Microbial biosurfactants: current trends and applications in agricultural and biomedical industries.

Synthetic surfactants are becoming increasingly unpopular in many applications due to previously disregarded effects on biological systems and this has led to a new focus on replacing such products with biosurfactants that are biodegradable and produced from renewal resources. Microbially derived biosurfactants have been investigated in numerous studies in areas including: increasing feed digestibility in an agricultural context, improving seed protection and fertility, plant pathogen control, antimicrobial activity, antibiofilm activity, wound healing and dermatological care, improved oral cavity care, drug delivery systems and anticancer treatments. The development of the potential of biosurfactants has been hindered somewhat by the myriad of approaches taken in their investigations, the focus on pathogens as source species and the costs associated with large-scale production. Here, we focus on various microbial sources of biosurfactants and the current trends in terms of agricultural and biomedical applications.

Identification and characterisation of short chain rhamnolipid production in a previously uninvestigated, non-pathogenic marine pseudomonad.

This study aimed to identify and characterise biosurfactant compounds produced by bacteria associated with a marine eukaryotic phytoplankton bloom. One strain, designated MCTG214(3b1), was isolated by enrichment with polycyclic aromatic hydrocarbons and based on 16S rDNA, and gyrB sequencing was found to belong to the genus Pseudomonas, however not related to P. aeruginosa. Cell-free supernatant samples of strain MCTG214(3b1) at stationary phase showed significant reductions in surface tension. HPLC-MS and NMR analysis of these samples indicated the presence of five different rhamnolipid (RL) congeners. Di-rhamnolipids accounted for 87% relative abundance and all congeners possessed fatty acid moieties consisting of 8-12 carbons. PCR screening of strain MCTG214(3b1) DNA revealed homologues to the P. aeruginosa RL synthesis genes rhlA and rhlB; however, no rhlC homologue was identified. Using the Galleria mellonella larvae model, strain MCTG214(3b1) was demonstrated to be far less pathogenic than P. aeruginosa. This study identifies for the first time a significantly high level of synthesis of short chain di-rhamnolipids by a non-pathogenic marine Pseudomonas species. We postulate that RL synthesis in Pseudomonas sp. MCTG214(3b1) is carried out by enzymes expressed from rhlA/B homologues similar to those of P. aeruginosa; however, a lack of rhlC potentially indicates the presence of a second novel rhamnosyltransferase responsible for the di-rhamnolipid congeners identified by HPLC-MS.

Rhamnolipids and lactonic sophorolipids: natural antimicrobial surfactants for oral hygiene.

AIMS: To assess the efficacy of rhamnolipid (mixture of monorhamnolipid and dirhamnolipid congeners), purified monorhamnolipid, dirhamnolipid and lactonic sophorolipid biosurfactants against pathogens important for oral hygiene. METHODS AND RESULTS: Acquired and produced biosurfactants were fully characterized to allow the antimicrobial activity to be assigned to the biosurfactant congeners. Antimicrobial activity was assessed using the resazurin-aided microdilution method. Mixed rhamnolipid JBR425 (MR) and lactonic sophorolipids (LSLs) demonstrated the lowest minimum inhibitory concentration (MIC) which ranged between 100 and 400 µg ml-1 against Streptococcus mutans, Streptococcus oralis, Actinomyces naeslundii, Neisseria mucosa and Streptococcus sanguinis. Combining these biosurfactants with standard antimicrobial agents namely chlorhexidine, sodium lauryl sulphate, tetracycline HCl and ciprofloxacin showed a dramatic drop in the MIC values. In addition, in vitro studies demonstrated the biosurfactants' ability to prevent and disrupt oral pathogens biofilms. The increased permeability of microorganisms treated with biosurfactant, as shown using bisbenzimide dye, in part explains the inhibition effect. CONCLUSION: The results demonstrate that rhamnolipids and LSLs have the ability to inhibit oral pathogens both in planktonic and oral biofilm states. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings indicate the potential value of biosurfactants for both oral hygiene and the pharmaceutical industries since there is a serious need to reduce the reliance on synthetic antimicrobials and antibiotics.

