Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

KL₄ is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL₄ binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL₄ is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL₄ is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL₄, and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL₄. Additionally, a clear effect of KL₄ on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL₄ causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL₄ to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment.     

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via ²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.     

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and ³¹P and ²H solid-state NMR spectroscopy. SP-B_59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B_59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B_59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B_59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL₄, a peptide mimetic of SP-B which was originally designed using SP-B_59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.     

Residue specific partitioning of KL4 into phospholipid bilayers

KL₄, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL₄ and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL₄ on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL₄, and the partitioning depths of specific KL₄ residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL₄ induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL₄ (V.C. Antharam et al., 2009), we are able to distinguish how KL₄ affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL₄ also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL₄ are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.     

Kinematic viscosity of therapeutic pulmonary surfactants with added polymers

The addition of various polymers to pulmonary surfactants improves surface activity in experiments both in vitro and in vivo. Although the viscosity of surfactants has been investigated, the viscosity of surfactant polymer mixtures has not. In this study, we have measured the viscosities of Survanta and Infasurf with and without the addition of polyethylene glycol, dextran or hyaluronan. The measurements were carried out over a range of surfactant concentrations using two concentrations of polymers at two temperatures. Our results indicate that at lower surfactant concentrations, the addition of any polymers increased the viscosity. However, the addition of polyethylene glycol and dextran to surfactants at clinically used concentrations can substantially lower viscosity. Addition of hyaluronan at clinical surfactant concentrations slightly increased Infasurf viscosity and produced little change in Survanta viscosity. Effects of polymers on viscosity correlate with changes in size and distribution of surfactant aggregates and the apparent free volume of liquid as estimated by light microscopy. Aggregation of surfactant vesicles caused by polymers may therefore not only improve surface activity as previously shown, but may also affect viscosity in ways that could improve surfactant distribution in vivo.     

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum

Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfactant proteins SP-B and SP-C obtained from pig lung lavage were added either singly or combined at two concentrations. Also, non-ionic polymers polyethylene glycol and dextran and the anionic polymer hyaluronan were added either singly or in pairs with hyaluronan included. Non-ionic polymers work by different mechanisms than anionic polymers, thus the purpose of placing them together in the same surfactant mixture was to evaluate if the combination would show enhanced beneficial effects. The resulting surfactant mixtures were studied in the presence or absence of serum. A modified bubble surfactometer was used to evaluate surface activities. Mixtures that included both SP-B and SP-C plus hyaluronan and either dextran or polyethylene glycol were found to be the most resistant to inhibition by serum. These mixtures, as well as some with either SP-B or SP-C with combined polymers were as or more resistant to inactivation than native surfactant. These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries.     

KL₄ peptide induces reversible collapse structures on multiple length scales in model lung surfactant

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).     

KL4 Peptide Induces Reversible Collapse Structures on Multiple Length Scales in Model Lung Surfactant

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).     

Calcium Ions as “Miscibility Switch”: Colocalization of Surfactant Protein B with Anionic Lipids under Absolute Calcium Free Conditions

One of the main determinants of lung surfactant function is the complex interplay between its protein and lipid components. The lipid specificity of surfactant protein B (SP-B), however, and the protein's ability to selectively squeeze out lipids, has remained contradictory. In this work we present, for the first time to our knowledge, by means of time-of-flight secondary ion mass spectrometry chemical imaging, a direct evidence for colocalization of SP-B as well as its model peptide KL₄ with negatively charged dipalmitoylphosphatidylglycerol under absolute calcium free conditions. Our results prove that protein/lipid localization depends on the miscibility of all surfactant components, which itself is influenced by subphase ionic conditions. In contrast to our earlier studies reporting SP-B/KL₄ colocalization with zwitterionic dipalmitoylphosphatidylcholine, in the presence of even the smallest traces of calcium, we finally evidence an apparent reversal of protein/lipid mixing behavior upon calcium removal with ethylene diamine tetraacetic acid. In addition, scanning force microscopy measurements reveal that by depleting the subphase from calcium ions the protrusion formation ability of SP-B or KL₄ is not hampered. However, in the case of KL₄, distinct differences in protrusion morphology and height are visible. Our results support the idea that calcium ions act as a “miscibility switch” in surfactant model systems and probably are one of the major factors steering lipid/protein mixing behavior as well as influencing the protein's protrusion formation ability.     

Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.     

