Glycolipid Biosurfactants in Skincare Applications: Challenges and Recommendations for Future Exploitation.

The 21st century has seen a substantial increase in the industrial applications of glycolipid biosurfactant technology. The market value of the glycolipid class of molecules, sophorolipids, was estimated to be USD 409.84 million in 2021, with that of rhamnolipid molecules projected to reach USD 2.7 billion by 2026. In the skincare industry, sophorolipid and rhamnolipid biosurfactants have demonstrated the potential to offer a natural, sustainable, and skin-compatible alternative to synthetically derived surfactant compounds. However, there are still many barriers to the wide-scale market adoption of glycolipid technology. These barriers include low product yield (particularly for rhamnolipids) and potential pathogenicity of some native glycolipid-producing microorganisms. Additionally, the use of impure preparations and/or poorly characterised congeners as well as low-throughput methodologies in the safety and bioactivity assessment of sophorolipids and rhamnolipids challenges their increased utilisation in both academic research and skincare applications. This review considers the current trend towards the utilisation of sophorolipid and rhamnolipid biosurfactants as substitutes to synthetically derived surfactant molecules in skincare applications, the challenges associated with their application, and relevant solutions proposed by the biotechnology industry. In addition, we recommend experimental techniques/methodologies, which, if employed, could contribute significantly to increasing the acceptance of glycolipid biosurfactants for use in skincare applications while maintaining consistency in biosurfactant research outputs.

Characterisation of cytotoxicity and immunomodulatory effects of glycolipid biosurfactants on human keratinocytes.

Skin irritation and allergic reactions associated with the use of skincare products formulated with synthetically derived surfactants such as sodium lauryl ether sulphate (SLES) have encouraged the search for naturally derived and biocompatible alternatives. Glycolipid biosurfactants such as sophorolipids (SL) and rhamnolipids (RL) offer a potential alternative to SLES. However, most studies on the bioactive properties of microbial glycolipids were determined using their mixed congeners, resulting in significant inter-study variations. This study aims to compare the effects of highly purified SL (acidic and lactonic) and RL (mono-RL and di-RL) congeners and SLES on a spontaneously transformed human keratinocyte cell line (HaCaT cells) to assess glycolipids' safety for potential skincare applications. Preparations of acidic SL congeners were 100% pure, lactonic SL were 100% pure, mono-RL were 96% pure, and di-RL were 97% pure. Cell viability using XTT assays, cell morphological analyses, and immunoassays revealed that microbial glycolipids have differing effects on HaCaT cells dependent on chemical structure. Compared with SLES, acidic SL and mono-RL have negligible effects on cell viability, cell morphology, and production of pro-inflammatory cytokines. Furthermore, at non-inhibitory concentrations, di-RL significantly attenuated IL-8 production and CXCL8 expression while increasing IL-1RA production and IL1RN expression in lipopolysaccharide-stimulated HaCaT cells. Although further studies would be required, these results demonstrate that as potential innocuous and bioactive compounds, microbial glycolipids could provide a substitute to synthetic surfactants in skincare formulations and perform immunopharmacological roles in topical skin infections such as psoriasis. KEY POINTS: • Purified glycolipid congeners have differing effects on human keratinocytes. • Compared with SLES, acidic sophorolipids and mono-rhamnolipids have innocuous effects on keratinocytes. • Di-rhamnolipids and mono-rhamnolipids modulate cytokine production in lipopolysaccharide stimulated human keratinocytes.

Mono-Rhamnolipid Biosurfactants Synthesized by Pseudomonas aeruginosa Detrimentally Affect Colorectal Cancer Cells.

