The Effects of N-Butonol on the Model and Reconstituted Membrane of Clostridium Pasteurianum

During n-butanol fermentation, n-butanol partitions into microbial membranes result indestabilizing cellular lipid membranes by altering their lipid composition(generally the ratio of saturated to unsaturated lipids) whereas adapted microorganisms respond by altering the ratio of unsaturated to saturated lipids. The mechanism of how microbes achieve a high adaptation in response to n-butanol is barely known. This dissertation describes the role of unsaturated lipids and charged lipid composition in modulating n-butanol partition into membrane using model bacteria (i.e. lipid bilayer vesicles or liposomes and Langmuir monolayers) and those studies were compared with reconstituted membranes (Clostridium pasteurianum) that represented an original sample which was collected during batch fermentation as a function of different fermentation conditions. Calorimetric, spectroscopic, Langmuir balance and chromatographic techniques were used to examine the effects of unsaturated lipid, charged lipid and nbutanol on membrane phase behavior, membrane packing, and membrane structure. The effects of n-butanol on heterogeneous membrane phase behavior was dependent onn-butanol concentration and which phase was continuous (saturated or unsaturated lipids). An increase of unsaturated lipid ratios increased n-butanol partitioning into the membranes due to “binding pocket” on acyl chain of unsaturated lipid and increased area per molecule resulting in enhancing membrane elasticity. Heterogenous monolayer membrane of DPPC/DOPC with n-butanol was also examined using Langmuir balance trough and fluorescence microscope. Lipid phase behavior, lipid packing, and monolayer elasticity were evaluated by surface pressurearea (Π-A) analysis. This study shows that n-butanol partitioning in DPPC, DOPC domain and at DPPC/DOPC interface. n-Butanol partitioning into DPPC monolayers led to lipid expansion and decreased elasticity. Lipid expansion became greater when DOPC content increased. n-Butanol accumulation at equimolar DPPC/DOPC was amplified at the interface between coexisting liquid expanded (LE, DOPC-rich) phases and liquid condensed (LC, DPPC-rich)domains. The accumulation of n-butanol also reduced LE-LC line tension and changed the domain size and morphology of LC domains. The integrity of charged lipid membrane was driven by electrostatic interactions between cations and negatively charged lipid headgroups and hydrophobic effects on lipid tails. However, above interdigitation concentration (0.13 M) of nbutanol, n-butanol partitioning into membrane transformed the gel phase to the interdigitated phase disregarding DPPG content and salt concentration. Increasing DPPG content in the DPPC/DPPG membrane and salts above 0.13 M of n-butanol concentration, aggregation/ fusion could be prevented and the transformation of LUVSUVs could be observed. Increasing salt and DPPG concentration, screening electrostatic repulsion between PG headgroups was apparent to promote more rigid bilayer structures and reduced butanol partition. Reconstituted membrane of C.pasteurianum have been examined to determine membrane composition, membrane phase behavior, and membrane fluidization using different techniques such as chromatographic, spectroscopic, and Langmuir balance. n-Butanol adapted membrane was the result of lipid modification by increasing longer fatty acids and decreasing the amount of unsaturation and protein improvement that increased membrane rigidity that counter-acted the fluidizing effect of butanol. Model and reconstituted membrane studies revealed that membrane rigidity and stability were promoted by decreasing unsaturated lipids, increasing the length of lipid tails and increasing charged lipid ratios in the electrolyte solution. The accumulation of n-butanol within membrane influenced membrane fluidity and membrane packing. These results demonstrate a fundamental link of the disordering effects of butanol and lipid compositions on cell membranes.


INTRODUCTION
Interest in n-butanol as a potential renewable biofuel has increased during the last four years due to its favorable physicochemical properties (hydrophobicity, high energy density, low vapor pressure) that enable it to be used directly or blended with gasoline in combustion engines. 1 Fermentation is the preferred route for n-butanol synthesis because it utilizes cellulosic (e.g. lignocellulose), sugar (e.g. glucose), and/or sugar-alcohol (e.g. glycerol) feedstocks that are available or can be derived naturally.
For example, Clostridium pasteurianum is a potential microorganism that can convert crude glycerol, a waste product of biodiesel production, to n-butanol. 2 However, as with most fermentation processes, n-butanol product yields are low (typically lower than 2 % by weight 3,4 ) due to low n-butanol-tolerance of the microorganism. The cause for this low tolerance lies within the cellular membrane. n-Butanol is a shortchain aliphatic alcohol consisting of a polar hydrophilic hydroxyl group and a nonpolar hydrophobic hydrocarbon chain (Figure 1.1).Thus, n-butanol can be described as an amphiphilic molecule that has high affinity for lipid membranes (lipophilic). 5 When n-butanol partitions into cellular membranes, it accumulates within the lipid headgroup region near the membrane/water interface destabilizing the membrane 6,7 and adversely affecting the structure and function of the cell. 3,8 Compared to ethanol, which has been extensively examined because of its relevance to human physiology and fermentation, 9 relatively little is known on how nbutanol interacts with cellular membranes on a biophysical level. Biophysical studies are conducted primarily on lipid bilayers or monolayers employed as model cellular membranes to eliminate complexities associated with cellular metabolism and growth -they provide a platform for quantifying membrane restructuring in response to nbutanol. However, most biophysical studies on membrane response to alcohols have relied on homogeneous single-lipid membranes that do not adequately account for the compositional and phase heterogeneity of actual cellular membranes. Quantifying restructuring of heterogeneous membranes in response to n-butanol would have direct implications to understanding/enhancing bacterial tolerance during n-butanol fermentation, which underpins the motivation of this dissertation. Connecting model membranes to reconstituted membranes comprised of cellular membrane extracts brings us one step closer to understanding the effects of n-butanol on membrane phenomena in C. pasteurianum.