A freeze-fracture transmission electron microscopy and small angle x-ray diffraction study of the effects of albumin, serum, and polymers on clinical lung surfactant microstructure

Freeze-fracture transmission electron microscopy shows significant differences in the bilayer organization and fraction of water within the bilayer aggregates of clinical lung surfactants, which increases from Survanta to Curosurf to Infasurf. Albumin and serum inactivate all three clinical surfactants in vitro; addition of the nonionic polymers polyethylene glycol, dextran, or hyaluronic acid also reduces inactivation in all three. Freeze-fracture transmission electron microscopy shows that polyethylene glycol, hyaluronic acid, and albumin do not adsorb to the surfactant aggregates, nor do these macromolecules penetrate the interior water compartments of the surfactant aggregates. This results in an osmotic pressure difference that dehydrates the bilayer aggregates, causing a decrease in the bilayer spacing as shown by small angle x-ray scattering and an increase in the ordering of the bilayers as shown by freeze-fracture electron microscopy. Small angle x-ray diffraction shows that the relationship between the bilayer spacing and the imposed osmotic pressure for Curosurf is a screened electrostatic interaction with a Debye length consistent with the ionic strength of the solution. The variation in surface tension due to surfactant adsorption measured by the pulsating bubble method shows that the extent of surfactant aggregate reorganization does not correlate with the maximum or minimum surface tension achieved with or without serum in the subphase. Albumin, polymers, and their mixtures alter the surfactant aggregate microstructure in the same manner; hence, neither inhibition reversal due to added polymer nor inactivation due to albumin is caused by alterations in surfactant microstructure.     

Probing the steric barrier of nonionic surfactant vesicles with melittin

The role of the surface polymer brush of nonionic surfactant vesicles (NSV) in inhibiting interactions with small membrane-perturbing molecules was investigated using the bee venom peptide melittin as a probe. The interaction between melittin and NSV was compared with that of distearoylphosphatidylcholine (DSPC) vesicles and sterically stabilised liposomes (SSL) containing 5 mol% pegylated distearoylphosphatidylethanolamine (DSPE.E₄₄). The degree of melittin interaction with the various vesicles was determined by measuring peptide binding and folding, using intrinsic tryptophan fluorescence and circular dichroism respectively, in addition to monitoring the release of encapsulated carboxyfluorescein dye. NSV composed of 1,2-di-O-octadecyl-rac-glyceryl-3-(ω-dodecaethylene glycol) (2C₁₈E₁₂) showed a strong affinity for melittin, whilst exhibiting ~ 50% less bound peptide than SSL. 2C₁₈E₁₂:Chol vesicles showed reduced melittin interaction, in a manner consistent with Chol incorporation into DSPC vesicles. These results are discussed with respect to the effect of Chol on the in-plane order of 2C₁₈E₁₂ bilayers and consequent attenuation of hydrophobic interactions with the peptide. NSV formed from equimolar mixtures of polyoxyethylene-n-stearoyl ethers C₁₈E₂ and C₁₈E₂₀ showed a greater interaction with melittin than 2C₁₈E₁₂. However, replacing C₁₈E₂₀ with C₁₈E₁₀ was sufficient to achieve an attenuation of melittin interaction similar to that observed in 2C₁₈E₁₂:Chol vesicles. This indicates that the presence of surface polymer brush alone may confer resistance to melittin, provided hydrophobic interactions between the peptide and the vesicles can be minimised, through improved in-plane bilayer order.     