Over the past 15 years, glycolipid-type biosurfactant compounds have been postulated as novel, naturally synthesized anticancer agents. This study utilized a recombinant strain of Pseudomonas aeruginosa to biosynthesize a preparation of mono-rhamnolipids that were purified via both liquid and solid-phase extraction, characterized by HPLC-MS, and utilized to treat two colorectal cancer cell lines (HCT-116 and Caco2) and a healthy colonic epithelial cell line CCD-841-CoN. Additionally, the anticancer activity of these mono-rhamnolipids was compared to an alternative naturally derived anticancer agent, Piceatannol. XTT cell viability assays showed that treatment with mono-rhamnolipid significantly reduced the viability of both colorectal cancer cell lines whilst having little effect on the healthy colonic epithelial cell line. At the concentrations tested mono-rhamnolipids were also shown to be more cytotoxic to the colorectal cancer cells than Piceatannol. Staining of mono-rhamnolipid-treated cells with propidium iodine and acridine orange appeared to show that these compounds induced necrosis in both colorectal cancer cell lines. These data provide an early in vitro proof-of-principle for utilizing these compounds either as active pharmaceutical ingredient for the treatment of colorectal cancer or incorporations into nutraceutical formulations to potentially prevent gastrointestinal tract cancer.

Biogenic propane production by a marine Photobacterium strain isolated from the Western English Channel.

Propane is a major component of liquefied petroleum gas, a major energy source for off-grid communities and industry. The replacement of fossil fuel-derived propane with more sustainably derived propane is of industrial interest. One potential production route is through microbial fermentation. Here we report, for the first time, the isolation of a marine bacterium from sediment capable of natural propane biosynthesis. Propane production, both in mixed microbial cultures generated from marine sediment and in bacterial monocultures was detected and quantified by gas chromatography-flame ionization detection. Using DNA sequencing of multiple reference genes, the bacterium was shown to belong to the genus Photobacterium. We postulate that propane biosynthesis is achieved through inorganic carbonate assimilation systems. The discovery of this strain may facilitate synthetic biology routes for industrial scale production of propane via microbial fermentation.

Microbial sophorolipids inhibit colorectal tumour cell growth in vitro and restore haematocrit in Apcmin+/- mice.

Sophorolipids are glycolipid biosurfactants consisting of a carbohydrate sophorose head with a fatty acid tail and exist in either an acidic or lactonic form. Sophorolipids are gaining interest as potential cancer chemotherapeutics due to their inhibitory effects on a range of tumour cell lines. Currently, most anti-cancer studies reporting the effects of sophorolipids have focused on lactonic preparations with the effects of acidic sophorolipids yet to be elucidated. We produced a 94% pure acidic sophorolipid preparation which proved to be non-toxic to normal human colonic and lung cells. In contrast, we observed a dose-dependent reduction in viability of colorectal cancer lines treated with the same preparation. Acidic sophorolipids induced apoptosis and necrosis, reduced migration, and inhibited colony formation in all cancer cell lines tested. Furthermore, oral administration of 50 mg kg-1 acidic sophorolipids over 70 days to Apcmin+/- mice was well tolerated and resulted in an increased haematocrit, as well as reducing splenic size and red pulp area. Oral feeding did not affect tumour numbers or sizes in this model. This is the first study to show that acidic sophorolipids dose-dependently and specifically reduces colon cancer cell viability in addition to reducing tumour-associated bleeding in the Apcmin+/- mouse model. KEY POINTS: • Acidic sophorolipids are produced by yeast species such as Starmerella bombicola. • Acidic sophorolipids selectively killed colorectal cells with no effect on healthy gut epithelia. • Acidic sophorolipids reduced tumour-associated gut bleed in a colorectal mouse model.

Achieving Commercial Applications for Microbial Biosurfactants.

There are numerous biosurfactant producing strains and products reported annually most of which aspiring to potential future industrial and environmental applications. Only a few of these compounds have reached commercial applications due to many impediments to large-scale production and applications. We investigate some of the important criteria and investigations required to achieve such future commercial application.

Biosurfactants as Anticancer Agents: Glycolipids Affect Skin Cells in a Differential Manner Dependent on Chemical Structure.