n-Butanol tolerance in Clostridium pasteurianum
When cell membranes of C. acetobutylicum, C. butyricum, or C. thermocellum are exposed to n-butanol, they are fluidized (i.e. disordered or less viscous)and, in response to counteract this fluidization, the lipid composition is altered. A similar PE PC PG DPG response is observed when C. pasteurianum is exposed to n-butanol during fermentation. However, C. pasteurianum, an anaerobic bacterium, has a natural ability to tolerate n-butanol toxicity at higher concentrations than other species of solventogenic clostridia and it can utilize biodiesel-derived crude glycerol or mixing purified glycerol as substrates to produce n-butanol. 3,11,12 These advantages make C.
pasteurianum a potential candidate for n-butanol fermentation and a model for studying the correlation between lipid membrane composition and n-butanol  By modifying the composition of the lipid membrane, C. pasteurianumadapts to tolerate the toxic solvents through homeoviscous adaptation. To counteract the fluidizing effects of n-butanol, Clostridia adjust the ratio of saturated to unsaturated fatty acids (SFA/UFA) and the chain length of SFA in the lipid membrane to yield a rigid membrane. [14][15][16] The fluidity of the lipid membrane is directly proportional to the amount of SFA in the tails of the lipid bilayer. 15 Hence, the n-butanol-tolerant bacteria may a much higher SFA/UFA ratio in the lipid bilayer. It is critical to understand how SFA/UFA ratio of homeoviscous microbes achieves their adaptation in response to nbutanol. Thus, a fundamental link between model and intact cell membranes has to be identified. The goal of this research is to identify and to understand how n-butanol restructures model membranes and to draw comparisons with reconstituted cell membranes of C. Pasteurianum.
It is very difficult to study the phenomena of membrane surface at molecular level due to the complexities of cell membranes. Reconstituted membranes of C.
pasteurianum are the best way to simplify experiment processes, reduce complexity in data interpretation, and improve greater experimental control. By extracting cell membrane, reconstituted membrane can be obtained and formed as lipid bilayer model membranes and lipid monolayer membranes. Reconstituted membranes are an excellent model membrane that can be studied as a comparison with model membrane.

Lipid bilayers as model membranes
Lipid bilayers are the scaffold for cellular membranes, which separate intracellular components from the extracellular environment and determine the cell shape 1 .The effect of n-butanol toxicity on bilayer membranes forces cells to adjust their membrane composition and intrinsic curvature, which are related to the function of several membrane proteins 2,3 Lipid bilayer model membranes (liposomes) have been used to mimic the complex biological membranes to understand membrane-alcohol interactions. By modifying lipid bilayer composition, fundamental insight into nbutanol-membrane interactions can be gained.
Phosphatidylglycerol (PG) lipid, negatively charged phospholipid, is the major component found in gram positive and gram negative bacteria. 10 A model consisting of PC and PG system is a suitable model to mimic cytoplasmic bacterial membrane to investigate the effect of n-butanol partitioning into negative charged lipid membranes.

Monolayers as model membranes
Lipid monolayer membranes are excellent as model membranes, represent a single membrane lipid leaflet, to investigate interactions between membrane components or the mechanism of solute partitioning into the membrane interface.
Comparatively, monolayers are more controllable than bilayers. The physicochemical properties of monolayer membrane can be carefully adjusted to define molecular density with altering molecular area and surface pressure; and the size and shape of the monolayer domain formation due to phase separation can be visualized analytically with Brewster angle microscopy (BAM) 19 or fluorescence microscopy. 20 The interactions between monolayer molecules can be represented with the surface pressure(Π) -area (A) isotherms under compressive or expansive force.
Dipalmitoylphosphatidylcholine (DPPC) has been used for many monolayer studies.  The surface pressure in G phase is very low. The lipid tails are not curled and lie extended on the surface. The lipid molecules also move freely at air/water interface. When a compressive force on water surface is increased, the surface pressure is also increased. Monolayers enter the G-LE phase transitions and then LE phase which lipid tails are still disordered. Further compressing the monolayer enters the LE-LC phase and the LC phase where lipid tails are tilted orderly. Further a high compression, monolayer experiences in collapse phase at which the monolayer films are collapsed and packed to their maximum density. 22 The interactions between DPPC monolayers and short chain alcohols such as methanol and ethanol have been previously studied. Increasing surface excess of alcohol molecules adsorbed at the interface promotes alcohol partitioning into monolayers and results in increases in area per molecule and surface pressure (see Figure 1.7), and decreases inelasticity and molecular packing density. 23 Molecular packing density, elasticity, and the excess free energy of mixing between lipid/alcohol are dependent on the particular alcohol and its concentration. Longer hydrocarbon chain length alcohols have a stronger impact on molecular arrangement and membrane structure, creating different sizes and shapes of monolayer domains at different surface pressures. 24 Even though the interactions of short chain alcohols (methanol, ethanol and propanol) 25 and homogeneous monolayers have been studied, understanding the interactions between mixed saturated/unsaturated monolayers with n-butanol are not well known. Thus, this dissertation will reveal the mechanism and the interactions between heterogeneous monolayers and n-butanol.

n-Butanol interactions with lipid membranes
Alcohol partitioning into membrane is correlated to hydrophobicity in the alkyl chain length of the alcohol molecule. 26 Longer chain alcohols are more hydrophobic and, hence, n-butanol that is more hydrophobic than methanol, ethanol or propanol interacts more strongly with lipid membranes. n-Butanol resides near the lipid polar headgroup and its alkyl chain extends parallel into the hydrophobic tails of the lipids.
The effects of n-butanol partitioning into membrane produce a large disorder in the glycerol backbone of the lipid membrane. 27 Previous studies show that incorporation of n-butanol in lipid bilayer membrane perturbs lipid-lipid and lipid-water interaction resulting in a disturbance of membrane functions. Zhang et al. found that n-butanol concentration affects on homogeneous membrane phase behavior of phosphatidylcholine and lipid ordering. 28 An increased fluidity of the lipid membrane reflects with decreasing the melting temperature of the membrane and loosing membrane packing. They also determined that restructuring membrane is dependent on n-butanol concentration exhibiting a changing membrane phase, gel phase to interdigitated phase (L β' L βI) (Figure 1.8). It is clear that determination of n-butanol concentration accumulated in the membrane (nbutanol partition coefficients) becomes more pronounce to maintain the membrane fluidity and stability. At low concentrations n-butanol disorders the lipid tails and promotes a lower transition temperature (T m ), a broader main phase transition P β' Lα, and a larger phase pretransition L β' P β' . At high concentrations of n-butanol molecular packing is tightened orderly due to interdigitation, resulting in an increase of T m and a disappearance of pretransition. When high n-butanol concentration is present, nbutanol replaces the interfacial lipid water molecules. The hydrophobic tail of nbutanol is aligned to the hydrophobic core of the bilayer and the hydroxyl group of nbutanol binds to the lipid headgroup, creating an expansion of lateral space or voids between the lipid headgroups. The voids are energetically unfavorable contributing the formation of an interdigitated phase (L βI ) to minimize the energy and the tail ends of the alcohol molecules shield the tail ends of the lipids. The interdigitated lipids gain energy due to stronger van der Waals interaction in the hydrophobic tails of lipids. 27-29

