Zdravko Lalchev

Rhamnolipid biosurfactants produced by Renibacterium salmoninarum 27BN during growth on n-hexadecane.

A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize nhexadecane as sole substrate. Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity. Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa. Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells.

Interactions of Meibomian gland secretion with polar lipids in Langmuir monolayers

The surface interactions of Meibomian gland secretion (MGS) with polar lipid (PL), Egg Sphingomyelin (SM) or Dipalmitoylphosphatidylcholine (DPPC), are studied in mixed pseudo-binary films formed at the air/water interface of Langmuir surface balance. The behavior of the mixed films during slow quasi-equilibrium compression and during fast dynamic compression-decompression is registered by measurements of surface pressure and surface potential, and by monitoring film morphology with Brewster Angle Microscopy (BAM). Quasi-equilibrium compression isotherms are used to calculate the excess Gibbs and Helmholtz energy of mixing between MGS and PLs and thus to evaluate the interactions between the lipid compounds at the interface. The effects of PLs on the mixed films elastic moduli of area compressibility, morphology and capability to attain high surface pressures are also examined. PLs interact with MGS with different strength and in different manner: MGS-SM interaction is weak and might lead to interfacial disaggregation of the thick meibium domains when SM is in excess, while MGS-DPPC interaction is strong and results in the formation of thick lipid aggregates. Both PLs increase the mixed films reciprocal compressibility and capability to achieve higher surface pressures. The results demonstrate that in vitro studies of the surface interactions between MGS and PLs might be beneficial in the selection of PLs for artificial tear formulations and for examination on molecular scale of the possible role of PLs at the ocular surface.

Surface chemistry study of the interactions of benzalkonium chloride with films of meibum, corneal cells lipids, and whole tears.

PURPOSE To perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents. METHODS The interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air-water interface were examined in vitro during controlled compression-expansion of the film area by a Langmuir surface balance, surface potential measurements, and pendant drop-axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the samples lateral elasticity and capability of compressing and spreading during dynamic area changes. Lipid film morphology was monitored by Brewster angle microscopy. The viability of BAC-treated Statens Seruminstitut rabbit cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001% to 0.02%. RESULTS In the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in (1) impaired lipid spread and formation of discontinuous nonuniform surface layers, (2) increased surface pressure-area hysteresis during compression and expansion, and (3) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure, and their magnitude increased with BAC concentration. CONCLUSIONS The surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well explain the TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.

MOLECULAR LATERAL DIFFUSION IN MODEL MEMBRANE SYSTEMS

Abstract The effect of both interface type and temperature on the lateral diffusion coefficient (D) of the fluorescent probe NBD-PE in egg PC films of different types has been studied by FRAP. Three model membrane systems (monolayer, black lipid membrane (BLM) and foam films (FF) of different thickness’) have been investigated. The monolayer at an air/water interface has been taken as a point of reference for the other two types of interface, probing the effect of introducing a second monolayer in close proximity to the first one. The results on monolayers showed an exponential relation between the area and diffusion. In the case of FFs going from thinner to thicker films saw a corresponding increase in the probe D, ultimately approaching its characteristic value in monolayers of similar free volume, at a macroscopic air/water interface above an infinitely thick subphase. The fact that the diffusion is clearly a function of thickness of FFs indicates a complex regime of modal coupling in which the headgroup interaction plays an important role. The diffusion coefficients in BLMs were found to be higher than those in the equivalent monolayers and FFs. A comparative analysis of D in monolayers, FFs of different thickness and BLMs has demonstrated the importance of monolayer orientation in the different model systems for the rate of molecular mobility in the plane of model interfaces. It also highlights the role of headgroup–headgroup interactions normal to the plane of the film which are predominant in FF systems.

Surfactant liquid and black foam film formation and stability in vitro and correlative conditions in vivo