Melanomas account for 80% of skin cancer deaths. Due to the strong relationship between melanomas and U.V. radiation, sunscreens have been recommended for use as a primary preventative measure. However, there is a need for targeted, less invasive treatment strategies. Glycolipids such as sophorolipids and rhamnolipids are microbially derived biosurfactants possessing bioactive properties such as antimicrobial, immunomodulatory and anticancer effects. This study aimed to ascertain the differing effects of glycolipids on skin cells. Highly purified and fully characterized preparations of sophorolipids and rhamnolipids were used to treat spontaneously transformed human keratinocyte (HaCaT) and the human malignant melanocyte (SK-MEL-28) cell lines. Cell viability and morphological analyses revealed that glycolipids have differential effects on the skin cells dependent on their chemical structure. Lactonic sophorolipids and mono-rhamnolipids were shown to have a significantly detrimental effect on melanoma cell viability compared to healthy human keratinocytes. These glycolipids were shown to induce cell death via necrosis. Additionally, sophorolipids were shown to significantly inhibit SK-MEL-28 cell migration. These findings suggest that glycolipids could be used as bioactive agents with selective inhibitory effects. As such, glycolipids could be a substitute for synthetically derived surfactants in sunscreens to provide additional benefit and have the potential as novel anti-skin-cancer therapies.

Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing.

Pseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI's) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

Microbial biosurfactant research: time to improve the rigour in the reporting of synthesis, functional characterization and process development.

The demand for microbially produced surface-active compounds for use in industrial processes and products is increasing. As such, there has been a comparable increase in the number of publications relating to the characterization of novel surface-active compounds: novel producers of already characterized surface-active compounds and production processes for the generation of these compounds. Leading researchers in the field have identified that many of these studies utilize techniques are not precise and accurate enough, so some published conclusions might not be justified. Such studies lacking robust experimental evidence generated by validated techniques and standard operating procedures are detrimental to the field of microbially produced surface-active compound research. In this publication, we have critically reviewed a wide range of techniques utilized in the characterization of surface-active compounds from microbial sources: identification of surface-active compound producing microorganisms and functional testing of resultant surface-active compounds. We have also reviewed the experimental evidence required for process development to take these compounds out of the laboratory and into industrial application. We devised this review as a guide to both researchers and the peer-reviewed process to improve the stringency of future studies and publications within this field of science.

Microbial Biosurfactants in Cosmetic and Personal Skincare Pharmaceutical Formulations.

Cosmetic and personal care products are globally used and often applied directly on the human skin. According to a recent survey in Europe, the market value of cosmetic and personal care products in Western Europe reached about 84 billion euros in 2018 and are predicted to increase by approximately 6% by the end of 2020. With these significant sums of money spent annually on cosmetic and personal care products, along with chemical surfactants being the main ingredient in a number of their formulations, of which many have been reported to have the potential to cause detrimental effects such as allergic reactions and skin irritations to the human skin; hence, the need for the replacement of chemical surfactants with other compounds that would have less or no negative effects on skin health. Biosurfactants (surfactants of biological origin) have exhibited great potential such as lower toxicity, skin compatibility, protection and surface moisturizing effects which are key components for an effective skincare routine. This review discusses the antimicrobial, skin surface moisturizing and low toxicity properties of glycolipid and lipopeptide biosurfactants which could make them suitable substitutes for chemical surfactants in current cosmetic and personal skincare pharmaceutical formulations. Finally, we discuss some challenges and possible solutions for biosurfactant applications.

Biosynthesis of rhamnolipid by a Marinobacter species expands the paradigm of biosurfactant synthesis to a new genus of the marine microflora.

