Biosurfactants

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Significance of Microbial Products Biosynthesis: A multistep, enzyme-catalyzed process converting simple compounds or substrates into macromolecules or complex products. Accomplished by the action of living microorganisms. Green Chemistry Approach: Advantages include minimally processed, large-scale production, economic viability, and health safety. Applications: Natural products are highlighted in food, pharmacy, medicine, and agriculture due to their chemical stability and biocompatibility. Biosurfactant Definition & Properties Amphipathic Molecules: Possess a hydrophobic tail and a hydrophilic head, aggregating at phase boundaries (oil/water, air/water). Micelle Formation: When concentration exceeds the Critical Micelle Concentration (CMC), they form micelles, reducing surface tension and aiding in processes like oil removal from water or soil. Eco-friendly: Biosurfactants, produced by microorganisms (bacteria, fungi, yeast) as secondary metabolites, are eco-friendly, less toxic, biodegradable, and have lower CMC values compared to chemical surfactants. Applications: Used in bioremediation, healthcare, cosmetics, food, and oil industries due to enhanced properties. Cellular Communication: Facilitate cellular communication, including quorum sensing. Biosurfactant Interaction with Dirt & Viruses Biosurfactants have a specific structure that allows them to interact with various substances, including dirt and viruses. A. Biosurfactant Structure Hydrophilic head Hydrophobic tail B. Interaction with Dirt Dirt Biosurfactants surround dirt, lifting it away. C. Effect on Coronavirus Virus Micelle Biosurfactants encapsulate virus fragments into micelles. Biosurfactants in Cellular Communication Quorum Sensing: Biosurfactants enable bacteria to detect and regulate cell population density. Biofilm Adhesion & Dispersion: Assist in biofilm formation and detachment through cellular signaling. Antimicrobial Agents: Participate in microbial competition. Detoxification: Help microorganisms survive by binding and sequestering toxic compounds. Virulence: Can influence virulence factors in pathogenic bacteria. Quorum Sensing Signals detected Biofilm Activity Adhesion/Dispersion Antimicrobial Action Inhibits pathogens Detoxification Binds toxins Biosurfactant Classes Categorized by chemical components (hydrophilic part: amino acids, peptides, saccharides; hydrophobic part: fatty acid chains). Classified by molecular mass into: Low Molecular Mass: Glycolipids, lipopeptides, proteins (primarily reduce surface tension). High Molecular Mass: Polysaccharides, lipoproteins, polymeric particles (effective in forming stable oil-in-water emulsions). Classification Diagram Biosurfactant Glycolipids Rhamnolipids Sophorolipids Trehalolipids Lipopeptides Surfactins Iturins Pontifactins Fatty Acids Corynomycolic acids Spiculisporic acids Polymeric Emulsans Alasans Liposans Particulate Micro-emulsions Whole cell Vesicles Types of Biosurfactants by Biochemical Composition Glycolipids Carbohydrates linked to long-chain aliphatic acids or hydroxy aliphatic acids by an ester group. Majorly biosurfactants. Well-known examples: rhamnolipids , trehalolipids , and sophorolipids . Rhamnolipids: One or two rhamnose molecules linked to one or two hydroxy decanoic acid molecules. Trehalolipids: Associated with species like Mycobacterium and Nocardia . Sophorolipids: Produced by yeasts, consist of a dimeric carbohydrate sophorose linked to a long-chain hydroxyl fatty acid by glycosidic linkage. Lipopeptides and Lipoproteins Cyclic peptides linked to fatty acid chains. Surfactin: From Bacillus subtilis ; seven-amino-acid ring structure connected to a fatty acid chain via lactone linkage. Exhibits potent surfactant and antimicrobial activity. Lichenysin: Similar to surfactin, produced by Bacillus licheniformis . Iturin: Cyclic peptide with seven amino acids and an 11–12 carbon fatty acid chain; strong surfactant and antimicrobial properties. Other examples: viscosin ( Pseudomonas fluorescens ), serrawettin ( Serratia marcescens ), arthrofactin ( Arthrobacter sp. ), and polymyxin ( Bacillus sp. ). Fatty Acids, Phospholipids, and Neutral Lipids Surface-active molecules with both hydrophilic and lipophilic components, similar to phospholipids in cell membranes. Reduce surface tension at interfaces; used in gene delivery, bioremediation, and as emulsifiers. Synthesized by bacteria and yeasts growing on n-alkanes. Acinetobacter sp. produces phosphatidyl ethanolamine-rich vesicles that emulsify alkanes. Penicillium spiculisporum produces spiculisporic acid , a fatty acid-type biosurfactant. Corynomycolic acids: Variable carbon chain