Effect of short-chain alcohols on lipid membrane
Short chain alcohols (i.e ethanol and n-butanol) have important applications as a biofuel. 11 The fundamental understanding of the molecular mechanism of ethanol interacts on the lipid membrane is well studied through experimental and simulation results. 5,26,27 To date, homogenous model systems such as unilamellar vesicles, supported bilayers or monolayers have been used to elucidate the effects of ethanol or n-butanol on the physical and thermodynamic properties of lipid membranes. [23][24][25][26]28,30 Ethanol partitioning into the headgroup region disturbs the lipid membrane. In addition, ethanol partition can modulate properties of membranes. With higher ethanol concentration partitioning into membrane, the fluidity of the membrane increases; the main phase transition temperature decreases; the membrane thickness decreases; and it can induce the formation of interdigitated bilayer structures. 31 Pastworks have also shown that n-butanol decreases the main phase transition temperature, induces the formation of interdigitated bilayer and decreases membrane thickness. 28 However, there are a few studies of the interactions between n-butanol and lipid membrane and understanding of n-butanol interacts with heterogeneous membranes is limited. Thus, quantitative studies of the n-butanol effects on coexisting phase in membrane will be more realistic resembling the real cell membrane. Motivated by realistic model systems, this dissertation will expand the understanding of the interactions between nbutanol and heterogeneous membrane, and apply this understanding as the best approach for gaining insight into how C.pasteurianum membranes respond to nbutanol partitioning.

Dissertation objectives
Determining the effects of n-butanol on model and reconstituted cell Chapter 5 presents the effects of n-butanol on reconstituted membranes of C.
pasteurianum grown under different conditions. The study discusses stability and change in the cell membrane composition that constitute a homeoviscous response under different conditions. The mechanical properties of cell membrane monolayer producing solvents will be elaborated. This is the first study to our knowledge to analyze the homeoviscous adaptation to butanol by C. pasteurianum and this study is also the first study that quantifies lipid composition in C. pasteurianum.

Introduction
The partitioning of primary alcohols into homogenous lipids bilayer membranes has been well studied. Generally speaking, the partitioning of short alcohols (up to C 6 ) dehydrates lipid headgroups at the membrane/water interface, reduces membrane surface tension, leads to a reduction in lipid ordering (i.e. membrane fluidization), and, at high concentrations, causes lipid interdigitation. [1][2][3][4][5][6][7][8][9][10][11][12] As the carbon-chain length of the alcohol increases, the partitioning coefficient increases and fluidization or interdigitation occurs at lower alcohol concentrations. While well characterized in homogenous membranes, there has been relatively little work on alcohol partitioning and its effects on heterogeneous membranes that contain multiple lipid species and coexisting phase states.
This work examines n-butanol partitioning and membrane restructuring which, compared to ethanol, have not been examined extensively. n-Butanol is a viable biofuel and platform chemical for biorefining. It is also lipophilic and, when produced by fermentation, partitions into microbial membranes and compromises membrane integrity. 13,14 This can ultimately inhibit or eliminate microbial activity.
Microorganisms respond by altering their lipid composition, specifically the ratio of saturated to unsaturated lipids and/or lipid tail length, to maintain a homeoviscous membrane state. 14 Studying the effects of n-butanol on heterogeneous membranes composed of saturated and unsaturated lipids is an important step towards understanding this response mechanism.

Liposome preparation
Liposomes were prepared by the Bangham method. 17 Briefly, DPPC and DOPC dissolved in chloroform were mixed to achieve the desired ratio of DPPC to DOPC. The chloroform was then evaporated under a stream of nitrogen and the samples were dried under vacuum for at least 30 minutes. The dry films were then hydrated with 1.5 ml of deionized water to yield a total lipid concentration of 0.5 mM.  Table 1. Butanol was then added to the samples at the desired concentration. The samples were briefly vortexed and stored for 2 h after the butanol addition before further processing or analysis.

High performance liquid chromatography (HPLC)
HPLC was one of the three methods to determine the lipid/water n-butanol partitioning coefficient. Sample preparation began by taking liposomes (no n-butanol) and liposomes + n-butanol, and centrifuging the suspensions at 10,000 rpm for 30 min The partition coefficient, ‫ܭ‬ , was calculated as where‫ݔ‬ and ‫ݔ‬ ௪ are the mole fractions of n-butanol (b) in the membrane (m) and aqueous (w) phases. n-Butanol mole fractions were determined based on mole balance, where ݊ and ݊ ௪ are the number of moles of n-butanol in the membrane and aqueous phases, respectively. The number of moles of n-butanol in the membrane was calculated as ݊ = ݊ − ݊ ௪ where ݊ was the total number of moles of n-butanol in the system.

Differential scanning calorimetry (DSC)
DSC was performed using a TA Instruments Nano DSC with capillary cells.
The reference cell was filled with degassed water and the sample cell was filled with 760 µl of degassed sample. The cell chamber was sealed and pressurized to 3 atm under nitrogen. Samples were analyzed by consecutive heating/cooling cycles between 25 o C and 50 o C at a rate of 1 o C/min. Pretransition temperature, ܶ , melting temperature, ܶ , and melting enthalpy, ‫ܪ∆‬ , were determined using the Universal Analysis software. The melting temperature was taken as the temperature at maximum peak height.
‫ܭ‬ was calculated from DSC results as: where∆ܶ is the measured change in melting temperature (ܶ − ܶ , ), R is the gas constant, ܶ , is the melting temperature of DPPC without n-butanol, and ‫ܪ∆‬ is the lipid melting enthalpy. ‫ܥ‬ , ‫ܥ‬ ௪ , and ‫ܥ‬ are the n-butanol, water, and lipid molar concentrations, respectively.

