Cooperative effects of fatty acids and n-butanol on lipid membrane phase behavior.

Biodiesel-derived crude glycerol can be fermented to produce n-butanol, which is a platform chemical for biorefining and a biofuel. One limitation to crude glycerol fermentation is the presence of long-chain fatty acids (FAs) that can partition into cellular membranes, leading to membrane fluidization and interdigitation, which can inhibit cellular function. In this work, we have examined the phase behavior of dipalmitoylphosphatidylcholine (DPPC, C16:0) membranes and the membrane partitioning of n-butanol as a function of FA degree of unsaturation (steric, oleic, and linoleic acids) using differential scanning calorimetry (DSC) and monolayer surface pressure studies. All three FAs at 15mol% (85mol% DPPC) prevented interdigitation by n-butanol based on the DSC results. n-Butanol partitioning and membrane expansion was greatest for DPPC/oleic acid membranes, where monounsaturated oleic acid (OA, C18:1) was miscible in gel and fluid phase DPPC. Saturated steric acid (SA, C18:0), which ordered the membranes and yielded a SA-rich phase during melting, led to a modest increase in n-butanol partitioning compared to DPPC alone. Di-unsaturated linoleic acid (LA, C18:2), which disordered the membranes and phase separated, had little affect on n-butanol partitioning into the DPPC-rich phases. The effects of OA and LA are attributed to the additional interfacial area provided by these FAs due to acyl tail 'kinks' at the carbon double bonds. These results show that exogenous FAs can partition into membranes, impacting n-butanol partitioning and acting cooperatively with n-butanol to alter membrane structure.

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

Clostridium pasteurianum ATCC 6013 achieves high n-butanol production when glycerol is used as the sole carbon source. In this study, the homeoviscous membrane response of C. pasteurianum ATCC 6013 has been examined through n-butanol challenge experiments. Homeoviscous response is a critical aspect of n-butanol tolerance and has not been examined in detail for C. pasteurianum. Lipid membrane compositions were examined for glycerol fermentations with n-butanol production, and during cell growth in the absence of n-butanol production, using gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H-NMR). Membrane stabilization due to homeoviscous response was further examined by surface pressure-area (π-A) analysis of membrane extract monolayers. C. pasteurianum was found to exert a homeoviscous response that was comprised of an increase lipid tail length and a decrease in the percentage of unsaturated fatty acids with increasing n-butanol challenge. This led to a more rigid or stable membrane that counteracted n-butanol fluidization. This is the first report on the changes in the membrane lipid composition during n-butanol production by C. pasteurianum ATCC 6013, which is a versatile microorganism that has the potential to be engineered as an industrial n-butanol producer using crude glycerol.

n-Butanol partitioning into phase-separated heterogeneous lipid monolayers.

Cellular adaptation to elevated alcohol concentration involves altering membrane lipid composition to counteract fluidization. However, few studies have examined the biophysical response of biologically relevant heterogeneous membranes. Lipid phase behavior, molecular packing, and elasticity have been examined by surface pressure-area (π-A) analysis in mixed monolayers composed of saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated dioleoylphosphatidylcholine (DOPC) as a function of DOPC and n-butanol concentration. n-Butanol partitioning into DPPC monolayers led to lipid expansion and increased elasticity. Greater lipid expansion occurred with increasing DOPC concentration, and a maximum was observed at equimolar DPPC:DOPC consistent with n-butanol partitioning between coexisting liquid expanded (LE, DOPC) phases and liquid condensed (LC, DPPC) domains. This led to distinct changes in the size and morphology of LC domains. In DOPC-rich monolayers the effect of n-butanol adsorption on π-A behavior was less pronounced due to DOPC tail kinking. These results point to the importance of lipid composition and phase coexistence on n-butanol partitioning and monolayer restructuring.

Role of ionic strength on n-butanol partitioning into anionic dipalmitoyl phosphatidylcholine/phosphatidylglycerol vesicles.

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 (PGs), but little is known regarding alcohol partitioning into anionic membranes, particularly for n-butanol. This work examines the effects of lipid charge on n-butanol partitioning into anionic membrane vesicles composed of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) in the absence and presence of salt (phosphate-buffered saline, PBS; 0.152 and 1.52 M). Above 0.135 M n-butanol, the membranes were interdigitated irrespective of DPPG or salt concentration, consistent with previous results for neutral membranes, such as DPPC. Increasing salt concentration led to greater n-butanol partitioning in DPPC membranes and caused aggregation/fusion. However, aggregation/fusion was prevented with increasing DPPG concentration (i.e., increasing membrane charge) and 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 the best of our knowledge to examine the effects of n-butanol partitioning on model cell membranes composed of negatively charged lipids in the presence of salts.

n-Butanol partitioning and phase behavior in DPPC/DOPC membranes.

Membrane phase behavior and fluidization have been examined in heterogeneous membranes composed of dipalmitoylphosphatidylcholine (DPPC, a saturated lipid) and dioleoylphosphatidylcholine (DOPC, an unsaturated lipid) at n-butanol concentrations below and above the interdigitation threshold of DPPC. Our results show that the presence of DOPC did not influence the interdigitation concentration of n-butanol on DPPC (0.1-0.13 M) despite the fact that DOPC increased n-butanol partitioning into the membranes. When DPPC was the continuous phase, up to equimolar DPPC:DOPC, n-butanol partitioning into gel or interdigitated DPPC was only slightly affected by the presence of DOPC. In this case a "cooperative effect" of DOPC + n-butanol eliminated the DPPC pretransition phase and yielded an untilted gel-like phase. When DOPC was the continuous phase, more n-butanol was needed to cause DPPC interdigitation (0.2 M), which was attributed to n-butanol residing at the interface between DOPC and DPPC domains. To our knowledge, this is the first study to examine the effects of n-butanol partitioning on membranes composed of saturated and unsaturated lipids that exhibit coexisting phase states.

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by Clostridium pasteurianum ATCC 6013.

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.