lengths, influenced by growth medium substrate. Polymeric Biosurfactants Well-known examples: alasan , liposan , lipomannan , emulsan , and polysaccharide-protein complexes. Liposan: Produced by Candida lipolytica ; water-soluble, 83% carbohydrate and 17% protein. Emulsan: Synthesized by Acinetobacter calcoaceticus ; three unbranched amino sugars and a fatty acid chain (10–22 carbons), molecular weight ~1,000 kDa. Alasan: Produced by Acinetobacter radioresistens ; complex of alanine, polysaccharides, and proteins, acting as a strong emulsifier. Mannoproteins: Produced by Acinetobacter sp. and Saccharomyces cerevisiae ; glycoproteins forming stable emulsions with oils and hydrocarbons, with antimicrobial properties. Particulate Biosurfactants Include extracellular vesicles and whole microbial cells. Vesicles: Consist of proteins, phospholipids, and lipopolysaccharides. Acinetobacter sp. accumulates vesicles (20–50 nm) on its cell surface. Whole microbial cells: Possess both hydrocarbon and non-hydrocarbon degrading properties, e.g., Acinetobacter calcoaceticus 2CA2, acting as an emulsifier. Biosurfactant Production Process Biosurfactants can be produced through fermentation, followed by downstream processing and purification steps. A. General Production Flow Substrate, Inoculum Fermentation Downstream Processing Purification Final Biosurfactant Product B. Detailed Purification Steps BS production (bioreactor) Removal of Bacteria (centrifugation) Solvent extraction of BS crude extract Solvent removal (Evaporation) Purified BS product (Column Chromatography) Polishing steps Detailed Biosurfactant Production Process Microorganism Selection: Identify strains known for high biosurfactant production (e.g., Pseudomonas aeruginosa for rhamnolipids, Bacillus subtilis for surfactin, yeast like Candida bombicola for sophorolipids). Consider factors like growth rate, metabolic pathway efficiency, and safety (GRAS status for food/cosmetic applications). Genetic engineering or random mutagenesis can enhance production yields and properties. Medium Optimization: Carbon Source: Hydrophobic: n-alkanes, crude oil, vegetable oils, fatty acids. Often results in higher biosurfactant yields but can be more expensive or toxic. Hydrophilic: Glucose, sucrose, glycerol, molasses, starch. Generally cheaper and easier to handle. Often leads to lower yields but better cell growth. Waste substrates (e.g., molasses, whey, industrial effluents, waste cooking oil) are preferred for cost-effectiveness and sustainability. Nitrogen Source: Organic (peptone, yeast extract, corn steep liquor) or inorganic (ammonium salts, nitrates). The C/N ratio is crucial; a high C/N ratio (carbon excess, nitrogen limitation) often triggers secondary metabolite production, including biosurfactants. Other Nutrients: Phosphate (e.g., $\text{K}_2\text{HPO}_4$, $\text{KH}_2\text{PO}_4$) for energy metabolism and cell structure. Trace elements (Mg, Fe, Mn, Zn, Ca) as cofactors for enzymes involved in biosynthesis. Fermentation Conditions (Bioreactor Operation): Temperature: Optimal range depends on the microorganism (typically $25-37^\circ\text{C}$ for mesophiles). Affects enzyme activity and cell growth. pH: Maintained within a specific range (e.g., $6.0-7.5$). Influences enzyme activity, nutrient solubility, and biosurfactant stability. Can be controlled by adding acid/base. Aeration and Agitation: Aeration: Crucial for aerobic microorganisms, providing oxygen for metabolic processes. Airflow rate is a key parameter. Agitation: Ensures uniform mixing of nutrients, cells, and oxygen, and reduces mass transfer limitations. Impeller type and speed are important. Inoculum Size: Sufficient inoculum ensures rapid onset of fermentation. Fermentation Strategy: Batch Fermentation: All nutrients added at the beginning. Simple but can suffer from substrate depletion and product inhibition. Fed-Batch Fermentation: Nutrients (especially carbon source) are added incrementally during the process. Allows for higher cell densities and prolonged biosurfactant production, reducing substrate inhibition. Continuous Fermentation: Fresh medium is continuously fed, and product is continuously harvested. Achieves high productivity but requires precise control and can be prone to contamination. Downstream Processing (Separation and Recovery): Cell Separation: Biosurfactants can be extracellular (secreted into medium) or cell-associated. Centrifugation or Microfiltration: Used to separate microbial cells from the cell-free supernatant containing extracellular biosurfactants. Precipitation: Acid Precipitation: Lowering the pH (e.g., to $2.0$) causes many biosurfactants (especially lipopeptides and some glycolipids) to precipitate due