With rising environmental concerns and the urgent need for sustainable agricultural practices, biosurfactants have garnered significant attention. These naturally occurring, surface-active compounds produced by microorganisms offer eco-friendly alternatives to synthetic chemicals.
Definition, Classification, and Structure of Microbial Surfactants
Biosurfactants are surfactants derived from biological sources, such as bacteria, fungi, and algae. These amphiphilic compounds are composed of two parts: a hydrophilic (polar) section, including amino acids, cations, peptide anions, mono-, di-, or polysaccharides, and a hydrophobic (non-polar) section consisting of unsaturated and saturated fatty acids. Recognized as metabolic products or the actual cell surface chemistry of microorganisms, biosurfactants are produced by various microorganisms, including Pseudomonas, Bacillus, Acinetobacter, and Candida lipolytica.
Biosurfactants possess important benefits, including biodegradability, lower toxicity, and diverse structural possibilities compared to chemically synthesized surfactants. The term “microbial surfactants” refers to compounds secreted by microorganisms in cultivation environments, such as bacteria or fungi. Biological surfactants are compounds derived from living sources that alter the surface properties of water. The cultivation environment typically contains carbon sources required by these microorganisms for growth and the production of surfactants.
Microbial surfactants serve as surface-active agents that facilitate the solubilization of water-insoluble compounds, such as hydrocarbons, fats, and oils. By reducing the surface tension of water, they enable hydrophobic substances to dissolve and disperse more effectively in an aqueous environment. In this environment, hydrophobic compounds can serve as a source of nutrients for organisms. Due to their microbial origin and access to nutrients, biosurfactants can be categorized into two groups based on their molecular weight and chemical composition. There are two types of biosurfactants: molecules with low molecular weights and molecules with higher molecular weights. Those with low molecular weights are primarily composed of hydrophobic lipid segments, which facilitate their solubility in aqueous environments. Biosurfactants with a low molecular weight are produced by bacteria, such as Bacillus and Pseudomonas. On the other hand, biosurfactants with higher molecular weights can form stable emulsions when combined with proteins, polysaccharides, lipopolysaccharides, or mixed biopolymer complexes.
The primary criterion for classifying biosurfactants is their chemical structure. Consequently, fatty acids, neutral lipids, lipopeptides, phospholipids, polymeric surfactants, and glycolipids are among the types of biosurfactants available (Figure 1).
Glycolipids
Glycolipids consist of two main components: carbohydrates and hydroxy fatty acids. Carbohydrates, such as rhamnose, sophorose, and trehalose, serve as the polar part of glycolipids, while hydroxy fatty acids provide the hydrophobic properties. Researchers have extensively studied glycolipids due to their diverse surface and physical properties resulting from the carbohydrate moieties they contain.
The polar carbohydrate components in glycolipid structures allow these compounds to be classified into various groups, including sulfolipids, sophorolipids, trehalolipids, rhamnolipids, lipomannans, and lipids. Each subgroup has its own specific characteristics and applications, utilized in industries, such as chemicals, biotechnology, and others. These applications highlight the versatility and importance of glycolipids in various industrial processes.
Glycolipids serve as surface-active agents, facilitating the solubilization of water-insoluble compounds, like hydrocarbons, fats, and oils. This capability is crucial in environmental applications, such as bioremediation, where glycolipids help disperse and degrade pollutants in soil and water environments.
Lipopeptides
Lipopeptides are biosurfactants produced by certain bacteria, especially Gram-positive bacteria, such as Bacillus species. Surfactin, a key lipopeptide, consists of two primary components: a hydrophilic section and a hydrophobic section. The hydrophilic part, known as the polar or water-attracting peptide ring, is formed by a cyclic peptide of seven consecutive amino acids. The hydrophobic section comprises a fatty acid chain with 13 to 15 carbon atoms, suitable for adsorption on non-aqueous surfaces, like oils and fats. These characteristics give lipopeptides specific biological and physical properties.
Phospholipids
Phospholipids are amphipathic molecules consisting of a hydrophilic phosphate group on one end and hydrophobic fatty acids on the other. These biosurfactants are essential components of microbial plasma membranes. When microbial strains grow in the presence of hydrocarbons, the surface area of phospholipids increases.