Lipid Polymorphism Induced by Surfactant Peptide SP-B1-25

Pulmonary surfactant protein B (SP-B) is an essential protein for lowering surface tension in the alveoli. SP-B_1-25, a peptide comprised of the N-terminal 25 amino-acid residues of SP-B, is known to retain much of the biological activity of SP-B. Circular dichroism has shown that when SP-B_1-25 interacts with negatively charged lipid vesicles, it contains significant helical structure for the lipid compositions and peptide/lipid ratios studied here. The effect of SP-B_1-25 on lipid organization and polymorphisms was investigated via DSC, dynamic light scattering, transmission electron microscopy, and solid-state NMR spectroscopy. At 1-3 mol% peptide and physiologic temperature, SP-B_1-25 partitions at the interface of negatively charged PC/PG lipid bilayers. In lipid mixtures containing 1-5 mol% peptide, the structure of SP-B_1-25 remains constant, but ²H and ³¹P NMR spectra show the presence of an isotropic lipid phase in exchange with the lamellar phase below the T_m of the lipids. This behavior is observed for both DPPC/POPG and POPC/POPG lipid mixtures as well as for both the PC and PG components of the mixtures. For 1-3 mol% SP-B_1-25, a return to a single lamellar phase above the lipid mixture T_m is observed, but for 5 mol% SP-B_1-25 a significant isotropic component is observed at physiologic temperatures for DPPC and exchange broadening is observed in ²H and ³¹P NMR spectra of the other lipid components in the two mixtures. DLS and TEM rule out the formation of micellar structures and suggest that SP-B_1-25 promotes the formation of a fluid isotropic phase. The ability of SP-B_1-25 to fuse lipid lamellae via this mechanism, particularly those enriched in DPPC, suggests a specific role for the highly conserved N-terminus of SP-B in the packing of lipid lamellae into surfactant lamellar bodies or in stabilizing multilayer structures at the air-liquid interface. Importantly, this behavior has not been seen for the other SP-B fragments of SP-B_8-25 and SP-B_59-80, indicating a critical role for the proline rich first seven amino acids in this protein.     

Exposure to Polymers Reverses Inhibition of Pulmonary Surfactant by Serum,Meconium, or Cholesterol in the Captive Bubble Surfactometer

Dysfunction of pulmonary surfactant in the lungs is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome. Serum, cholesterol, and meconium have been described as inhibitory agents of surfactant’s interfacial activity once these substances appear in alveolar spaces during lung injury and inflammation. The deleterious action of these agents has been only partly evaluated under physiologically relevant conditions. We have optimized a protocol to assess surfactant inhibition by serum, cholesterol, or meconium in the captive bubble surfactometer. Specific measures of surface activity before and after native surfactant was exposed to inhibitors included i), film formation, ii), readsorption of material from surface-associated reservoirs, and iii), interfacial film dynamics during compression-expansion cycling. Results show that serum creates a steric barrier that impedes surfactant reaching the interface. A mechanical perturbation of this barrier allows native surfactant to compete efficiently with serum to form a highly surface-active film. Exposure of native surfactant to cholesterol or meconium, on the other hand, modifies the compressibility of surfactant films though optimal compressibility properties recover on repetitive compression-expansion cycling. Addition of polymers like dextran or hyaluronic acid to surfactant fully reverses inhibition by serum. These polymers also prevent surfactant inhibition by cholesterol or meconium, suggesting that the protective action of polymers goes beyond the mere enhancement of interfacial adsorption as described by depletion force theories.     

Inhibition of surfactant function by copper-zinc superoxide dismutase (CuZn-SOD)

Haddad, Imad Y., Bedford Nieves-Cruz, and Sadis Matalon.Inhibition of surfactant function by copper-zinc superoxide dismutase (CuZn-SOD). J. Appl.Physiol. 83(5): 1545-1550, 1997.The efficacy ofantioxidant enzymes to limit oxidant lung injury by instillation withsurfactant mixtures in preterm infants with hyaline membrane disease isunder investigation. However, there is concern that instillation ofproteins in the alveolar space may inactivate pulmonary surfactant. Westudied the effects of bovine copper-zinc superoxide dismutase(CuZn-SOD) on the biophysical properties of two distinct surfactantpreparations. Incubation of calf lung surfactant extract (CLSE, 1 mgphospholipid/ml) and Exosurf (0.1 mg phospholipid/ml) with CuZn-SOD(1-10 mg/ml) prevented the fall of surface tension at minimalbubble radius (T_min) to lowvalues with dynamic compression in a pulsating bubble surfactometer. CuZn-SOD also enhanced the sensitivity to inactivation by albumin, normal human serum, and after treatment with peroxynitrite. The inhibitory effects of CuZn-SOD on CLSE, but not Exosurf, were abolishedat high lipid concentrations (3 mg/ml) and after the addition of humansurfactant protein A (by weight). We conclude that CuZn-SOD mayinterfere with the surface activity of surfactant mixtures, leading todecreased effectiveness of surfactant replacement therapy.

