Bioemulsifier Essentials

Cheatsheet Content

Bioemulsifier vs. Biosurfactant Biosurfactants: Primarily reduce surface and interfacial tension. Typically low molecular weight. Bioemulsifiers: Primarily stabilize emulsions by forming a barrier around droplets. High molecular weight, complex compounds. Bioemulsifiers are not necessarily effective at reducing tension. Strong biosurfactants may not be good emulsion stabilizers. Some molecules can act as both. Emulsion Basics Mixture of oil and water. Emulsifiers prevent emulsion breaking and oil droplet separation. Emulsifiers efficiently emulsify immiscible liquids (e.g., hydrocarbons) even at low concentrations, but are less effective at surface tension reduction. Bioemulsifiers are higher molecular weight than biosurfactants, often complex mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins, and proteins. Classification of Microbial Surfactants Biosurfactants (Low Molecular Mass) Lipoproteins, Lipopeptides: e.g., Surfactin, Viscosin, Iturin, Fengycin. Glycolipids: e.g., Rhamnolipids, Sophorolipids, Trehalolipids, Cellobiolipids. Polypeptides: e.g., Hydrophobins. Fatty acids and other lipids: e.g., Phospholipids, Neutral lipids, Polyol lipids. Bioemulsifiers (High Molecular Mass) Polymeric biosurfactants: e.g., Emulsan, Biodispersan, Alasan, Liposan, Yansan. Physicochemical Properties pH Stability: Maintains emulsifying properties across pH 2-12 (acidic to basic). Temperature Stability: Stable from $5^\circ C$ to $70^\circ C$. Salinity Tolerance: Stable in high salt concentrations (1-35% NaCl w/v), useful in marine/industrial settings. Production & Screening Methods Screening for Bioemulsifier (BE) Production Hemolytic Activity (HA): Evaluates cell membrane lysis. Modified Drop Collapse (MDC) Method: Observe if a drop on an oily surface destabilizes within 1 minute. Parafilm M Test: Similar to MDC, observe drop shape on parafilm. Lipase Activity: Observe zone of clearance around colony on tributyrin agar. Oil Spread Method (OSM): Observe displacement of oil and clear zone formation on a water/oil surface. Blue Agar Plate (BAP) Method: Reaction with cationic CTAB and methylene blue dye forms a dark blue halo around colonies. Hydrocarbon Overlay Agar (HOA) Method: Observe emulsified halos around colonies on hydrocarbon-coated agar. Emulsification Assay (EA) & Emulsification Index (EI): Measures emulsion stability. $EI (E24\%) = \frac{\text{Height of the emulsion layer}}{\text{The total height of the liquid column}} \times 100$ Tensiometric Measurement: By pendant drop technique. Production Conditions Minimal medium with oil. Optimization of C:N ratio. Physico-chemical conditions of growth. Time course and inoculum size. Product recovery and quantification. Examples of Bioemulsifiers Derived from Yeast Microorganism Bioemulsifiers Torulopsis petrophilum Sophorolipids Pseudozyma rugulosa Mannosylerythritol lipids Saccharomyces cerevisiae Mannoprotein Rhodotorula glutinis Polymeric bioemulsifier Phaffia rhodozyma Produces Astaxanthin (indirect emulsifier), grows on carbohydrates/hydrocarbons. Derived from Bacteria Microorganism Bioemulsifiers Pseudomonas aeruginosa Rhamnolipids Bacillus subtilis Surfactin/Iturin, Subtilisin Acinetobacter calcoaceticus Emulsan, Alasan Acinetobacter radioresistens KA53 Alasan (anionic polysaccharide + protein), highly stable. Pseudomonas spp. Produce Lauryl Fructose (LF), a fat-sugar ester with emulsifying properties. Applications of Bioemulsifiers Biomedical Applications Cell Membrane Permeabilization: Forms pores that destabilize lipid bilayer. Antimicrobial Activity: Exopolysaccharides (EPS) inhibit pathogens by interacting with cell wall structures or blocking receptors. Anticancer Properties: P. chlororaphis -produced BEs show cytotoxic effects on melanoma cells (MNT-1) at 150-200 µg/mL ($IC_{50}$), with selective toxicity (spares normal skin cells). Fatty acid-derived BEs from Halomonas sp. demonstrate antimicrobial and anticancer properties. Drug Delivery: Can form micelles to encapsulate oil-soluble or water-soluble drugs, enhancing solubility and bioavailability. Food Industry Food Additives: Can alter sensory and physical properties of food. Examples: Mannoproteins from Saccharomyces cerevisiae for salad dressings. Mannoproteins from spent brewer's yeast for mayonnaise stabilization. Candida bombicola BE in cupcake formulations as fat substitute (thermally stable, non-cytotoxic). Glycerol monolaurate (GML) and Lecithin are common emulsifiers. Oil Industry Sustainable Alternative: To synthetic emulsifiers in petroleum industry. Applications: Oil recovery, bioremediation, equipment cleaning. Examples: Alcaligenes sp. PHY 9L-86 degrades 98% of tetradecane. Halomonas sp. strains stabilize oil emulsions. Bacillus licheniformis K125 reduces surface tension, aids oil recovery. Cryptococcus sp. YLF shows high salt tolerance, removes sand contaminants. Stenotrophomonas maltophilia UCP 1601 disperses burnt engine oil, cleans sandy soil. Acinetobacter radioresistens KA53 enhances biodegradation of polyaromatic hydrocarbons. Emulsan from Acinetobacter calcoaceticus PTCC1318 removes crude oil from surfaces.