A case-control study to identify risk factors for acute salmonellosis in New Zealand dairy herds, 2011-2012.

In late 2011 the New Zealand Ministry for Primary Industries reported an increase in confirmed laboratory diagnoses of salmonellosis in dairy herds. To identify risk factors for herd-level outbreaks of salmonellosis we conducted a case-control study of New Zealand dairy herds in 2011-2012. In a multivariable analysis, use of continuous feed troughs [adjusted odds ratio (aOR) 6·2, 95% confidence interval (CI) 2·0-20], use of pelletized magnesium supplements (aOR 10, 95% CI 3·3-33) and use of palm kernel meal as a supplementary feed (aOR 8·7, 95% CI 2·5-30) were positively associated with a herd-level outbreak of salmonellosis between 1 July 2011 and 31 January 2012. We conclude that supplementary feeds used on dairy farms (regardless of type) need to be stored and handled appropriately to reduce the likelihood of bacterial contamination, particularly from birds and rodents. Magnesium supplementation in the pelletized form played a role in triggering outbreaks of acute salmonellosis in New Zealand dairy herds in 2011-2012.

Pseudomonas aeruginosa biofilm disruption using microbial surfactants.

AIMS: To establish the ability of the rhamnolipids biosurfactants from Pseudomonas aeruginosa, in the presence and absence of caprylic acid and ascorbic acid, to disrupt bacterial biofilms, compared with the anionic alkyl sulphate surfactant Sodium dodecyl sulphate (SDS). METHODS AND RESULTS: Pseudomonas aeruginosa ATCC 15442 biofilms were disrupted by rhamnolipids at concentrations between 0·5 and 0·4 g l(-1) and with SDS at 0·8 g l(-1) . The combination of rhamnolipids 0·4 g l(-1) and caprylic acid at 0·1 g l(-1) showed a remarkable effect on biofilm disruption and cell killing. After 30 min of treatment most of the biofilm was disrupted and cell viability was significantly reduced. Neither caprylic acid nor ascorbic acid has any effect on biofilm disruption at 0·1 g l(-1) . SDS is an effective antimicrobial agent; however, in the presence of caprylic acid its effect was neutralized. CONCLUSIONS: The results show that rhamnolipids at low concentration in the presence of caprylic acid are promising molecules for inhibition/disruption of biofilms formed by Ps. aeruginosa ATCC 15442. SIGNIFICANCE AND IMPACT OF THE STUDY: The disruption of biofilms has major significance in many industrial and domestic cleaning applications and in medical situations.

Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds.

Solution self-assembly and adsorption at the air-water interface of the monorhamnose and dirhamnose rhamnolipids and their mixtures.

The self-assembly in solution and adsorption at the air-water interface, measured by small-angle neutron scattering, SANS, and neutron reflectivity, NR, of the monorhamnose and dirhamnose rhamnolipids (R1, R2) and their mixtures, are discussed. The production of the deuterium-labeled rhamnolipids (required for the NR studies) from a Pseudomonas aeruginosa culture and their separation into the pure R1 and R2 components is described. At the air-water interface, R1 and R2 exhibit Langmuir-like adsorption isotherms, with saturated area/molecule values of about 60 and 75 Å(2), respectively. In R1/R2 mixtures, there is a strong partitioning of R1 to the surface and R2 competes less favorably because of the steric or packing constraints of the larger R2 dirhamnose headgroup. In dilute solution (<20 mM), R1 and R2 form small globular micelles, L(1), with aggregation numbers of about 50 and 30, respectively. At higher solution concentrations, R1 has a predominantly planar structure, L(α) (unilamellar, ULV, or bilamellar, BLV, vesicles) whereas R2 remains globular, with an aggregation number that increases with increasing surfactant concentration. For R1/R2 mixtures, solutions rich in R2 are predominantly micellar whereas solutions rich in R1 have a more planar structure. At an intermediate composition (60 to 80 mol % R1), there are mixed L(α)/L(1) and L(1)/L(α) regions. However, the higher preferred curvature associated with R2 tends to dominate the mixed R1/R2 microstructure and its associated phase behavior.

