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 (1H-NMR). Membrane stabilization due to homeoviscous response was further examined by surface pressure–area (π–A) analysis of membrane extract monolayers. C. pasteurianum was found to exert a homeoviscous response that was comprised of an increase lipid tail length and a decrease in the percentage of unsaturated fatty acids with increasing n-butanol challenge. This led to a more rigid or stable membrane that counteracted n-butanol fluidization. This is the first report on the changes in the membrane lipid composition during n-butanol production by C. pasteurianum ATCC 6013, which is a versatile microorganism that has the potential to be engineered as an industrial n-butanol producer using crude glycerol

Role of lipid saturation in modulating the effects of n-butanol on membrane phase behavior

Butanol has been identified by the US Department of Energy as an alternative fuel and a platform chemical for biorefining. When produced by fermentation, butanol concentrations are often low because of the low butanol tolerance of microorganisms. Low tolerance is attributed in part to butanol fluidization of cell membranes. One mechanism by which microbes adapt to butanol is by changing the ratio of saturated to unsaturated membrane lipids. To date, little is known regarding how the ratio of saturated to unsaturated lipids modulates the effects of butanol fluidization. In this research we examined the effects of n-butanol on the fluidity and phase behavior of synthetic lipid membranes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) using fluorescence anisotropy and differential scanning calorimetry (DSC). For this system, temperatures below the melting temperature of DPPC (T m = 42 °C) yielded coexisting gel DPPC and fluid DOPC phases, while temperatures above T m yielded a fluid phase. DPPC alone exhibited membrane fluidization at butanol concentrations \u3c 10 g/l and interdigitation at concentrations \u3e 10 g/l butanol. These results, coupled with the large melting hysteresis that was observed between heating and cooling scans due to interdigitation, are consistent with previous studies. With increasing DOPC concentration (up to a 1:3 molar ratio of DPPC/DOPC) the fluidizing effects of n-butanol were enhanced in the coexisting gel DPPC phases. This effect may be attributed to greater partitioning of n-butanol via favorable packing within DOPC phases where the acyl tails are kinked due to the double bonds. The effects of DOPC concentration on DPPC interdigitation are less clear. T m hysteresis was reduced while the melting enthalpy hysteresis was increased with increasing DOPC concentration. This may reflect differences by which DOPC inhibits or enhances DPPC interdigitation with heating and cooling, or the partitioning behavior of n-butanol between DPPC and DOPC phases

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 nbutanol 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 nbutanol 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 nbutanol partitioning on membranes composed of saturated and unsaturated lipids that exhibit coexisting phase states. © 2012 American Chemical Society

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. © 2013 American Chemical Society

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. © 2011 Springer-Verlag

Role of Ionic Strength on <i>n</i>‑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 <i>n</i>-butanol. This work examines the effects of lipid charge on <i>n</i>-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 <i>n</i>-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 <i>n</i>-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 <i>n</i>-butanol partitioning, and subsequent changes in membrane and vesicle structure, was driven by a balance between the “salting-out” of <i>n</i>-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 <i>n-</i>butanol partitioning on model cell membranes composed of negatively charged lipids in the presence of salts