to reduced solubility. Salt Precipitation: Adding salts (e.g., ammonium sulfate) can cause salting out of biosurfactants. Solvent Extraction: Commonly used to extract biosurfactants from the aqueous phase using organic solvents (e.g., chloroform/methanol mixtures, ethyl acetate, butanol). The choice of solvent depends on the polarity of the biosurfactant. Foam Fractionation: Biosurfactants tend to concentrate at the air-liquid interface due to their surface-active properties. Bubbling air through the broth creates foam rich in biosurfactant, which can then be collected. Membrane Separation: Ultrafiltration/Diafiltration: Used to concentrate and purify biosurfactants based on molecular size. Purification: Adsorption/Desorption: Using activated carbon or resins. Chromatography: Column Chromatography: (e.g., silica gel, reverse-phase, ion-exchange) for high-purity biosurfactant isolation. HPLC (High-Performance Liquid Chromatography): For analytical separation and further purification. Drying: Evaporation, lyophilization (freeze-drying), or spray drying to obtain a solid biosurfactant product. Challenges in Biosurfactant Production Low Yields: Compared to synthetic surfactants, biosurfactant yields are often lower, making them less competitive. High Production Costs: Raw material costs, fermentation conditions, and complex downstream processing contribute to high production expenses. Purification Difficulty: Obtaining high-purity biosurfactants can be challenging due to their diverse chemical structures and similarities to other microbial products. Lack of Standardization: Variability in production processes and product characteristics can hinder widespread industrial adoption. Biosurfactant Biosynthesis ( de novo pathway) The de novo pathway refers to synthesizing complex molecules from simple precursors. Involves the biosynthesis of surface-active molecules from simpler building blocks within a microorganism. Typically involves synthesizing fatty acids, sugars, and other precursors, which are then assembled into the final biosurfactant structure. De novo pathway for biosurfactant production Simple Precursors Enzymatic Reactions Intermediate Compounds Assembly Final Biosurfactant Hydrophilic Head Synthesis: Carbohydrates, amino acids, phosphates. Hydrophobic Tail Synthesis: Fatty acids (e.g., long-chain fatty acids, alpha-alkyl betahydroxy fatty acids). These moieties are then joined to form the amphipathic structure. Molecular Biology of Surfactin Synthesized via a non-ribosomal pathway involving multi-enzyme complexes. srfA operon: Contains multiple genes ( srfAA, srfAB, srfAC, srfAD , and sfp gene), transcribed together in Bacillus subtilis . SrfA, SrfB, SrfD subunits: Key components of the synthetase complex. SrfD initiates the process, followed by SrfA and SrfB, which incorporate amino acids into the growing peptide chain. sfp gene: Encodes a phospho-pantetheinyl transferase (PPTase) that activates peptidyl carrier protein (PCP) domains of surfactin synthetase. Biosurfactants and Quorum Sensing Quorum Sensing (QS): Population density-based mechanism where bacteria use signaling molecules for communication. In Pseudomonas aeruginosa , QS regulates biosurfactant biosynthesis through three major mechanisms: las , rhl , and PQS systems. Las System: Transcriptional activators LasR and LasI produce autoinducer 3-oxo-C12HSL, which activates LasR to induce gene expression and PQS system. Rhl System: RhlA and RhlB produce rhamnosyltransferase; transcriptional activators RhlR and RhlI produce autoinducer C4–HSL. The C4-HSL-RhlR complex activates the rhlA promoter, leading to rhamnolipid production. Biosurfactant and Virulence Many microorganisms produce virulence factors (e.g., pyocyanin, elastases, proteases) that aid in biofilm adhesion, dispersion, and are linked to biosurfactant production. Examples: Pseudomonas aeruginosa and Klebsiella pneumoniae are pathogenic biosurfactant producers. In P. aeruginosa , biosurfactant production and virulence factor regulation are interconnected through the las/rhl quorum sensing system. Regulatory proteins: VqsR and Vfr influence virulence and biosurfactant production. VqsR mutants show reduced rhamnolipid synthesis, while Vfr mutations affect LasR/RhIR system, altering expression of proteases, exotoxins, and rhamnolipids. Rhamnolipids can disassemble biofilms and enhance virulence factors like protease and siderophore. Biosurfactants and Motility Many swarming bacteria produce biosurfactants to reduce surface tension, aiding in rapid movement across surfaces. Correlation with HAA production: FleQ transcriptionally activates FleSR (required for hook and basal body complex formation) and controls HAA formation. HAA is