BACKGROUND: In comparison to synthetically derived surfactants, biosurfactants produced from microbial culture are generally regarded by industry as being more sustainable and possess lower toxicity. One major class of biosurfactants are rhamnolipids primarily produced by Pseudomonas aeruginosa. Due to its pathogenicity rhamnolipid synthesis by this species is viewed as being commercially nonviable, as such there is a significant focus to identify alternative producers of rhamnolipids. RESULTS: To achieve this, we phenotypically screened marine bacteria for biosurfactant production resulting in the identification of rhamnolipid biosynthesis in a species belonging to the Marinobacter genus. Preliminary screening showed the strain to reduce surface tension of cell-free supernatant to 31.0 mN m-1. A full-factorial design was carried out to assess the effects of pH and sea salt concentration for optimising biosurfactant production. When cultured in optimised media Marinobacter sp. MCTG107b produced 740 ± 28.3 mg L-1 of biosurfactant after 96 h of growth. Characterisation of this biosurfactant using both HPLC-MS and tandem MS showed it to be a mixture of different rhamnolipids, with di-rhamnolipid, Rha-Rha-C10-C10 being the most predominant congener. The strain exhibited no pathogenicity when tested using the Galleria mellonella infection model. CONCLUSIONS: This study expands the paradigm of rhamnolipid biosynthesis to a new genus of bacterium from the marine environment. Rhamnolipids produced from Marinobacter have prospects for industrial application due to their potential to be synthesised from cheap, renewable feed stocks and significantly reduced pathogenicity compared to P. aeruginosa strains.

Quorum sensing as a potential target for increased production of rhamnolipid biosurfactant in Burkholderia thailandensis E264.

Burkholderia thailandensis E264 is a potential non-pathogenic substitute for producing rhamnolipid biosurfactant, replacing the pathogenic Pseudomonas aeruginosa. However, it has low rhamnolipid production and longer fermentation time. We have earlier suggested that media supplementation with exogenous quorum sensing (QS) molecules could lead to early onset of biosynthesis and increased rhamnolipid yield. Here, we assessed the effect of single, double or triple mutations in the various QS systems of B. thailandensis on rhamnolipid production, with the view to see which system(s) have the most impact on rhamnolipid yield and subsequently use the QS molecule to potentially increase yield in the wild-type B. thailandensis. The triple mutant strain had a rhamnolipid yield of 4.46 ± 0.345 g/l at 240 h of fermentation which was significantly higher than that of the wild type (0.94 ± 0.06 g/l), an unexpected outcome. To gain more insight as to how this might occur, we studied substrate metabolism and energy storage in the form of polyhydroxyalkanoate (PHA) by both the triple mutant and the wild type. We observed increased glycerol metabolism and reduced PHA production in the triple mutant compared with the wild type. Glycerol concentration at 240 h and maximum PHA productivity (g/gDCB) were 8.76 g/l or 16.19 g/l and 21.80% or 31.4% in either the triple mutant or the wild type respectively. Complementation of the triple-mutant cultures with exogenous QS molecules restored rhamnolipid production to similar levels as the wild type. QS therefore is a potential target for increased rhamnolipid production in B. thailandensis.

Natural quorum sensing inhibitors effectively downregulate gene expression of Pseudomonas aeruginosa virulence factors.

At present, anti-virulence drugs are being considered as potential therapeutic alternatives and/or adjuvants to currently failing antibiotics. These drugs do not kill bacteria but inhibit virulence factors essential for establishing infection and pathogenesis through targeting non-essential metabolic pathways reducing the selective pressure to develop resistance. We investigated the effect of naturally isolated plant compounds on the repression of the quorum sensing (QS) system which is linked to virulence/pathogenicity in Pseudomonas aeruginosa. Our results show that trans-cinnamaldehyde (CA) and salicylic acid (SA) significantly inhibit expression of QS regulatory and virulence genes in P. aeruginosa PAO1 at sub-inhibitory levels without any bactericidal effect. CA effectively downregulated both the las and rhl QS systems with lasI and lasR levels inhibited by 13- and 7-fold respectively compared to 3- and 2-fold reductions with SA treatment, during the stationary growth phase. The QS inhibitors (QSI) also reduced the production of extracellular virulence factors with CA reducing protease, elastase and pyocyanin by 65%, 22% and 32%, respectively. The QSIs significantly reduced biofilm formation and concomitantly with repressed rhamnolipid gene expression, only trace amount of extracellular rhamnolipids were detected. The QSIs did not completely inhibit virulence factor expression and production but their administration significantly lowered the virulence phenotypes at both the transcriptional and extracellular levels. This study shows the significant inhibitory effect of natural plant-derived compounds on the repression of QS systems in P. aeruginosa.