Fluorescence anisotropy
Liposome preparation for fluorescence anisotropy was similar to that for DSC.
In this case, DPH was added to the lipids in chloroform as a probe at a DPH:lipid molar ratio of 1:400. The samples were hydrated with 3 ml of DI water to yield a total lipid concentration of 0.04 mM. DPH anisotropy, <r>, in DPPC/DOPC bilayers was measured as a function of n-butanol concentration using a LS55 Luminescence Spectrometer with a Peltier system (Perkin-Elmer, Shelton,CT). Heating and cooling scans were conducted between 25 o C and 50 o C at a rate of 1 o C/min, and the sample was continuously mixed with a magnetic stirrer. Steady-state DPH anisotropy was determined at an excitation wavelength of 350 nm and an emission wavelength of 452 nm with a 10 nm slit width. The anisotropy was calculated as where I represents the fluorescence intensity, the subscripts V and H represent the vertical and horizontal orientation of the excitation and emission polarizers, respectively, and G is the grating factor ‫ܩ(‬ = ‫ܫ‬ ு ‫ܫ/‬ ு ), which accounts for the correction factor of the sensitivity of the instrument towards vertically and horizontally polarized light. Anisotropy is dependent upon the fluorescence lifetime of DPH (τ), which changes with temperature and lipid composition. 18,19 However, in this work steady-state anisotropy was measured and changes in τ are not considered.

Membrane phase behavior
Results are first presented for DPPC with n-butanol, which has been previously studied. 4,8,12 DSC was conducted by adding n-butanol at room temperature followed by sequential heating and cooling scans (

4.3.Membrane fluidization
Fluorescence anisotropy was conducted using the probe DPH to assess membrane fluidization due to lipid disordering. Anisotropy, <r>, is shown in Figure   2.

Discussion
For DPPC, it has been reported that n-butanol partitioning is greater in ‫ܮ‬ ఈ phases than in ‫ܮ‬ ఉ ᇲ phases. 12 Although the same information is not available for membranes with saturated verses unsaturated lipids, previous work for ethanol has shown that partitioning into unsaturated ‫ܮ‬ ఈ membranes is approximately 4-fold greater than into saturated ‫ܮ‬ ఈ membranes. 10  contradict these results and indicate that, at equimolar DPPC:DOPC, the gel DPPC phase does not form a ‫ܮ‬ ఉ ᇲ phase, but rather an untilted ‫ܮ‬ ఉ -like phase. 24 Our DSC results show that a pretransition did occur at all DPPC:DOPC ratios in the absence of n-butanol, but quickly disappeared with increasing DOPC and n-butanol concentration. We do not attribute the disappearance of the pretransition to interdigitation, but rather a cooperative effect of DOPC + n-butanol that restricts lipid tilt and eliminates the ‫ܮ‬ ఉ ᇲ → ܲ ఉ ᇲ transition. This is based in part on evidence of ܶ and ‫ܪ∆‬ hysteresis that infers a common interdigitation concentration between 0.1 M and 0.13 M n-butanol when gel DPPC is the continuous phase.
Surface tension (ߛ) in lipid membranes is related to the area compressibility modulus (ߛ ∝ ‫ܭ‬ ) and, with solute concentration (ܿ), the partition coefficient (݀∆ߛ/ ݀ܿ ≡ ‫ܭ‬ ). ‫ܭ‬ is lower for fluid phases and unsaturated lipids than for gel phases and saturated lipids. Hence, increasing DOPC and/or n-butanol concentrations would lower membrane surface tension and reduce ܶ . This was not observed in the absence of n-butanol, consistent with the observation that DPPC (continuous or as domains) was not influenced by DOPC at the conditions examined. However, a reduction in ܶ was observed in the presence of n-butanol below the interdigitation concentration. In this case, increasing the DOPC concentration up to an equimolar DPPC:DOPC ratio did not affect this ܶ reduction. This suggests that increasing DOPC concentration did not increase n-butanol partitioning into gel DPPC and that increases in ‫ܭ‬ , when gel DPPC was the continuous phase, can be attributed to additional partitioning into fluid DOPC. This is in agreement with ‫ܭ‬ calculated from DSC.
‫ܭ‬ increased linearly with DOPC concentration below the n-butanol interdigitation concentration and appeared to be independent of which phase was continuous. The linear relationship suggests that greater partitioning correlated with greater fluid phase fraction. Above the interdigitation concentration, ‫ܭ‬ was dependent upon which phase was continuous. Up to a DPPC:DOPC ratio of 1:1, interdigitated DPPC was the continuous phase and there was a modest increase in ‫ܭ‬ .
This indicates that n-butanol had a similar affinity for the interdigitated and fluid phases, which has been shown for ethanol. 20 At a DPPC:DOPC ratio of 1:3, fluid DOPC was the continuous phase and there was a notable increase in ‫ܭ‬ . At this stage we attribute the increase in ‫ܭ‬ to n-butanol partitioning at the ‫ܮ‬ ఉூ ‫ܮ/‬ ఈ interface. This concept is supported by theoretical 25 and experimental [25][26][27] evidence that the dynamic wetting layer at gel/fluid interfaces become more influential when the fluid phase fraction is increased and becomes the continuous phase.
Fluorescence anisotropy provides additional insight into membrane structure.
Assuming that DPH has equal affinity for gel and fluid phases, 28   there was little change in the partition coefficient when DPPC was the continuous phase. However, when DOPC was the continuous phase, n-butanol partitioning increased due to the dynamic wetting effect at the gel/fluid interface.