transported outside through the cell membrane. Early flagellar assembly activates HAA production, but late stages and flagellar maturation restrict it. Biosurfactant: Antitumor Agents Biosurfactant Cell line Description Activity Mannosylerythritol lipids (MELs) K562 Myelogenous leukemia Growth inhibition, differentiation Succinoyl trehalose lipids (STLs) HL60, KU812 Promyelocytic leukemia, Basophilic leukemia Growth inhibition, differentiation Sophorolipids HL60, H7402, A549, HPAC, KYSE109/KYSE450 Promyelocytic leukemia, Liver cancer, Lung cancer, Pancreatic cancer, Esophageal cancer Growth inhibition, Interaction with plasma membrane, Growth inhibition, cell cycle arrest, apoptosis induction, Necrosis Surfactin or surfactin-like biosurfactants BEL7402, K562, LoVo, MCF7, T47D/MDA-MB231 Hepatocellular carcinoma, Myelogenous leukemia, Colon adenocarcinoma, Breast cancer Growth inhibition, apoptosis induction, cell cycle arrest e-poly-L-lysine Caco2, HCT15/HT29, HeLaS3, HepG2, PC3M Colorectal cancer, Colon cancer, Cervix adenocarcinoma, Hepatocellular liver carcinoma, Metastatic prostate cancer Growth inhibition, apoptosis induction, migration inhibition Viscosin BCLL B-Chronic lymphocytic leukemia Apoptosis induction Serratamolide HeLa Cervical cancer Growth inhibition Monoolein U937 Leukemia cancer Growth inhibition Glycoprotein from Lactobacillus paracasei T47D/MDA-MB231 Breast cancer Growth inhibition, cell cycle arrest Applications of Biosurfactants for Industrial Uses Industry Application Role of Biosurfactants Environment Bioremediation; Oil spill cleanup; Soil remediation and flushing Emulsification of oils, lowering interfacial tension, dispersion of oils, solubilization of oils, wetting, spreading, detergency, foaming, corrosion inhibition Petroleum Enhanced oil recovery; De-emulsification Emulsification of oils, lowering interfacial tension, de-emulsification of oil emulsions, solubilization of oils, viscosity reduction, dispersion of oils, wetting of solid surfaces, spreading, detergency, foaming, corrosion inhibition Mining Heavy metal cleanup; Soil remediation; Flotation Wetting and foaming, collectors and frothers, removal of metal ions from aqueous solutions, soil and sediments, heavy metals sequestrants, spreading, corrosion inhibition Food Emulsification and de-emulsification; Functional ingredient Solubilization of flavored oils, control of consistency, emulsification, wetting agent, spreading, detergency, foaming, thickener Medicine Microbiological; Pharmaceuticals and therapeutics Anti-adhesive agents, antifungal agents, antibacterial agents, antiviral agents, vaccines, gene therapy, immunomodulatory molecules Biosurfactant: Bioremediation Enhance pollutant bioavailability through pseudo-solubilization and emulsification. Assist in heavy metal recovery by chelation and micelle formation. Increase degradation of hydrocarbons like phenanthrene, anthracene, and fluorene. Phormidium sp. showed significant capacity to remove hexadecane and diesel from water. Developing bacterial-algal consortia for pollutant removal is a promising approach. Pseudomonas aeruginosa SR17 rhamnolipid achieved high degradation of total petroleum hydrocarbons (TPH) in soil. Outperformed synthetic surfactant sodium dodecyl sulfate (SDS) in degradation. GC-MS analysis revealed presence of polycyclic aromatic hydrocarbons (PAHs) after treatment. Rhamnolipid treatment eliminated floranthene, benz(b)fluorene, and benz(d)anthracene, and reduced other PAHs. MEOR (Microbial Enhanced Oil Recovery) Process where microorganisms and/or their metabolic by-products are injected into mature oil reservoirs to recover residual crude oil. Introduced microbes grow exponentially, and their metabolic products mobilize residual oil. MEOR: Fundamental Principles First principle: Oil movement through porous media is facilitated by altering interfacial properties of oil-water-minerals, decreasing interfacial tension (IFT), and reducing viscosity. Second principle: Degradation and removal of sulfur and heavy metals from heavy oils by microbial activity. Injection of pre-cultured bacteria or consortia with nutrients (oxygen, nitrogen) is preferred, as bacteria produce biosurfactants in-situ , reducing IFT and facilitating extraction. MEOR Mechanism Injection Well Bacteria, Nutrients Pressing water + microorganisms Enhanced mobility Biodegradation of crude oil Production Well Crude Oil Microbial metabolites/biosurfactants: Gas Acid Biomass Polymer Crude oil + Advanced water Improved crude oil mobility Improved reservoir percolation Enhanced oil recovery MEOR: Before and After Before MEOR After MEOR Oil trapped in rock pores Biosurfactants mobilize oil Injector Producer Injector Producer