Abstract We studied foam film formation by three preparations that are used as surfactant replacement therapy by injection into the lungs of neonatal infants with surfactant insufficiency (“respiratory distress syndrome”). The preparations contain either all putative hydrophobic components of the normal surfactant system (prepared from bovine lung lavage, Infasurf (IN); prepared from minced bovine lung tissue, Survanta (SU)) or a single component of the system, dipalmitoylphosphatidylcholine (Exosurf Neonatal (EX)). Foam film formation is relevant directly to normal surfactant structure and function in vivo in the neonatal period (Pediatr. Res. 12 (1978) 1070–1076). We assessed morphology and stability of liquid to black film formation and its dependence on substrate concentration C, adsorption time, temperature T, “capillary pressure” Pc, film drainage time τ0–1 and black film formation times τ1–2. We found that (1) in contrast with the other preparations, IN regularly formed stable black films at the lowest C (“threshold C” Ct) under all conditions of T (22 and 37°C) and Pc (0.3, 0.4, 1.2 and 2.4 × 103 dyn cm−2), (2) EX also formed black films but differed from IN in that Ct was higher, film integrity required a longer adsorption time and black film formation was blocked (instability) at Pc values of 1.2 and 2.4 × 103 dyn cm−2 and (3) SU did not form black films but instead formed slowly (long τ0–1 and τ1–2), at relatively high Ct, viscosity-dependent inhomogeneous atypical (“rheological”) films containing aggregates of material in the preparation. Extrapolation to in-vivo conditions of C, T and Pc indicates that both IN and SU (but not EX) may form stable foam films in situ, that film formation by IN is the more efficient process and that stable surfactant foam films, formed naturally in vivo, are consistent with both the in-vitro characteristics described here and correlative lung function in vivo.

Pure and mixed lipid black foam films as models of membrane fusion

Black foam films (BFF) from water solutions of the phospholipid dilauroyl lecithin (DLL) with admixtures of palmitoyl lysolecithin (Lyso) were formed. Microscopic BFF were studied by the method of Scheludko and Exerowa. The formation probability for BFF and the BFF lifetime in a black state before film rupture were measured as functions of the film composition. At a fixed overal lipid concentration it was shown that an increased percentage of Lyso exponentially increased the lifetime of the film up to the CMC of Lyso. This stabilizing Lyso effect nicely corresponds with its stabilizing action on the waiting time for fusion of two contacting black lipid membranes (BLM), as found by Chernomordik et al. In contrast, Lyso is known to destabilize a single BLM. In this way we have found experimental proof of our earlier prediction that Lyso should have opposite effects on the lifetimes of BLM and BFF. In addition, we have shown for the first time that foam films made of lipids are a convenient model for monlayer membrane fusion studies. This model is characterized by its simplicity and experimental reliability and provides a means for quick screening of the fusogenic capacity of various amphiphilic and hydrophilic admixtures.

Effects of pulmonary surfactant proteins SP-B and SP-C and calcium ions on the surface properties of hydrophobic fractions of lung surfactant

Abstract This study focused on two hydrophobic fractions (HF-A and HF-B) isolated from porcine lung surfactant (LS) that had similar phospholipid composition, but HF-A consisted of the hydrophobic LS specific proteins (SP-B and SP-C), in contrast to HF-B. Monolayers spread in a Langmuir trough were formed at the air/water interface of both fractions and the rate of adsorption-desorption and the respreading potential of the LS constituents was studied during six consecutive compression/decompression cycles of the monolayers. By drawing a comparison between the behavior of HF-A and HF-B monolayers on the subphase of 150 mm NaCl, either with or without additional Ca2+, we estimated the role of hydrophobic LS proteins and Ca2+ ions for LS surface activity. The results demonstrated much higher ability of the HF-A sample, compared to HF-B, to maintain lower surface tension (γ) during monolayer compression and its better respreading capacity during decompression. For instance, at a surface concentration corresponding to 80 Å2 per phospholipid molecule, the HF-A monolayers showed a much lower γmax value (surface tension at 100% of the trough area), being ca. 31.0 mN/m, compared to the HF-B monolayers (γmax≅ 62.0 mN/m). The surface tension after compression to 20% of the initial area (γmin) reached ca. 7.0 and 19.0 mN/m in the HF-A and HF-B monolayers, respectively. Better respreading of the HF-A monolayers compared to the HF-B monolayers was due to the faster adsorption and spreading of LS phospholipids during decompression, facilitated by the hydrophobic proteins. As the phospholipid composition of both fractions was similar, we showed that the hydrophobic surfactant proteins were responsible also for the prevention of the irreversible loss of material from the surface during monolayer compression/decompression. The effects observed demonstrated also that the hydrophobic surfactant proteins were the stronger determinant, compared with Ca2+ ions, for the surface tension decrease and respreading of the monolayers during film compression/decompression. For instance, when the HF-A monolayers were spread on a subphase with an additional 5 mm Ca2+ ion content, no significant changes were detected in the γmin and γmax values between the first and sixth cycle, compared to the monolayers spread on a subphase of 150 mm NaCl only. However, in the absence of positively charged SP-B and SP-C (HF-B sample) in highly compressed monolayers, Ca2+ ions were able to cause the effects shown by SP-B and SP-C, although to a less extent. The role of the electrostatic and hydrophobic interactions is discussed for the better respreading of LS components in the presence of LS proteins and Ca2+ ions.