Mixing behavior of the biosurfactant, rhamnolipid, with a conventional anionic surfactant, sodium dodecyl benzene sulfonate.

The use of small angle neutron scattering, SANS, neutron reflectivity, NR, and surface tension to study the mixing properties of the biosurfactant rhamnolipid with a conventional anionic surfactant, sodium dodecyl 6-benzene sulfonate, LAS, is reported. The monorhamnose rhamnolipid, R1, mixes close to ideally with LAS at the air-water interface, whereas for mixtures of LAS with the dirhamnose rhamnolipid, R2, the LAS strongly partitions to the air-water interface relative to R2, probably because of the steric hindrance of the larger R2 headgroup. These trends in the binary mixtures are also reflected in the ternary R1/R2/LAS mixtures. However, for these ternary mixtures, there is also a pronounced synergy in the total adsorption, which reaches a maximum for a LAS/rhamnolipid mole ratio of about 0.6 and a R1/R2 mol ratio of about 0.5, an effect which is not observed in the binary mixtures. In solution, the R1/LAS mixtures form relatively small globular micelles, L(1), at low surfactant concentrations (<20 mM), more planar structures (lamellar, L(α), unilamellar/multilamellar vesicles, ulv/mlv) are formed at higher surfactant concentrations for R1 and LAS rich compositions, and a large mixed phase (L(α)/L(1) and L(1)/L(α)) region forms at intermediate surfactant compositions. In contrast, for the R2/LAS mixtures, the higher preferred curvature of R2 dominates the phase behavior. The predominant microstructure is in the form of small globular micelles, except for solution compositions rich in LAS (>80 mol % LAS) where more planar structures are formed. For the ternary mixtures, there is an evolution in the resulting phase behavior from one dominated by L(1) (R2 rich) to one dominated by planar structures, L(α), (R1, LAS rich), and which strongly depends upon the LAS/rhamnolipid and R1/R2 mole ratio.

The performance of surfactant mixtures at low temperatures.

Optimising detergency at lower temperatures is of increasing interest due to environmental and economic factors, and requires a greater understanding of the effects of temperature on the adsorption of surfactant mixtures at interfaces. The adsorption properties of surfactant mixtures and biosurfactant/surfactant mixtures have been studied at room temperatures and at temperatures below ambient using surface tension and neutron reflectivity measurements. For the ternary surfactant mixture of octaethylene monododecyl ether, C12E8, sodium dodecyl 6-benzene sulfonate, LAS, and sodium dioxyethylene glycol monododecyl sulfate, SLES, the surface tension at the air-water interface increases with decreasing temperature. In contrast, there is a notable reduction in the increase in the surface tension with a decrease in temperature from 25 °C to 10 °C for the 5 component rhamnolipid/surfactant mixture of the mono-rhamnose, R1, and di-rhamnose, R2, with C12E8/LAS/SLES. The associated neutron reflectivity data for the ternary C12E8/LAS/SLES mixture and the significant observation is that the 3, 4, and 5-component mixtures containing rhamnolipids in conjunction with the other surfactants show changes in composition and adsorbed amounts of the individual components which are close to the experimental error. However the significant observation is that the neutron reflectivity data indicate that the improved surface tension tolerance at lower temperatures is associated with the dominance of the rhamnolipid adsorption in such mixtures. Hence the introduction of the rhamnolipids provides a tolerance to the adverse effects associated with reduced temperatures, and a potential for improved detergency at relatively low temperatures.