Conclusions
Results from this work depict a cooperative effect of DOPC + n-butanol on membrane phase behavior that was dependent on n-butanol concentration and which phase (DPPC or DOPC) was continuous. Below the interdigitation concentration with a continuous gel DPPC phase, the total n-butanol partitioning increased proportional to the fluid phase fraction (DOPC), but partitioning into DPPC was unchanged. In this case DOPC alone did not eliminate ripple gel formation by DPPC, but DOPC + nbutanol did by promoting a ‫ܮ‬ ఉ -like DPPC phase. Fluidization results showed that the fluorophore preferentially partitioned into gel or interdigitated gel phases at low nbutanol concentrations, but distributed more evenly between DPPC and DOPC at high n-butanol concentrations (twice that for interdigitation). We propose that this was achieved by n-butanol filling the membrane/water interface and reducing unfavorable tail packing conditions normally experienced DOPC that expose the hydrophobic region to water. This study infers a cooperative effect of unsaturated lipids and alcohols in heterogeneous membranes, which aids our understanding of how membranes with mixtures of saturated to unsaturated lipids restructure in response to alcohols. Ethanol, like other short chain alcohols (up to C 6 ), partitions to the membrane/water interface, hydrogen bonds to carbonyl groups in the lipid headgroups, leads to lipid expansion and reduced interlipid van der Waals attraction, and increases membrane elasticity. [6][7][8][9] Above a critical concentration, short chain alcohols also induce lipid interdigitation in gel-phase membranes composed of saturated phosphatidylcholine (PC) such as dipalmitoyl PC (DPPC). 2,[9][10][11] Recent studies on alcohol-induced membrane fluidization have focused on heterogeneous membranes comprised of different lipid species and phase states (i.e.

References
lipid domains in membranes containing saturated and unsaturated lipids), which better reflect the complex structure of cellular membranes. 7,12,13 Marques et al. 13

Fluorescence microscopy.
A hydrophilic glass slide pretreated with piranha solution (2.54 cm x 1.27 cm) was immersed vertically into the subphase using a dipper assembly (KSV Nima).
Monolayers were then formed as described in the preceding section with RhPE at 3 mol%.The labeled monolayer films were compressed at 10 cm 2 /minto a surface pressure of 30 mN/m. Monolayers were transferred to the slides during withdrawal from the subphase at a rate of 3 mm/min.Imaging was performed at 100× magnification using a Nikon Diaphot-TMD inverted Epi-fluorescence microscope (Nikon, Japan). The microscope was equipped with phase contrast-2 ELWD 0.52 phase-contrast condenser, a12 V 100W mercury lamp (Nikon, Japan), and a digital sight DS-L2 camera (Nikon, Japan)equipped with G-2B filter cube.NIS-element software was used to capture the images and ImageJ was used for processing and analysis. 23

Monolayer compressibility
The bulk elastic moduli, ‫ܥ‬ ௦ ିଵ , were determinedbased on the Π-A isotherms from where A is the area per molecule at a given surface pressure. 30,31 .  Ethanol partitioning into PC monolayers has been shown to reduce hydrogenbonding between water and PC headgroups. 34 n-Butanol presumably has the same effect on PC monolayers but, due to its longer tail, exhibits greater van der Waals attraction between neighboring lipid tails. The combined effects of lipid dehydration, n-butanol hydrogen bonding with PC headgroups, and van der Waals attraction between PC tails may explain the increases in ‫ܥ‬ ெ௫ ିଵ when DPPC was the main component.

3. Monolayer structure and n-butanol partitioning
Additional analysis was conducted on the Π-A isotherms in the region between

Conclusions
To the author's knowledge this is the first study depicting the effects of nbutanol partitioning on heterogeneous monolayers with coexisting LE and LC phases arising from a mixture of unsaturated and saturated lipids, respectively. Approaches

Abstract
Bacteria adjust their membrane lipid composition to counteract the fluidizing effects of alcohol and to adapt to elevated alcohol concentrations during fermentation.
Bacterial membranes are rich in anionic phosphatidylglycerols (PG), but little is known regarding alcohol partitioning into anionic membranes, particularly for n- small vesicles were observed. The results suggest that n-butanol partitioning, and subsequent changes in membrane and vesicle structure, was driven by a balance between the 'salting-out' of n-butanol, interlipid electrostatic interactions, and interfacial cation binding and hydration. This is the first study to our knowledge to examine the effects of n-butanol partitioning on model cell membranes composed of negatively charged lipid in the presence of salts.

Introduction
The partitioning of n-butanol into zwitterionic lipid bilayer membranes (e.g. n-Butanol is a biorefinery key platform chemical that can be used to produce polymers and resins. 7 n-Butanol also can be used for a viable biofuel. 8 However, the lipophilic solvent n-butanol is toxic during fermentation, destabilizing cell membranes and disrupting membranecomponents. 9 Bacteria, which generally have negatively charged membranesand exist in electrolyte media, respond to butanol toxicity by altering their lipid composition to compensate the fluidizing effects of butanol. 10 Studying the effects of n-butanol and electrolytes on charged lipids membranes is important to understand bacterial response mechanism. n-Butanol partitioning can restructure lipid molecules resulting in changes inPC lipid phase behavior such as a decrease in the pretransition (T p )and melting temperatures (T m ), which reflect tilted gel to rippled gel ‫ܮ(‬ ఉᇱ → ܲ ఉᇱ ) and rippled gel to fluid (ܲ ఉᇱ → ‫ܮ‬ ఈ ) phase transitions, respectively. 1-3 Previous resultshave shown that above 0.13 M (10 g/l),n-butanol can promote the transition from a gel to an interdigitated gel ‫ܮ(‬ ఉᇱ → ‫ܮ‬ ఉூ )phase. 1,3 This behavior can persist even when DPPC is present as domains within a continuous unsaturated lipid phase; however, the presence of unsaturated lipids can reduce or even eliminate interdigitation at high concentrations. 11,12 In addition to lipid composition, electrolytes play a central role in determining lipid membrane phase behavior and agglomeration in vesicle dispersions. [13][14][15][16][17] With increasing electrolyte concentration from 0 to 3M, T p andT m of DPPC phase transition increases due to ion binding to PC headgroups. 6

3.1.Vesicle preparation
LUVs were prepared by thin-film hydration method. 21  and stored for 30 min after n-butanol addition before further processing or analysis.

