Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.

Bacteria can synthesize a broad spectrum of multifunctional polysaccharides including extracellular polysaccharides (EPS). Bacterial EPS can be utilized in the food, pharmaceutical, and biomedical areas owing to their physical and rheological properties in addition to generally presenting low toxicity. From an ecological viewpoint, EPS are biodegradable and environment compatible, offering several advantages over synthetic compounds. This study investigated the EPS produced by Klebsiella oxytoca (KO-EPS) by chemically characterizing and evaluating its properties. The monosaccharide components of the KO-EPS were determined by HPLC coupled with a refractive index detector and GC-MS. The KO-EPS was then analyzed by methylation analysis, FT-IR and NMR spectroscopy to give a potential primary structure. KO-EPS demonstrated the ability to stabilize hydrophilic emulsions with various hydrophobic compounds, including hydrocarbons and vegetable and mineral oils. In terms of iron chelation capacity, the KO-EPS could sequester 41.9 % and 34.1 % of the most common iron states, Fe2+ and Fe3+, respectively. Moreover, KO-EPS exhibited an improvement in the viscosity of aqueous dispersion, being proportional to the increase in its concentration and presenting a non-Newtonian pseudoplastic flow behavior. KO-EPS also did not present a cytotoxic effect indicating that the KO-EPS could have potential applications as a natural thickener, bioemulsifier, and bioremediation agent.

Microbial biosurfactant is an emerging vital biomolecule of the 21st century. They are amphiphilic compounds produced by microorganisms and possess unique properties to reduce surface tension activity. The use of microbial surfactants spans most of the industrial fields due to their biodegradability, less toxicity, being environmentally safe, and being synthesized from renewable sources. These would be highly efficient eco-friendly alternatives to petroleum-derived surfactants that would open up new approaches to research on the production of biosurfactants. In the upcoming era, biobased surfactants will become a dominating multifunctional compound in the world market. Research on biosurfactants ranges from the search for novel microorganisms that can produce new molecules, structural and physiochemical characterization of biosurfactants, and fermentation process for enhanced large-scale productivity and green applications. The main goal of this review is to provide an overview of the recent state of knowledge and trends about microbially derived surfactants, various aspects of biosurfactant production, definition, properties, characteristics, diverse advances, and applications. This would lead a long way in the production of biosurfactants as globally successful biomolecules of the current century.

Biosurfactants are in demand by the global market as natural commodities suitable for incorporation into commercial products or utilization in environmental applications. Fungi are promising producers of these molecules and have garnered interest also for their metabolic capabilities in efficiently utilizing recalcitrant and complex substrates, like hydrocarbons, plastic, etc. Within this framework, biosurfactants produced by two Fusarium solani fungal strains, isolated from plastic waste-contaminated landfill soils, were analyzed. Mycelia of these fungi were grown in the presence of 5% olive oil to drive biosurfactant production. The characterization of the emulsifying and surfactant capacity of these extracts highlighted that two different components are involved. A protein was purified and identified as a CFEM (common in fungal extracellular membrane) containing domain, revealing a good propensity to stabilize emulsions only in its aggregate form. On the other hand, an unidentified cationic smaller molecule exhibits the ability to reduce surface tension. Based on the 3D structural model of the protein, a plausible mechanism for the formation of very stable aggregates, endowed with the emulsifying ability, is proposed. KEY POINTS: • Two Fusarium solani strains are analyzed for their surfactant production. • A cationic surfactant is produced, exhibiting the ability to remarkably reduce surface tension. • An identified protein reveals a good propensity to stabilize emulsions only in its aggregate form.