Fatty Acids
The microbial oxidation of alkanes can produce fatty acids that act as surfactants. These fatty acids may have linear or complex hydrocarbon chains with alkyl branches and hydroxyl groups. The balance between the hydrophilic and hydrophobic properties of fatty acids is closely related to the length and complexity of the hydrocarbon chain.
Polymeric Biosurfactants
Polymeric biosurfactants are composed of various chemical structures, including heteropolysaccharides, exopolysaccharides, carbohydrates, lipids, proteins, and other polysaccharide-protein complexes. The most extensively studied polymeric biosurfactants include emulsan and liposan, produced by Acinetobacter radioresistens, A. calcoaceticus, and Candida lipolytica.
Screening, Extraction, Purification, and Assessment of Biosurfactant Activity
Screening Methods for Biosurfactants
Promising microbial strains or their supernatants are subjected to various tests to evaluate biosurfactant activity. These tests include assays for hemolytic activity, surface tension measurements, the drop collapse test, oil displacement assessments, emulsification activity [44], the emulsification index, the CTAB/methylene blue agar test, and high-throughput screening for penetration assessments.
Identifying microorganisms that produce biosurfactants typically involves several steps. Initially, microorganisms are isolated from a culture medium suspected of biosurfactant production. These isolates are then purified to obtain individual microbial strains. Next, DNA is extracted from the isolated microorganisms, followed by polymerase chain reaction (PCR) to amplify specific DNA regions indicative of genes associated with biosurfactant production. The amplified DNA fragments are sequenced, and the sequence data are subjected to phylogenetic analysis to identify the closest relatives or classify the microorganisms.
Extraction of Biosurfactants
Various techniques are used to extract biosurfactants, each with its own advantages and limitations based on the nature of the biosurfactant, the complexity of the culture medium, and the desired purity and yield. Common techniques include centrifugation, which separates biosurfactants based on density differences, and methods involving the addition of solvents or acids to precipitate biosurfactants from the culture broth, followed by filtration or centrifugation. Ion exchange chromatography and isoelectric focusing are also used to separate biosurfactants based on charge and isoelectric points, respectively. Ultrasonication employs high-frequency sound waves to disrupt microbial cells and release biosurfactants, while dialysis uses a semipermeable membrane to separate biosurfactants based on molecular size or charge differences.
Purification of Biosurfactants
Purification is crucial for the characterization and application of biosurfactants. Several techniques are notable for this purpose:
Thin-Layer Chromatography: This method separates raw biosurfactants on a silica gel plate using a solvent mixture. The type of biosurfactant is identified using a developing solvent system and color indicators, such as ninhydrin, which produces a red spot for lipopeptides.
Dialysis and Ultrafiltration: These cost-effective methods enhance the purity of biosurfactants. Dialysis, using cellulose bags, removes impurities and salts, resulting in a more refined product.
Isoelectric Focusing: A sophisticated purification technique that involves filling a column with gradient density solutions, electrolytes, and non-ionic conducting polymers. Biosurfactants migrate through the column until they reach a neutral pH, influenced by an electric field, pH, density gradient, and ampholytes. Once separation is complete, the purified biosurfactant is compared with the crude form in terms of activity.
Assessment of Biosurfactant Activity
Evaluating biosurfactant activity typically involves measuring its ability to alter the surface tension and hydrophilic-lipophilic balance. These parameters provide insights into the effectiveness of purified biosurfactants in various applications.
Factors Influencing Biosurfactant Production
Various factors, including genetic, physiological, nutritional, and environmental elements, significantly affect the production of microbial biosurfactants (Figure 2). Optimizing these factors is essential to maximize biosurfactant production, which typically occurs under specific conditions. Nutritional components, such as nitrogen and carbon sources, trace elements, and vitamins greatly influence the quantity and type of biosurfactants produced. Environmental factors, such as the pH, temperature, and aeration levels, also play a critical role in biosurfactant production dynamics.
Nutritional Factors
Carbon Source
Nutritional factors significantly influence microbial biosurfactant production, with carbon sources being particularly crucial. Biosurfactant-producing microorganisms are predominantly heterotrophic, meaning they depend on external carbon sources for growth and metabolite synthesis, including biosurfactants. Carbon sources used for biosurfactant production typically fall into three main categories: hydrocarbons, oils and fats, and carbohydrates. The type and concentration of the carbon source can vary depending on the microbial species and the biosurfactant being produced. The metabolic pathways involved in biosurfactant precursor generation are closely linked to the availability and composition of carbon sources in the production environment.