Evaluation of Different Carbon Sources for Growth and Biosurfactant Production by Pseudomonas fluorescens Isolated from Wastewaters

The indigenous strain Pseudomonas fluorescens, isolated from industrial wastewater, was able to produce glycolipid biosurfactants from a variety of carbon sources, including hydrophilic compounds, hydrocarbons, mineral oils, and vegetable oils. Hexadecane, mineral oils, vegetable oils, and glycerol were preferred carbon sources for growth and biosurfactant production by the strain. Biosurfactant production was detected by measuring the surface and interfacial tension, rhamnose concentration and emulsifying activity. The surface tension of supernatants varied from 28.4 mN m-1 with phenanthrene to 49.6 mN m-1 with naphthalene and heptane as carbon sources. The interfacial tension has changed in a narrow interval between 6.4 and 7.6 mN m-1. The emulsifying activity was determined to be highest in media with vegetable oils as substrates. The biosurfactant production on insoluble carbon sources contributed to a signifi cant increase of cell hydrophobicity and correlated with an increased growth of the strain on these substrates. Based on these results, a mechanism of biosurfactant-enhanced interfacial uptake of hydrophobic substrates could be proposed as predominant for the strain. With hexadecane as a carbon source, the pH value of 7.0 - 7.2 and temperature of (28 ± 2) °C were optimum for growth and biosurfactant production by P. fluorescens cells. The increased specific protein and biosurfactant release during growth of the strain on hexadecane in the presence of NaCl at contents up to 2% could be due to increased cell permeability. The capability of P. fluorescens strain HW-6 to adapt its own metabolism to use different nutrients as energy sources and to keep up relatively high biosurfactant levels in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

Production and properties of biosurfactants from a newly isolated Pseudomonas fluorescens HW-6 growing on hexadecane

The newly isolated from industrial wastewater Pseudomonas fluorescens strain HW-6 produced glycolipid biosurfactants at high concentrations (1.4-2.0 g l-1) when grown on hexadecane as a sole carbon source. Biosurfactants decreased the surface tension of the air/ water interface by 35 mN m-1 and possessed a low critical micelle concentration value of 20 mg l-1, which indicated high surface activity. They efficiently emulsified aromatic hydrocarbons, kerosene, n-paraffins and mineral oils. Biosurfactant production contributed to a significant increase in cell hydrophobicity correlated with an increased growth of the strain on hexadecane. The results suggested that the newly isolated strain of Ps. fluorescens and produced glycolipid biosurfactants with effective surface and emulsifying properties are very promising and could find application for bioremediation of hydrocarbon-polluted sites.

Thin liquid films and monolayers of DMPC mixed with PEG and phospholipid linked PEG

In this work thin liquid films (TLFs) and monolayers at the air/water interface formed by dimyristoylphosphatidylcholine (DMPC) and by DMPC mixed with poly ethylene glycols (PEGs) and dimyristoylphosphatidylethanolamine (DMPE) linked PEGs were studied. Film forming dispersions were composed of two types of particles: liposomes and micelles. TLFs stability, threshold concentration Ct (i.e., the minimum one for stable film formation), and hydrodynamic behavior were measured. At equivalent conditions, DMPC films were Newton black films (real bilayers), while DMPE-PEGs films were much thicker with free water between the monolayers. DMPE-PEG addition to DMPC films caused both Ct decrease (depending on PEG moiety length and Mw) and change of TLF formation mechanism. TLFs’ hydrodynamic behavior also strongly depended on DMPE-PEG content and Mw. It was observed that thinning of the DMPC and DMPE-PEGs films continued to different film types and thickness, being much thicker for the latter films. Addition of free PEGs (PEG-200/6000) did not alter TLF type or stability, but changed TLF thinning time, confirming that free PEGs with Mw<8000 could not penetrate in the membrane and alter “near-membrane” water layer viscosity. Monolayer studies showed improved formation kinetics of both adsorbed and spread films, decrease of surface tension (equilibrium and dynamic), and of film compression/decompression histeresis area in DMPE-PEGs monolayers compared with DMPC pure films. Our study shows that combining the models of phospholipid TLFs and monolayers provide the opportunity to investigate the properties of membrane surface and to clarify some mechanisms of its interactions with membrane-active agents.