Lactic acid production by mixed cultures of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus.

Lactic acid production using Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) individually or as mixed culture on cheese whey in stirred or static fermentation conditions was evaluated. Lactic acid production, residual sugar and cell biomass were the main features examined. Increased lactic acid production was observed, when mixed cultures were used in comparison to individual ones. The highest lactic acid concentrations were achieved when K. marxianus yeast was combined with L. delbrueckii ssp. bulgaricus, and when all the strains were used revealing possible synergistic effects between the yeast and the two lactic acid bacteria. The same synergistic effects were further observed and verified when the mixed cultures were applied in sourdough fermentations, proving that the above microbiological system could be applied in the food fermentations where high lactic acid production is sought.

Characterization of a fungal strain isolated from a polyphenol polluted site.

A group of fungal strains were isolated from a polyphenol polluted soil, taken from an olive oil processing plant in Attica, Greece. The fungi were tested for their ability to decolorize a polyaromatic dye Poly R-478, which was used as a model compound to test their ligninolytic activities. The strain K1.1 decolorized efficiently the dye on agar plates and was further studied. PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA genes from the genomic DNA isolated from mycelium grown in liquid culture resulted in amplified fragments. Via BLASTN search, the length of a 773 base pairs was identified as the basidiomycetes Coprinellus xanthothrix. The growth rates and the tolerance of the fungus were compared on solid media, containing four different concentrations of pentachlorophenol. Extracellular enzyme activities (lignin peroxidase, manganese peroxidase and laccase) were determined in defined liquid medium. The isolate expressed laccase and manganese peroxidase but not lignin peroxidase. The removal of the dye was also estimated in liquid medium. The fungus showed biosorption and biotransformation as removal mechanisms.

Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for alcoholic fermentation.

A biocatalyst was prepared by immobilizing a commercial Saccharomyces cerevisiae strain (baker's yeast) on orange peel pieces for use in alcoholic fermentation and for fermented food applications. Cell immobilization was shown by electron microscopy and by the efficiency of the immobilized biocatalyst for alcoholic fermentation of various carbohydrate substrates (glucose, molasses, raisin extracts) and at various temperatures (30-15 degrees C). Fermentation times in all cases were low (5-15 h) and ethanol productivities were high (av. 150.6 g/ld) showing good operational stability of the biocatalyst and suitability for commercial applications. Reasonable amounts of volatile by-products were produced at all the temperatures studied, revealing potential application of the proposed biocatalyst in fermented food applications, to improve productivities and quality.

Self-assembly in dilute mixtures of non-ionic and anionic surfactants and rhamnolipd biosurfactants.

The self-assembly of dilute aqueous solutions of a ternary surfactant mixture and rhamnolipid biosurfactant/surfactant mixtures has been studied by small angle neutron scattering. In the ternary surfactant mixture of octaethylene glycol monododecyl ether, C12E8, sodium dodecyl 6-benzene sulfonate, LAS, and sodium dioxyethylene monododecyl sulfate, SLES, small globular interacting micelles are observed over the entire composition and concentration range studied. The modelling of the scattering data strongly supports the assumption that the micelle compositions are close to the solution compositions. In the 5-component rhamnolipid/surfactant mixture of the mono-rhamnose, R1, di-rhamnose, R2, rhamnolipids with C12E8/LAS/SLES, globular micelles are observed over much of the concentration and composition range studied. However, for solutions relatively rich in rhamnolipid and LAS, lamellar/micellar coexistence is observed. The transition from globular to more planar structures arises from a synergistic packing in the 5 component mixture. It is not observed in the individual components nor in the ternary C12E8/LAS/SLES mixture at these relatively low concentrations. The results provide an insight into how synergistic packing effects can occur in the solution self-assembly of complex multi-component surfactant mixtures, and give rise to an unexpected evolution in the phase behaviour.