Abundant and Cost-Effective Substrates
Recently, there has been an increased focus on using abundant and cost-effective substrates as viable carbon sources for biosurfactant production. When selecting these substrates, factors, such as variability, stability, raw material availability, and waste management, should be considered. Additionally, factors like purity, storage conditions, packaging, and transportation methods significantly impact their suitability for biosurfactant production.
Nitrogen Source
In addition to carbon sources, nitrogen is vital for synthesizing primary and secondary metabolites, such as biosurfactants, proteins, and enzymes. Various nitrogen sources, including nitrate, amino acids, and ammonia, are commonly used by researchers. These nitrogen sources have proven to be effective in supporting biosurfactant production, particularly in microorganisms like Pseudomonas aeruginosa. Selecting suitable nitrogen sources is crucial for optimizing biosurfactant-production processes and enhancing the yield and quality of the final product.
Mineral Components
Mineral components, such as phosphates, calcium, and trace elements, play a significant role in facilitating microbial activity and growth, thereby influencing biosurfactant production. KH2PO4 and K2HPO4 act as buffers in biosurfactant environments, maintaining optimal pH conditions for microbial growth. Calcium, often added as chloride salts or hydrated chlorides, supports microbial growth and biosurfactants. Trace elements or micronutrients also play a crucial role in biosurfactant production by positively influencing microbial growth when added in small quantities to the production environment. The specific micronutrients required can vary depending on the microorganism used.
Environmental Factors
Environmental factors significantly impact the quantity and characteristics of biosurfactants, highlighting the importance of optimizing these conditions for efficient production. Most microorganisms studied for biosurfactant production are mesophiles, thriving within a temperature range of 20 to 40 ℃. The optimal temperature for biosurfactant production generally falls between 25 and 37 ℃. Additionally, while neutral or near-neutral pH levels are preferred for bacterial biosurfactant production, acidic conditions are more conducive to biosurfactant production by algae and fungi. Understanding and controlling these environmental factors are essential for maximizing the biosurfactant yield and quality in various industrial and environmental applications.
Cultivation Strategy
Two bioprocess strategies, submerged fermentation and solid-state fermentation, are utilized at laboratory and pilot scales. These strategies are crucial for the economical production of biosurfactants and optimizing the production environment, thus playing a vital role in establishing commercial and economical biosurfactant production.
Molecular Characteristics of Biosurfactants
Biosurfactants, derived from bacteria, yeasts, and fungi, are recognized for their environmentally friendly nature and high biodegradability. Their diverse functionalities, phase behaviors, extensive structural variations, and biodegradable properties open up numerous possibilities for innovative applications. In agriculture, the microbial aspects of biosurfactants can be broadly summarized into three dimensions, as illustrated in Figure 3. The amphiphilic nature plays a pivotal role, creating two immiscible surfaces that reduce surface tension and enhance the solubility of water-repellent compounds. Biosurfactants are currently extensively utilized for soil improvement by boosting trace element concentrations. They are applied either in conjunction with pesticides or independently on plant surfaces to address plant diseases.
According to their molecular weight, biosurfactants can be classified as neutral or anionic molecules, depending on their chemical properties. These versatile compounds display a range of fundamental properties, including surface tension reduction, emulsification, absorption, micelle formation, and more, making them valuable in various applications. Microbial genera, such as Bacillus spp., Pseudomonas spp., Candida spp., and Pseudozyma spp., have garnered significant attention for their potential in biosurfactant production. Moreover, The use of rhamnolipid at a concentration of 25 μg/mL has demonstrated significant effectiveness in mitigating diseases caused by the Phytophthora cryptogea fungus in chicory. Within Verticillium, where storage compounds are fats, surfactants can infiltrate the membrane, leading to the disappearance of these storage compounds and preventing germination. Pseudomonas fluorescens produces a surfactant that has antifungal properties, particularly against potato pathogens, such as Pythium ultimum, Fusarium oxysporum, and Phytophthora cryptogea. This finding is significant considering the widespread occurrence of Verticillium wilt, a highly destructive fungal disease that has inflicted substantial damage on crops globally.