Differential scanning calorimetry (DSC)
Lipid bilayer phase behavior in the DPPC/DPPG vesicles was analyzed by DSC (TA Instruments Nano DSC). Samples were equilibrated at 25 o C, and the cell chamber was sealed and pressurized to 3 atm under nitrogen. Samples were analyzed by heating between 25 o C and 50 o C at a rate of 1 o C/min. Pretransition temperature, ܶ , melting temperature, ܶ , and melting enthalpy, ‫ܪ∆‬ , were determined using the Universal Analysis software. The melting temperature was taken at maximum peak height.
The membrane/water n-butanol partitioning coefficient, ‫ܭ‬ , was calculated from DSC results as: where∆ܶ is the measured change in melting temperature (ܶ − ܶ , ), R is the gas constant, ܶ , is the melting temperature of DPPC without n-butanol, and ‫ܪ∆‬ is the lipid melting enthalpy. ‫ܥ‬ ‫ܥ,‬ ௪ , and ‫ܥ‬ are the n-butanol, water, and lipid molar concentrations, respectively.

High performance liquid chromatography (HPLC)
Sample preparation began by taking liposomes without n-butanol and liposomes with n-butanol, and centrifuging the suspensions at 10,000 rpm for 30 min The partition coefficient, ‫ܭ‬ , was calculated as wherea b , γ b , and x b are the activity, activity coefficient, and mole fraction of n-butanol, respectively, and the superscript m denotes the membrane phase and w the water phase. n-Butanol mole fractions were determined directly based on a mole balance.

Dynamic Light Scattering (DLS)
Average size and zeta potential of vesicles at different DPPC:DPPG ratios and n-butanol concentration were measured by Dynamic Light Scattering (DLS) with a Zetasizer Nano ZS instrument (Malvern Instruments Ltd.). This instrument is equipped with 5mW He-Ne laser operating at 633nm. A quartz cuvette was used to determine the vesicle hydrodynamic diameter a folded capillary cell was used to measure the vesicle zeta potential. The cells were sealed with a cap and secured in the sample chamber of the instrument. DLS was conducted in backscattering mode at an angle of 173 o and zeta potential was measured based on laser Doppler electrophoresis.

Partitioning of n-butanol into membranes (۹ ‫ܘ‬ )
n-Butanol partitioning coefficients,K p , were determined from DSC (equation 1) and by HPLC (Figure 4.4). It should be noted that K p from DSC were calculated based on the linear portion of the T m graphs before interdigitation while K p by HPLC was determined below and above the n-butanol interdigitation concentration (0.13 M).
Calculated and measured values of K p for gel phase ‫ܮ(‬ ఉᇱ ) DPPC in DI water (71 and 141, respectively) were in general agreement with reported values. 23 Collectively, trends for K p from DSC and HPLC were consistent and showed that (i) K p increased for DPPC with increasing salt concentration (ca. 2-fold from DI water to 10x PBS),  Increases in K p with PBS concentration (i) can be partially explained by the 'salting out' effect of cations on n-butanol water solubility. 24 ASPEN simulation was conducted to determine the effect of Na + and K + on the activity coefficient of nbutanol in water ( Figure F.1). Na + reduced the activity coefficient by as much as 37% at NaCl concentrations representing 10x PBS. The effect of K + was less pronounced.
Considering equation 2 for K p , this reduction may explain in part why butanol partitioning increased with PBS concentration assuming that the activity coefficient of n-butanol in the membrane was unchanged. However, trends observed with DPPG concentration are still unclear and will be discussed in more detail below.

Vesicle hydrodynamic size
The effect of DPPG and electrolyte concentration on vesicle size are shown in repulsive inter-vesicle force that prevents aggregation. Na + and K + did not perturb the hydration of the bound water layer, as previously reported, 25 but rather reduced the activity coefficient of n-butanol in water and increased the concentration of n-butanol in the membranes (as discussed for K p ). It is unlikely that cation adsorption played a role DPPC vesicle aggregation or fusion. 26 Not surprisingly, cation adsorption did influence vesicle size in DPPC/DPPG vesicles as Na + and K + bound to anionic DPPG. This is clearly depicted by zeta potential measurements at 1:3 DPPC:DPPG in DI water and 10x PBS ( Figure F

Discussion
Interdigitated structures have been studied intensively for PC/short alcohol systems. 2  that the formation of DPPG-rich interdigitated vesicles or micelles as previously observed for bile salts. 27 It is unclear why this did not occur at low salt conditions. Regarding n-butanol partitioning, DPPG appears to influence K p through collective hydration and electrostatic interactions. PG headgroups mimic water and exhibit increased solvation and hydrogen bonding capacity compared to PC. 28 Considering that n-butanol displaces interfacial water and itself hydrogen bonds to lipid headgroups, it is expected that K p would decrease with DPPG concentration. This was observed in PBS and total salt concentration was not a significant factor at or above X DPPG = 0.5. At X DPPG = 0.25, which is representative of PG content in bacterial membranes, there is discrepancy between K p values determined by DSC and HPLC, particularly at high salt conditions. At low salt conditions, which mimic physiological Na + concentrations, K p did decrease with increasing DPPG concentration (X DPPG from 0 to 0.25).Reductions in electrostatic repulsion between DPPG molecules via salt screening would have increased lipid packing (i.e. reduced area per lipid), which may also have contributed to the decrease in K p .
The mechanism involved in increasing K p in DI water with increasing DPPG concentration is more elusive. 'Salting out' is a possible factor that may have been driven by the Na + counter ion of DPPG, but this cannot be the primary cause. It is possible that the increase in K p arose from difference in lipid packing. In pure bilayers PG posses a larger area per molecule (48.7 Å 2 ) than PC (47.2 Å 2 ) due in part to electrostatic repulsion between PG headgroups. 29 Without charge screening additional area would be available for n-butanol partitioning with increasing DPPG concentration (1.5 Å 2 per DPPG). Geometrically, twelve DPPG molecules would be needed to create the additional space needed to accommodate one n-butanol molecule. This simple analysis neglects membrane expansion due to n-butanol partitioning, which would reduce electrostatic repulsion between DPPG.