Glycocalyx-mimetic dextran-modified poly(vinyl amine) surfactant coating reduces platelet adhesion on medical-grade polycarbonate surface.

A dextran-modified poly(vinyl amine) comb-like surfactant polymer, poly(N-vinyl dextran aldonamide-co-N-vinyl hexanamide), that can surface-adsorb on hydrophobic polymeric substrates, was designed to improve the interfacial blood-compatibility of polymeric biomaterials. Medical-grade polycarbonate was selected as a model substrate because of its extensive use in blood-contacting biomedical devices like hemodialyzers, blood pumps and oxygenators. The surfactant polymer was physisorbed from aqueous solution onto the polycarbonate substrate. The surfactant coating was stable under dynamic shear conditions in whole blood, as confirmed by fluorescence microscopy and total internal reflection fluorescence (TIRF) experiments with fluorescein-labeled surfactant polymer. The coated disks and uncoated control disks were exposed to platelet-rich plasma (PRP) and whole human blood in a rotating disk system (RDS) to study platelet-adhesion under dynamic shear stress environments. Adhered platelets were stained with fluorescein isothiocyante (FITC)-tagged anti-CD41a monoclonal antibody and imaged by epifluorescence microscopy. Complimentary images were obtained by phase-contrast microscopy. Platelet adhesion on the surfactant-coated disks was reduced by approximately 90%, compared with uncoated disks. The images also showed a concomitant reduction in platelet-derived microparticles on surfactant-coated disks, compared with uncoated disks. The results suggest potential application of carbohydrate-modified surfactant polymers as a glycocalyx-mimetic non-thrombogenic interfacial coating for blood-contacting biomaterials.

Pulsed-field gel electrophoresis of Campylobacter jejuni sheep abortion isolates.

Campylobacter species are a significant cause of sheep abortion in most sheep-raising countries. In New Zealand, Campylobacter fetus subsp. fetus is the leading cause of diagnosed sheep abortion and the species C. jejuni and C. coli have also been implicated. To date, strain typing information of C. jejuni sheep abortion isolates is limited. The objective of the present study was to genotype C. jejuni isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the 2000 breeding season, using pulsed-field gel electrophoresis (PFGE). In this study, C. jejuni isolates were cultured from approximately 10% of farms from which Campylobacter species were isolated from sheep abortions in the year 2000. This equated to 25 C. jejuni isolates from 21 farms. These isolates were obtained from the veterinary diagnostic laboratories and strain typed using the molecular typing technique PFGE. Ten distinct PFGE types were identified amongst the isolates. No particular PFGE type was found most frequently amongst these C. jejuni sheep abortion isolates. However, indistinguishable or similar C. jejuni PFGE types were identified from different aborted foetuses from the same flock, consistent with the role of C. jejuni as an infectious cause of abortion in sheep. These strain types were similar or indistinguishable from C. jejuni sheep abortion isolates obtained in 1999 in a smaller study (Mannering, S.A., Marchant, R.M., Middelberg, A., Perkins, N.R., West, D.M., Fenwick, S.G., 2003. Pulsed-field gel electrophoresis typing of C. fetus subsp. fetus from sheep abortions in the Hawke's Bay region of New Zealand. NZ Vet. J. 51, 33-37).

Biodegradation of lindane by Pleurotus ostreatus via central composite design.

The degradation of lindane was studied in liquid-agitated cultures using a commercial strain of the fungus Pleurotus ostreatus as the biodegrading organism. The biodegradation was accomplished with the action of extracellular oxidative enzymes, produced by the fungus to decompose woody substrates. Enzyme activities of manganese peroxidase and laccase were measured in a liquid mineral medium. An orthogonal Central Composite Design of experiments was used to construct second-order response surfaces with the fungus growth, final pH and the lindane biodegradation as optimization parameters. The initial lindane concentration, the nitrogen content, the incubation time and the temperature were used as design factors. Optimal conditions found for all these parameters will be used for the continuation of this project aiming at the bioremediation of contaminated sites with persistent organic pollutants such as lindane.