Marine derived biosurfactants: a vast potential future resource.

Surfactants and emulsifiers are surface-active compounds (SACs) which play an important role in various industrial processes and products due to their interfacial properties. Many of the chemical surfactants in use today are produced from non-renewable petrochemical feedstocks, while biosurfactants (BS) produced by microorganisms from renewable feedstocks are considered viable alternatives to petroleum based surfactants, due to their biodegradability and eco-friendly nature. However, some well-characterised BS producers are pathogenic and therefore, not appropriate for scaled-up production. Marine-derived BS have been found to be produced by non-pathogenic organisms making them attractive possibilities for exploitation in commercial products. Additionally, BS produced from marine bacteria may show excellent activity at extreme conditions (temperature, pH and salinity). Despite being non-pathogenic, marine-derived BS have not been exploited commercially due to their low yields, insufficient structural elucidation and uncharacterised genes. Therefore, optimization of BS production conditions in marine bacteria, characterization of the compounds produced as well as the genes involved in the biosynthesis are necessary to improve cost-efficiency and realise the industrial demands of SACs.

Fatty acid synthesis pathway provides lipid precursors for rhamnolipid biosynthesis in Burkholderia thailandensis E264.

Rhamnolipid production was monitored for a period of 216 h using different substrates in Pseudomonas aeruginosa PAO1 and Burkholderia thailandensis E264 which showed comparable crude yields attained by both after 216 h. The crude yield for P. aeruginosa, however, was significantly higher at the early stages of fermentation (72 or 144 h). Additionally, P. aeruginosa produced rhamnolipid with odd and even carbon chain lipid moieties using odd carbon chain fatty acid substrates (up to 45.97 and 67.57%, respectively). In contrast, B. thailandensis produced rhamnolipid with predominantly even carbon chain lipid moieties (up to 99.26). These results indicate the use of the fatty acid synthesis (FAS II) pathway as the main source of lipid precursors in rhamnolipid biosynthesis by B. thailandensis. Isotope tracing using 0.25% stearic acid - d 35 + 1% glycerol as carbon substrate showed a single pattern of deuterium incorporation: with predominantly less than 15 deuterium atoms incorporated into a single Di-C14-C14 rhamnolipid molecule. This further indicates that the FAS II pathway is the main source of the lipid precursor in rhamnolipid biosynthesis by B. thailandensis. The pathogenicity of these strains was also assessed, and results showed that B. thailandensis is significantly less pathogenic than P. aeruginosa with an LC50 at 24 h > 2500, approximately three logs higher than P. aeruginosa using the Galleria mellonella larva model.

Going Green and Cold: Biosurfactants from Low-Temperature Environments to Biotechnology Applications.

Approximately 80% of the Earth's biosphere is cold, at an average temperature of 5°C, and is populated by a diversity of microorganisms that are a precious source of molecules with high biotechnological potential. Biosurfactants from cold-adapted organisms can interact with multiple physical phases - water, ice, hydrophobic compounds, and gases - at low and freezing temperatures and be used in sustainable (green) and low-energy-impact (cold) products and processes. We review the biodiversity of microbial biosurfactants produced in cold habitats and provide a perspective on the most promising future applications in environmental and industrial technologies. Finally, we encourage exploring the cryosphere for novel types of biosurfactants via both culture screening and functional metagenomics.