Conclusions
This study illustrates the role of ionic strength on n-butanol partitioning in neutral and anionic membranes, which is important to understanding how heterogeneous model bacterial membranes restructure in response to n-butanol. This work has shows that DPPG concentration and ionic strength have a significant effect on DPPC/DPPG membrane phase behavior, size and n-butanol partitioning. Cation adsorption on DPPC membranes and high n-butanol partitioning (> 0.13M n-butanol) led to interdigitation and a fusion. However, increasing DPPG content in DPPC/DPPG membrane and salts with high n-butanol partitioning did not prevent interdigitation but it did prevented aggregation/fusion and caused a LUV-to-SUV transformation. n-Butanol partitioning was reduced likely due to charge screening via cation adsorption, which packed and rigidified the membrane. To our knowledge this is the first study depicting the effects of ionic strength and n-butanol partitioning intoa mixture of negatively charged and neutral lipids.

Abstract
Clostridium pasteurianum has been shown to ferment glycerol into butanol at higher yields than when sugars are used as the carbon source. C. pasteurianum's potential to use biodiesel-derived crude glycerol as the carbon source has been gaining importance in the recent past. This study investigated the homeoviscous response of C.
pasteurianum during butanol stress. C. pasteurianum's lipid composition of the plasma membrane during butanol challenge was analyzed. C. pasteurianum was found to exert two different homeoviscous responses by altering the composition of the lipid membranes in an attempt to counteract the butanol toxicity. Addition of exogenous butanol to glycerol fermentation when C. pasteurianum produced endogenous butanol led to an increase in the ratio of saturated to unsaturated lipids.
On the contrary, addition of exogenous butanol to fermentation when C. pasteurianum did not produce any endogenous butanol led to a decrease in the ratio of saturated to unsaturated lipids. This counterintuitive finding of an alternate response to butanol toxicity, which is similar to a response observed in gram negative microbes, was verified for the presence of hydrophobic membrane proteins and the ability of the cells to retain butanol productivity. The differential responses for exogenous butanol during the presence and absence of an active butanol biosynthesis indicates that C.
pasteurianum is a versatile micro-organism that has the potential to be engineered as an industrial butanol producer using crude glycerol, a promising low cost feedstock for butanol production.

Introduction
Clostridium pasteurianum, an anaerobic spore-forming firmicute, ferments glycerol as the sole substrate, resulting in a mixture of butanol, ethanol, and 1,3propanediol (PDO) along with acetate and butyrate. 1-4 C. pasteurianum is of particular interest for butanol production, as it has also been shown to ferment biodiesel-derived crude glycerol as the sole substrate. 3,4 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, the partition coefficient. Solvents with a log P value less than 4 partition 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 lipids (S/U) 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 inversely related to the amount of saturated fatty acids in the tail of the lipid bilayer. Hence, the bacteria that tolerate more butanol have a much higher S/U 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][7][8]10,12,13 It has been established that composition and distribution of lipids in cell membrane play an essential role not only in maintaining membrane stability, curvature and membrane fluidity but also in modulating protein function and insertion of membrane proteins into 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 with an increase in S/U has been observed in Clostridia, particularly, C. acetobutylicum 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 the carbon source (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, C. pasteurianum membrane lipid composition with or without homeoviscous adaptation has not been examined. Furthermore, the effect of lipid composition on the membrane structure of butanol-tolerant solvent producing bacteria have are unknown. In this study, membrane extracts of C.pasteurianum exposed to exogenous addition of different butanol concentrations were analyzed and reconstituted as dispersed bilayer vesicles or monolayers to investigate membrane composition, membrane fluidity, and membrane compressibility.

Materials
All chemicals were purchased from Fisher Scientific and Sigma-Aldrich.

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 bacterial growth and the stability and changes in membrane composition 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 C. pasteurianum does not produce butanol when grown 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 reinforced clostridial media (RCM).

Extraction of the Cell Membrane
Cell membranes were extracted using the modified protocol of Bligh and Dyer using dichloromethane/methanol mixtures. 28 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 CDCl 3 for 1 H-NMR analysis.

Liposome Preparation and Fluorescence Anisotropy
The dry film of the reconstituted cell membrane and 0.1 mM 1,6-diphenyl 1,3,5-hexatriene (DPH) dissolved in chloroformwere 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 for 60 min at 50°C, which has been previously shown to yield unilamellar liposomes. A 10 mM DPPC liposomal solution was used as control.
Fluorescence anisotropy(Perkin Elmer 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 90ºC 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 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 borontrifluoride-methanol complex at 60ºC 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 containing 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 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 aSHR5xLB silica capillary column (30m× 0.25mm ID, composed of 100% dimethyl polysiloxane).Manufacturer's instructuion were followed for FAME analysis.The compoundswere identified by comparison with the data in theNIST libraries.The FAME distribution was determined using equation 2: % FAME = ∑ (Eq 2)

Elastic modulus / area compressibility modulus
Mechanical properties of the monolayer films were determined by the compressibility modulus. The elastic modulus ‫ܥ(‬ ௦ ିଵ ) / area compressibility modulus is the reciprocal of the compressibility ‫ܥ(‬ ௦ ). 31 The elastic modulus corresponds to the elasticity of the Langmuir films under the compression force. ‫ܥ‬ ௦ ିଵ values were defined as: where, A is the area per molecule at the particular surface pressure and Π is the corresponding surface pressure. A maximum in the elastic modulus ‫ܥ(‬ ௦ெ௫ ିଵ )denotes the maximum molecular 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. This study is focused on investigating the effect of butanol inC. pasteurianum leading to the tolerance response, which involves changes in the lipid membrane composition. These changes in the physical and structural compositions of the membranes were analyzed to examine the relationship between the composition and function of the lipids as it provides C. pasteurianum cells an ability to use two different homeoviscous responses to tolerate the toxic effects of butanol.

Effect of Exogenous Butanol
The homeoviscous response of C. pasteurianum to the addition of exogenous butanol was studied at two conditions, first by adding exogenous butanol during the endogenous production of butanol by C. pasteurianum, i.e., glycerol fermentation (EB1), and second by adding exogenous butanol while no butanol was produced, i.e.
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. which consisted of fatty acids from C 12 to C 19. 10 The ratio of the unsaturated to saturated fatty acids was found to be close to 1 in the absence of butanol exposure but the ratio reduced to 0.87 and 0.77 respectively with an exposure to 4 g/L and 8 g/L butanol respectively. This reduction in unsaturation and the concurrent increase in saturated fatty acids in the membrane lipid constitutes the organism's homeoviscous response.