At the best conditions of the bioprocess (30 °C, pH 7.0, 3.0 g/L NaCl) were obtained 0.66 g/L cell concentration, 3.3 g/L of bioemulsifier, which showed high emulsifying activity (53 % ± 2), reducing the surface tension of the water in 47.2 % (38 mN/m). The polymeric structure of the purified bioemulsifier comprised a carbohydrate backbone composed of hexose-based amino sugars with a monomeric mass of 1099 Da, structurally similar to emulsan. A. venetianus bioemulsifier is non-phytotoxic (GI% > 80 %) against Ocimum basilicum and Brassica oleracea and non-cytotoxic (LC50 5794 mg/L) against Artemia salina, being safe local organisms in comparison to other less eco-friendly synthetic emulsifiers. This bioemulsifier effectively dispersed spilled oil in vitro (C22-C33), reducing oil mass by 12 % (w/w) and dispersing oil in a displacement area of 75 cm2 (23.8 % of the spilled area). Thus, the isolated A. venetianus AMO1502 produced a bioemulsifier potentially applicable for environmentally friendly oil spill remediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers.
RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number \"OR643436\". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts.
CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

The emulsifying ability of SA01-OmpA (outer membrane protein A from Acinetobacter sp. SA01) was found to be constrained by challenges like low production efficiency and high costs associated with protein recovery from E. coli inclusion bodies, as described in our previous study. The present study sought to benefit from the advantages of the targeted truncating of SA01-OmpA protein, taking into account the reduced propensity of protein expression as inclusion bodies and cytotoxicity. Here, the structure and activity relationship of two truncated recombinant forms of SA01-OmpA protein was unraveled through a hybrid approach based on experimental data and computational methodologies, representing an innovative bioemulsifier with advantageous emulsifying activity. The recombinant truncated SA01-OmpA variants were cloned and heterologously expressed in E. coli host cells and subsequently purified. The results showed increased emulsifying activity of N-terminally truncated SA01-OmpA (NT-OmpA) compared to full-length SA01-OmpA. Molecular dynamics (MD) simulations analysis demonstrated a direct correlation between the C-terminally truncated SA01-OmpA (CT-OmpA) and its expression as inclusion bodies. Analysis of the structure-activity relationship of truncated variants of SA01-OmpA revealed that, compared to the full-length protein, deletion of the β-barrel portion from the N-terminal of SA01-OmpA increased the emulsifying activity of NT-OmpA while lowering its expression as inclusion bodies. Contrary to the full-length protein, the N-terminally truncated SA01-OmpA was not as cytotoxic, according to the MTT assay, FCM analysis, and AO/EB staining. The findings of this extensive study advance our knowledge of SA01-OmpA at the molecular level as well as the design and development of efficient bioemulsifiers.IMPORTANCEPrevious research (Shahryari et al. 2021, mSystems 6: e01175-20) introduced and characterized the SA01-OmpA protein as a multifaceted protein with a variety of functions, including maintaining cellular homeostasis under oxidative stress conditions, biofilm formation, outer membrane vesicles (OMV) biogenesis, and beneficial emulsifying capacity. By truncating the SA01-OmpA protein, the current study presents a unique method for developing protein-type bioemulsifiers. The findings indicate that the N-terminally truncated SA01-OmpA (NT-OmpA) has the potential to fully replace full-length SA01-OmpA as a novel bioemulsifier with significant emulsifying activity. This study opens up a new frontier in bioemulsifiers, shedding light on a possible relationship between the structure and activity of SA01-OmpA truncated forms.

Surface-active compounds (SACs) of microbial origin are an active group of biomolecules with potential use in the formulation of emulsions. In this sense, the present study aimed to isolate and select yeasts from fruits that could produce SACs for essential oil emulsions. The Candida krusei M4CK was isolated from the Byrsonima crassifolia fruit to make SACs. This emulsification activity (E24) was equal to or greater 50% in all carbon sources, such as olive oil, sunflower oil, kerosene, hexane, and hexadecane. E24 followed exponential growth according to the growth phase. The stability of emulsions was maintained over a wide range of temperatures, pH, and salinity. The OMBE4CK (melaleuca essential oil emulsion) had better and more significant inhibitory potential for biofilm reduction formation. In addition, bioemulsifier BE4CK alone on Escherichia coli and Pseudomonas aeruginosa biofilm showed few effective results, while there was a significant eradication for Staphylococcus aureus biofilms. The biofilms formed by S. aureus were eradicated in all concentrations of OMBE4CK. At the same time, the preformed biofilm by E. coli and P. aeruginosa were removed entirely at concentrations of 25 mg/mL, 12.5 mg/mL, and 6.25 mg/mL. The results show that the bioemulsifier BE4CK may represent a new potential for antibiofilm application.