Adsorption at the Air-Water Interface in Biosurfactant-Surfactant Mixtures: Quantitative Analysis of Adsorption in a Five-Component Mixture.

The composition of the air-water adsorbed layer of a quinary mixture consisting of three conventional surfactants, octaethylene glycol monododecyl ether (C12E8), dodecane-6-p-sodium benzene sulfonate (LAS6), and diethylene glycol monododecyl ether sodium sulfate (SLE2S), mixed with two biosurfactants, the rhamnolipids l-rhamnosyl-l-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoyl, R2, and l-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoyl, R1, has been measured over a range of compositions above the mixed critical micelle concentration. Additional measurements on some of the subsets of ternary and binary mixtures have also been measured by NR. The results have been analyzed using the pseudophase approximation (PPA) in conjunction with an excess free energy, GE, that depends on the quadratic and cubic terms in the composition. The compositions of the binary, ternary, and quinary mixtures could all be fitted to two sets of interaction parameters between the pairs of surfactants, one for micelles and one for adsorption. No ternary interactions or ternary corrections were required. Because the system contains two strongly anionic surfactants, the PPA can be extended, in practice, to ionic surfactants, contrary to the prevailing view. The values of the interaction parameters show that the quinary mixture, SLE2S-LAS6-C12E8-R1-R2, which is known to be a highly effective surfactant system, is characterized by a sequence of strong surface but weak micellar interactions. About half of the minima in GE for the strong surface interactions occur well away from the regular solution value of 0.5.

Enhanced rhamnolipid production in Burkholderia thailandensis transposon knockout strains deficient in polyhydroxyalkanoate (PHA) synthesis.

Microbially produced rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa, is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa. Multiple knockout strains for the phbA, phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (ΔphbA1, ΔphbB1 and ΔphbC1) with the best enhancement of rhamnolipid production were selected for detailed study. ΔphbB1 produced the highest level of purified RL (3.78 g l-1) compared to the wild-type strain (1.28 g l-1). In ΔphbB1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa, knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway.

Antibacterial properties of sophorolipid-modified gold surfaces against Gram positive and Gram negative pathogens.

Sophorolipids are bioderived glycolipids displaying interesting antimicrobial properties. We show that they can be used to develop biocidal monolayers against Listeria ivanovii, a Gram-positive bacterium. The present work points out the dependence between the surface density and the antibacterial activity of grafted sophorolipids. It also emphasizes the broad spectrum of activity of these coatings, demonstrating their potential against both Gram-positive strains (Enteroccocus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes) and Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhymurium). After exposure to sophorolipids grafted onto gold, all these bacterial strains show a significant reduction in viability resulting from membrane damage as evidenced by fluorescent labelling and SEM-FEG analysis.

Microbial rhamnolipid production: a critical re-evaluation of published data and suggested future publication criteria.

High production cost and potential pathogenicity of Pseudomonas aeruginosa, commonly used for rhamnolipid synthesis, have led to extensive research for safer producing strains and cost-effective production methods. This has resulted in numerous research publications claiming new non-pathogenic producing strains and novel production techniques many of which are unfortunately without proper characterisation of product and/or producing strain/s. Genes responsible for rhamnolipid production have only been confirmed in P. aeruginosa, Burkholderia thailandensis and Burkholderia pseudomallei. Comparing yields in different publications is also generally unreliable especially when different methodologies were used for rhamnolipid quantification. After reviewing the literature in this area, we strongly feel that numerous research outputs have insufficient evidence to support claims of rhamnolipid-producing strains and/or yields. We therefore recommend that standards should be set for reporting new rhamnolipid-producing strains and production yields. These should include (1) molecular and bioinformatic tools to fully characterise new microbial isolates and confirm the presence of the rhamnolipid rhl genes for all bacterial strains, (2) using gravimetric methods to quantify crude yields and (3) use of a calibrated method (high-performance liquid chromatography or ultra-performance liquid chromatography) for absolute quantitative yield determination.