The Differential Response to Butanol Toxicity
The effect of butanol under EB1 conditions showed a conventional homeoviscous response by increasing the fatty acid chain length and the ratio of saturated to unsaturated lipids. To further investigate the sole effect of butanol toxicity,cells were grown in glucose and exposed to exogenous butanol (EB2 conditions). Initially, the EB2 experiment was conducted to match the butanol stress concentration of 0 g/L to 10 g/L, but experiments at higher butanol concentrations of up to 20 g/L were also performed. 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 32 . 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. 33  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. 36 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. 36 However, overexpression of butanol and iso-butanol efflux pumps did not improve butanol tolerance,leading to the conclusion that the toleration of butanol is a complex phenomenon. 36

Π-Aisotherms ofreconstituted cell membrane (RM) monolayers
The surface pressure and elasticity modulus of RM monolayers were examined to gain more understanding of lipid-lipid and (possible) lipid-protein interactions. Π-Aisotherms of RM monolayers spread at air/water interfaces are shown in Figure 5.5 for the first compression cycle where A (cm 2 /µg) was based on the mass of the membrane extract. Fluorescence anisotropy data indicated that the membranes (EB1) were in a fluid state, which, in a two-dimensional lipid monolayer, is analogous to a liquid expanded (LE) or disordered state. Π of RM monolayers was lower than Π of monolayers of saturated/unsaturated phospholipid 33,37 and E.coli lipid membrane extract. 17 In addition to 37°C, temperatures at 25°C and 50°C were employed to determine if two-dimensional lipid phase changes could be elicited.  Based on our interpretation data, the effect of protein can be interpreted as shown in Figure 5.5. Temperature affected the structure of membrane proteins and lipid. At low temperature (T< 37°C), proteins remained in native state and lipid tails were ordered. At low compression (ߎ<ߎ ௦ ௧ ) and low temperature, foldedhydrophobic protein at 0 g/l butanol displaced within lipid membrane monolayers and water molecules remained between the hydrophilic lipid head groups. As a result, repulsive contribution at polar headgroup was high, and attractive van der Waals interactions between lipid tails were low promoting a weak molecular packing. At high surface pressure, ߎ>ߎ ௦ ௧ external pressure compressed the folded protein network until it failed and displaced from the interface, forming collapsed protein multilayers in the aqueous bulk phase near to the interface; thus, the collapse phase appeared ( Figure 5.5B).
Protein content in RM increased with increasing EB2. As a result, the increased amount of the hydrophobic proteins at 10 g/l adsorbed at air/water interface increasingߎ. At high temperature (T> 37°C) lipid tails were disordered and proteins were unfolded increasing the protein relaxation, the displacement and the elasticity.
Even though monolayer was under high surface pressure, the membrane elasticity at 10 g/l butanol was higher than the membrane elasticity at 0 g/l butanol. This indicates that the protein at air/water interface did not occur due to the orogenic displacement of unfolded protein ( Figure 5.6).  to endogenous, exogenous and

Conclusions
This study is the first report of the analysis of the homeoviscous adaptation to butanol by C. pasteurianumand 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 under two different conditions (i) butanol production by glycerol fermentation (EB1) and (ii) no butanol production by glucose fermentation (EB2), 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 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 and metabolic information from closely related species can be explored to establish a platform that can be used to perform transcriptional analysis of C. pasteurianum during butanol stress. Collectively, this work shows a truly novel aspect inclusive of the importance of gel-fluid or LE-LC interfaces on partitioning; membranes containing DOPC or DPPG are still interdigitated; salt can enhance interdigitation and/or change liposome structure; C. pasteurianum responds by increasing lipid tail length and protein content; and C. pasteurianum monolayers can be used to probe homeoviscous response. To our knowledge, these works are the first study that has ever been done.

Future work
As introduced in chapter 1, the major lipid components of bacterial cell membranes consist of phosphoethanolamine (PE), phosphoglycerol (PG), and diphosphatidylglycerol (DPG).Thus, unilamellar vesicles and monolayer membranes composed of PE or DPG will be well-suited to improve our understanding of bilayer perturbations in a more realistic system. pasteurianum. However, the expression of protein in the homeoviscous C.
Pasteurianum has not been evaluated. Thus, comprehensive analysis on the membrane proteome of C. pasteurianum wild type strain and its butanol-tolerant mutant will be very useful to reveal the protein expression related to n-butanol tolerance. This comparative membrane proteomics study, together with lipid membrane study will bring a systemic understanding of the n-butanol effects on cellular physiology of C.pasteurianum.
The main focus of this dissertation was to evaluate the interaction of n-butanol with liposomes, monolayers, reconstituted membranes, and use that fundamental  where ζ is the zeta potential, V p is the electrophoretic velocity, ε is the permittivity, and η is the viscosity of the medium. 101

Fluorescence anisotropy (FA)
The changes of membrane fluidity are determined quantitatively through steady-state fluorescence spectroscopy by using a probe molecule to penetrate into hydrophobic part of the lipid membrane. 102

APPENDIX D Langmuir Blodgett (LB)
A Langmuir Blodgett monolayer technique is used to measure the surface pressure of amphiphilic molecules in monolayer at air/water interface. 104 The molecular density at the air/water interface with constant temperature is altered with moveable barriers in a Langmuir-Blodgett (LB) film balance. By moving barriers the film density decreases (compression) or increases (expansion), which changes the surface tension, γ, of the air-water interface which is measured by Wilhelmy plate

Effect of Endogenous Butanol
The effect of endogenously produced butanol on the bacterial lipid membrane was studied by growing the bacteria in conditions that resulted in the formation of butanol at different concentrations from 0 g/L to 10 g/L (0.134 M). Growing the bacteria in a dual carbon source of glycerol (butanol producer) and glucose (not a butanol producer) resulted in the formation of higher butanol titers. Hence, for the studies on the homeoviscous response during endogenous butanol formation, cells were grown in glucose (10 g/L) and glycerol (10 g/L to 50 g/L), resulting in butanol formation between 0 g/L and 10 g/L. The membranes were extracted from the stationary phase cultures and the extracted membranes were analyzed using 1 H-NMR