BACKGROUND: Bioemulsifiers are natural or microbial-based products with the ability to emulsify hydrophobic compounds in water. These compounds are biodegradable, eco-friendly, and find applications in various industries.
RESULTS: Thirteen yeasts were isolated from different sources in Alexandria, Egypt, and evaluated for their potential to produce intracellular bioemulsifiers. One yeast, isolated from a local market in Egypt, showed the highest emulsification index (EI24) value. Through 26S rRNA sequencing, this yeast was identified as Saccharomyces cerevisiae strain MYN04. The growth kinetics of the isolate were studied, and after 36 h of incubation, the highest yield of cell dry weight (CDW) was obtained at 3.17 g/L, with an EI24 of 55.6%. Experimental designs were used to investigate the effects of culture parameters on maximizing bioemulsifier SC04 production and CDW. The study achieved a maximum EI24 of 79.0 ± 2.0%. Furthermore, the crude bioemulsifier was precipitated with 50% ethanol and purified using Sephadex G-75 gel filtration chromatography. Bioemulsifier SC04 was found to consist of 27.1% carbohydrates and 72.9% proteins. Structural determination of purified bioemulsifier SC04 was carried out using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance spectroscopy (NMR). FTIR spectroscopy revealed characteristic bands associated with carboxyl and hydroxyl groups of carbohydrates, as well as amine groups of proteins. HPLC analysis of monosaccharide composition detected the presence of mannose, galactose, and glucose. Physicochemical characterization of the fraction after gel filtration indicated that bioemulsifier SC04 is a high molecular weight protein-oligosaccharide complex. This bioemulsifier demonstrated stability at different pH values, temperatures, and salinities. At a concentration of 0.5 mg/mL, it exhibited 51.8% scavenging of DPPH radicals. Furthermore, in vitro cytotoxicity evaluation using the MTT assay revealed a noncytotoxic effect of SC04 against normal epithelial kidney cell lines.
CONCLUSIONS: This study presents a new eco-friendly bioemulsifier, named SC04, which exhibits significant emulsifying ability, antioxidant and anticancer properties, and stabilizing properties. These findings suggest that SC04 is a promising candidate for applications in the food, pharmaceutical, and industrial sectors.

Bread undergoes physicochemical processes known as \'staling\', which limits shelf life and quality. Despite the fact that several chemical emulsifiers have been employed to combat this issue, they may offer risks to human health. In this investigation, the effects of bioemulsan, a natural bioemulsifier (BE), on bread quality and staleness were examined. The yield of emulsan generated by Acinetobacter calcoaceticus RAG-1 was 1.49 g/L. The presence of clear zones around colonies, high emulsification value of 100%, and remaining surface tension below 40 mN/m after heating (at 250 °C for 15-20 min) verified emulsan thermal stability. BE-supplemented bread had a greater moisture percentage than the control, resulting in reduced crumb hardening and improved bread quality during storage as measured by moisture content. The first day after adding 0.5% emulsan, the hardness rose from 90.45 N (for the control) to 150.45 N. Texture analysis showed that although the hardness increased during storage, adding emulsan allowed obtaining bread with clearly softer crumb after 2 and 3 days of baking, especially at 0.5% level (from 215.6 N for the control to 150.5 N for 0.5% BE-enriched bread after 2 days, and from 425.7 to 210.25 N after 3 days). Based on the sensory evaluation results, emulsan did not lead to any unpleasant changes on bread organoleptic parameters. Therefore, using bioemulsifier RAG-1 as a green emulsifier and anti-staling agent found to be more promising.