrane (Figure 7 and 8).This hypothesis was further confirmed using by Π-A isotherms and the presence of hydrophobic membrane proteins in the lipid membrane of C> apsteurinum during EB2.
At 25 °C Π-A isotherms of the membrane monolayer were observed in the LE phase and the collapse phase.The collapse phase on membrane monolayers was observed because of a greater attractive interaction of saturated lipids and a displacement of lipid monolayers on collapse protein network at the interface.At 50 °C Π-A isotherms membrane monolayer exhibited a G-LE phase transition and LE phase.The G-LE phase transition appeared due to a low interaction of lipid-lipid and lipid-protein associating with disordering protein and lipid structure.This study implies the effect of unsaturated lipids and proteins on membrane monolayers and helps our understanding of the mechanism of butanol tolerant membrane by C.
Pasteurianum could be utilized to develop cultures in higher concentrations of butanol leading to more cost-effective and more efficient on butanol production.
The result from this study substantiates the assumption that a correlation exists between the modes of homeoviscous response, which are in turn, dependent on the activation of butanol production pathway.Furthermore, the existence of two different homeoviscous adaptations to butanol challenge in C. pasteurianum, demonstrates the potential of this organism to be studied further in terms of proteomics, functional genomics and metabolic engineering for the development of an industrial strain.The unavailability of the genome sequence and the proteome data must be addressed to explore butanol production and tolerance in C.
pasteurianum.In the meantime, methods to use genomic data from closely related species can The dry film of the reconstituted cell membrane and 0.1 mM 1,6-diphenyl 1,3,5hexatriene (DPH) dissolved in chloroform were mixed and co-evaporated under a gentle stream of nitrogen until a dry reconstituted cell membrane/DPH film remained.Vacuum was used (~ 60 min) to remove residual solvent from the film.The film was then hydrated with distilled water and maintained at 50°C in a water bath for 1 hour before shaking.The reconstituted membrane/DPH film was suspended as multilamellar liposomes by vigorously shaking for approximately 1 hour.The liposomes were sonicated before use for 60 min at 50°C, which has been previously shown to yield unilamellar liposomes.DPPC liposomes at 10 mM lipid generated by the same procedure were used as control.Fluorescence anisotropy (Perkin Elmer compounds were analyzed with GC-MS solution and identified by comparison with the data in the NIST libraries.The distribution of FAMEs of C. Pasteurianum was determined by dividing area counts for each FAME species by the total FAME area, as defined below: area (Π-A) isotherms Π-A isotherms were conducted in a Langmuir trough (model 102M, Nima technology Ltd, UK) with a deposition area of 70 cm 2 at 25 o C, 37 o C and at 50 o C. A Wilhelmy plate was used to measure surface pressure with an accuracy of ± 1μN/m connected to an electronic microbalance.Isotherms were monitored by NIMA TR 7.4 .visoftware.The external water bath system was used to control the sub phase temperature.Monolayers were obtained by spreading diluted solutions of dry lipid sample in chloroform at the air/water interface using a 5 μl microsyringe.
5 g/L (0.105 M) and 10 g/L (0.134 M), respectively, once the cells reached mid exponential phase.The membranes were extracted after 24 hours of butanol exposure and analyzed using 1 H-NMR, fluorescent anisotropy and GC-MS .The 1 H-NMR and fluorescent anisotropy results are summarized in Figure1, while Figure2summarizes the data from the GC-MS analysis on the fatty acid composition of the membranes.The addition of butanol during glycerol fermentation resulted in a homeoviscous response due to a decrease in the degree of unsaturation in the membrane as determined by 1 H-NMR (Figure1) of the extracted lipid of cells exposed to different butanol concentrations.The fluorescent anisotropy of the reconstituted C. pasteurianum cell membranes supports the results obtained from 1 H-NMR.The fluorescence anisotropy data at 37°C shows a decrease in anisotropy with increase in the butanol dose.The anisotropy, <r>, of the unexposed control was found to be 0.120 and <r> was found to reduce as the butanol dose increased in the media.The anisotropy of the membrane had a relatively constant value with a minor increase at higher concentrations of butanol at 7.5 g/L and 10 g/L.The fluorescence anisotropy of DPH in the membranes is inversely related to the fluidity of the reconstituted lipid membranes.The decrease in the anisotropy indicates the fluidizing effect of butanol on the membrane lipids.The stabilization of the anisotropy at higher butanol concentrations can be explained as the response of the bacteria to tolerate the fluidizing effects of butanol.The overlap of the anisotropy data with the 1 H-NMR data substantiates the existence of a dominant homeoviscous response leading to a compositional change in the lipid membranes.This can be further confirmed by analyzing the fatty acid composition of the membrane.