     

Inhibition of pulmonary surfactant adsorption by serum and the mechanisms of reversal by hydrophilic polymers: theory

A theory based on the Smolukowski analysis of colloid stability shows that the presence of charged, surface-active serum proteins at the alveolar air-liquid interface can severely reduce or eliminate the adsorption of lung surfactant from the subphase to the interface, consistent with the observations reported in the companion article (pages 1769-1779). Adding nonadsorbing, hydrophilic polymers to the subphase provides a depletion attraction between the surfactant aggregates and the interface, which can overcome the steric and electrostatic resistance to adsorption induced by serum. The depletion force increases with polymer concentration as well as with polymer molecular weight. Increasing the surfactant concentration has a much smaller effect than adding polymer, as is observed. Natural hydrophilic polymers, like the SP-A present in native surfactant, or hyaluronan, normally present in the alveolar fluids, can enhance adsorption in the presence of serum to eliminate inactivation.     

Synthesis,copolymerization and peptide-modification of carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) for neural electrode interfaces

Background

Conjugated polymers have been developed as effective materials for interfacing prosthetic device electrodes with neural tissue. Recent focus has been on the development of conjugated polymers that contain biological components in order to improve the tissue response upon implantation of these electrodes.

Methods

Carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) monomer was synthesized in order to covalently bind peptides to the surface of conjugated polymer films. EDOTacid was copolymerized with EDOT monomer to form stable, electrically conductive copolymer films referred to as PEDOT-PEDOTacid. The peptide GGGGRGDS was bound to PEDOT-PEDOTacid to create peptide functionalized PEDOT films.

Results

The PEDOT-PEDOTacid-peptide films increased the adhesion of primary rat motor neurons between 3 and 9 times higher than controls, thus demonstrating that the peptide maintained its biological activity.

Conclusions

The EDOT-acid monomer can be used to create functionalized PEDOT-PEDOTacid copolymer films that can have controlled bioactivity.

General Significance

PEDOT-PEDOTacid-peptide films have the potential to control the behavior of neurons and vastly improve the performance of implanted electrodes. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Basis for formulating biosurfactant mixtures to achieve ultra low interfacial tension values against hydrocarbons

Biosurfactants could potentially replace or be used in conjunction with synthetic surfactants to provide for more cost-effective subsurface remediation. The design of surfactant formulations that are effective in lowering interfacial tension (IFT), which is necessary to mobilize entrapped hydrocarbons, requires information about the surface-active agent (surfactant) and the targeted non-aqueous phase liquids (NAPL). We hypothesized that biosurfactant and synthetic surfactant mixtures can be formulated to provide the appropriate hydrophobic/hydrophilic conditions necessary to produce low IFT against NAPLs, and that such mixtures will produce synergism that make them more effective than individual biosurfactants or synthetic surfactants. Our work tested the interfacial activity of biosurfactants from individual strains and mixtures of biosurfactants from different strains with and without a synthetic surfactant. Multiple regression analysis showed that, for lipopeptide biosurfactants produced by various Bacillus species, the interfacial activity against toluene depended on the relative proportions of 3-OH-C₁₄, C₁₅, C₁₆, and C₁₈ in the fatty acid tail. As the fatty acid composition became more heterogeneous the system produced lower IFT against toluene. In mixtures of lipopeptide biosurfactants with the more hydrophilic, rhamnolipid biosurfactant, the IFT against toluene decreased as the percentage of the 3-OH C₁₄ fatty acid increased in the lipopeptide. Mixtures of lipopeptide biosurfactants with the more hydrophobic synthetic surfactant, C12, C13-8PO SO₄Na, were able to produce low IFT against hexane and decane. In general, we found that lipopeptide biosurfactants with a heterogeneous fatty acid composition or mixtures of lipopeptide and rhamnolipid biosurfactants lowered the IFT against hydrophilic NAPLs. Conversely, mixtures of lipopeptide biosurfactants with a more hydrophobic synthetic surfactant lowered the IFT against hydrophobic NAPLs.     

Emulsifying and surface active agents from Corynebacterium hydrocarboclastus

A Corynebacterium hydrocarboclastus culture isolated in our laboratory (see, Zajic and Knettig, Developments in Industrial Microbiology, 1971, p. 87) has been shown to produce an extracellular biopolymer with emulsifying properties when grown on a mixture of linear hydrocarbons. This microorganism was found to grow well on a variety of carbohydrates and hydrocarbons. However, the best substrates were pure linear hydrocarbons and particularly, n-C₁₂, n-C₁₃, and n-C₁₄. The substrates supporting good growth gave good polymer production. Maximum cell mass of 10–11 g/liter and a maximum amount of polymer of 5–6 g/liter were recorded. The polymers recovered from the different substrates were found to be complex molecules or mixtures with a protein, a lipid, and a carbohydrate moiety. All the polymers are surface active and have two critical micelle concentrations.