Molecular views and measurements of hemostatic processes using atomic force microscopy.

Hemostasis and thrombosis are highly complex and coordinated interfacial responses to vascular injury. In recent years, atomic force microscopy (AFM) has proven to be a very useful approach for studying hemostatic processes under near physiologic conditions. In this report, we review recent progress in the use of AFM for studying hemostatic processes, including molecular level visualization of plasma proteins, protein aggregation and multimer assembly, and structural and morphological details of vascular cells under aqueous conditions. AFM offers opportunities for visualizing surface-dependent molecular and cellular interactions in three dimensions on a nanoscale and for sensitive, picoNewton level, measurements of intermolecular forces. AFM has been used to obtain molecular and sub-molecular, resolution of many biological molecules and assemblies, including coagulation proteins and cell surfaces. Surface-dependent molecular processes including protein adsorption, conformational changes, and subsequent interactions with cellular components have been described. This review outlines the basic principles and utility of AFM for imaging and force measurements, and offers objective perspectives on both the advantages and disadvantages. We focus primarily on molecular level events related to hemostasis and thrombosis, particularly coagulation proteins, and blood platelets, but also explore the use of AFM in force measurements and surface property mapping.

Surface-dependent conformations of human fibrinogen observed by atomic force microscopy under aqueous conditions.

Conformational differences in human fibrinogen under aqueous conditions on hydrophobic, positively charged and negatively charged surfaces, were examined by atomic force microscopy (AFM). Hydrophobic and positively charged surfaces were prepared by depositing octadecyltrichlorosilane (OTS) and 3-aminopropyltriethoxysilane (APTES) respectively on cleaned glass coverslips forming self-assembled monolayers. The negatively charged surface was prepared by freshly cleaving muscovite mica. AFM operated in fluid tapping mode with an ultrasharp carbon spike probe was used to obtain the molecular scale images. Fibrinogen displayed a characteristic trinodular structure on all three surfaces. although additional U-shaped conformations were observed on mica. In its native hydrated state, fibrinogen is well represented by three connected ellipsoids in close proximity. Quantitative dimensional analysis, which yielded structural information in three dimensions, indicates that surface-dependent structural deformation or spreading of fibrinogen increases according to the order: mica < APTES < OTS. Molecular length, and D and E domain widths of fibrinogen are increased, while the corresponding heights are decreased. The results provide direct evidence that material surface properties affect the conformational state of interacting fibrinogen.

Three dimensional structure of human fibrinogen under aqueous conditions visualized by atomic force microscopy.

Fibrinogen plays a central role in surface-induced thrombosis. However, the interactions of fibrinogen with different substrata remain poorly understood because of the difficulties involved in imaging globular proteins under aqueous conditions. We present detailed three dimensional molecular scale images of fibrinogen molecules on a hydrophobic surface under aqueous conditions obtained by atomic force microscopy. Hydrated fibrinogen monomers are visualized as overlapping ellipsoids; dimers and trimers have linear conformations predominantly, and increased affinity for the hydrophobic surface compared with monomeric fibrinogen. The results demonstrate the importance of hydration on protein structure and properties that affect surface-dependent interactions.

Factors affecting the genetic engineering of plants by microprojectile bombardment.

Since its development in the mid-1980s, microprojectile bombardment has been widely employed as a method for direct gene transfer into a wide range of plants, including the previously difficult-to-transform monocotyledonous species. Although the numerous instruments available for microprojectile-mediated gene delivery and their applications have been widely discussed, less attention has been paid to the critical factors which affect the efficiency of this method of gene delivery. In this review we do not wish to describe the array of devices used for microprojectile delivery or their uses which have already been definitively described, but instead wish to report on research developments investigating the factors which affect microprojectile-mediated transformation of plants.