It was found that 8 out of 12 bacteria producing surfactants exhibited antifungal properties against Fusarium sp., the causative agent of Pokkah disease, with one strain showing superior efficacy. Subsequent investigations unveiled that the surfactant from this strain was a rhamnolipid. It was demonstrated that lower concentrations of rhamnolipid induced the lysis or cessation of the movement of Phytophthora sojae zoospores, with concentrations ranging from 100 to 1000 mg/L inhibiting mycelium growth by up to 30%. Furthermore, the rhamnolipid produced by Pseudomonas aeruginosa hindered the mycelial growth of the pathogenic fungus Leptosphaeria maculans in rapeseed. Prolonging the interaction time between petroleum hydrocarbons and soil has been observed to diminish the biodegradability of these compounds. An increase in the organic carbon content within the soil enhances the desorption of petroleum hydrocarbons, impeding their breakdown by microorganisms. Surfactants facilitate the desorption of contaminants from the soil through mineralization and solutionization mechanisms. A study investigating two biosurfactants produced by C. sphaerica and Bacillus sp. revealed their high efficiency in releasing motor oil from the soil. It was conducted research on the impact of glycolipopeptide surfactant and sodium dodecyl sulfate (SDS) on oil release from a soil contaminated with 7000 mg/kg octane for 30 days. The results indicated that glycolipopeptide and SDS achieved octane release rates of 65.1% and 37.2%, respectively. The maximum octane release after 30 days reached 92% (591 mg/kg) and 94% (430 mg/kg) for glycolipopeptide and sodium dodecyl sulfate, respectively.
It was explored various surfactant concentrations to assess the performance of rhamnolipid and surfactin in releasing total petroleum hydrocarbon from soils containing 3000 and 9000 g TPH/kg soil. Higher surfactant concentrations (both rhamnolipid and surfactin) appeared to enhance of total petroleum hydrocarbon (TPH) release from both soils. In soil with lower contamination percentages (3000 g/kg), both rhamnolipid and surfactin achieved maximum oil-release efficiencies. For soils with higher contamination percentages (9000 g/kg), increasing the surfactant concentration from 0 to 0.2% resulted in release efficiencies exceeding 62% for both biosurfactants. It was highlighted that the main process contributing to oil release from contaminated soil using the surfactant produced by P. aeruginosa L2-1 is solubilization, and increasing the concentration enhances the efficiency of oil release from the soil. It was demonstrated that elevating the concentration of the lipopeptide biosurfactant significantly improved the oil-release efficiency from the soil, increasing it from 0.005 to 0.1 g/L.
The affinity between metals and surfactants surpasses the bond between metals and the load-bearing surfaces of the soil, attributed to the reduction in the surface tension leading to the adsorption of metals from soil surfaces to the soil solution. Cationic surfactants compete with metals for negatively charged adsorption surfaces, causing metals to be adsorbed into the soil solution. Soil washing with a 25 mM rhamnolipid solution resulted in desorption levels of 91.6% for cadmium and 87.2% for zinc. Research on the surfactant produced by Candida indicated its capability to desorb 98.9% zinc, 89.3% iron, and 81.1% lead. The surfactant produced by Starmerella bombicola CGMCC 1576 demonstrated superior abilities in the desorption of contaminants from the soil compared to synthetic surfactants and distilled water, increasing the desorption capacity of cadmium by 83.6% and 44.8% at an 8% concentration. In soil contaminated with 45 mg/g copper and 14 mg/g nickel, a 2 g/L saponin concentration exhibited a desorption capacity of 83% for copper and 85% for nickel. It was observed a decrease in the concentration of elements, such as copper, cadmium, and zinc, during soil washing with a saponin solution. A study on rhamnolipid, sophorolipid, surfactin, and lipopeptides produced by Pseudomonas aeruginosa ATCC 9027, Torulopsis bombicola ATCC 22214, and Bacillus subtilis ATCC 21332 found that rhamnolipid could desorb 65% of copper and 18% of zinc, whereas sophorolipid achieved 25% desorption for copper and 60% for zinc.
As evident from Table 1, the predominant biosurfactants employed in agriculture include rhamnolipid, sophorolipid, surfactin, and lipopeptides.
| Experiment Conditions | Surfactant Type | Results |
| Review study | Microbial activity derived biosurfactants | Increasing feed digestibility, improving seed protection and fertility |
| Microbial sources: Pseudomonas, Bacillus, and Candida | Rhamnolipid, sophorolipid and surfactin | Oil spill management |
| Analyze microbial biosurfactant production, focusing on the optimization of metabolic pathways and production | Rhamnolipids surfactin | Improved yield and reduced ATP |
| Review study | Rhamnolipids | Potential antimicrobials, immune modulators, virulence factors, and anticancer agent |
| Microbial sources: Pseudomonas, Burkholderia, and Bacilus species | Rhamnolipids and lipopeptides | Plant resistance to phytopathogens |
| Soil application study | Rhamnolipid and surfactin | Enhanced TPH release from soil |
| Soil washing study | Rhamnolipid solution | Desorption of cadmium and zinc |
| Heavy-metal-contaminated soil study | Surfactant produced by Pseudomonas aeruginosa | Enhanced efficiency of oil release from soil |
| Agricultural community cohort study | Rhamnolipid and other biosurfactants | Significant alterations in microbiome composition due to pesticide exposure |
| Investigation of biosurfactants for sustainable agriculture | Surfactin, lipopeptides | Improvement in nutrient availability and soil revitalization |
| Production of biosurfactants using agriculturalresidues | Rhamnolipids, sophorolipids | Potential in waste management and bioremediation applications |
| Study on farm work tasks | Multiple surfactants | Increased microbial diversity in indoorenvironments |
Biosurfactants in Soil Nutrient Availability and Soil Quality Improvement
Effects of Biosurfactants on Soil Nutrient Availability
Biosurfactants produced by bacteria in the soil’s rhizosphere enhance the availability of water-repellent molecules, potentially assisting in plant growth through various methods. These methods include acting as nutrient supplements, enhancing soil wettability, and facilitating the proper distribution of chemical fertilizers in the soil. The properties of biosurfactants indirectly influence nutrient availability. When combined with chemical compounds, biosurfactants serve as plant protectants, contributing to increased solubility and the powdered form of these compounds. Additionally, these substances exhibit valuable antimicrobial properties, aiding in the absorption of biogenic materials and promoting seed germination.
Classifying biosurfactants based on their molecular weight is indeed a common approach, dividing them into two main categories: high-molecular-weight biosurfactants and low-molecular-weight biosurfactants. Each category exhibits distinct properties and functions in various applications, particularly in biodegradation processes and emulsion stabilization. Low-molecular-weight biosurfactants typically have molecular weights below 1 kilodalton (kDa) and are known for their ability to reduce surface and interfacial tension between immiscible fluids, such as oil and water. This property allows them to enhance the solubility and dispersion of hydrophobic compounds, facilitating their degradation by microorganisms. On the other hand, high-molecular-weight biosurfactants, with molecular weights typically exceeding 1 kDa, are more effective at stabilizing emulsions, particularly oil-in-water emulsions. They form a protective layer around oil droplets, preventing their coalescence and promoting the formation of stable emulsions. This stabilization mechanism is valuable in various industrial processes, such as the production of food, cosmetics, and pharmaceuticals. Research has highlighted that the adoption of nutrient-management practices can lead to improved productivity and the reduced environmental impact of farming. However, the adoption of key practices remains below expectations globally. Nutrient management planning can significantly enhance soil nutrient availability, influencing farmers’ intentions to apply fertilizers based on soil test results. In urban gardening, the quality of soil can be influenced by the cultivation techniques, highlighting the role of biosurfactants in maintaining the soil quality. Overall, biosurfactants serve as valuable tools in enhancing soil nutrient availability and improving soil quality, contributing to sustainable agricultural practices.
Use of Biosurfactants to Improve Soil Quality
In the realm of agriculture, biosurfactants offer a versatile range of applications. They can be employed to combat plant pathogens, enhance the accessibility of nutrients for beneficial plant-related microorganisms, and improve soil quality through amendments. As eco-friendly alternatives, these bio-based molecules have the potential to replace industrial surfactants, contributing significantly to environmental pollution control. The amphiphilic nature of biosurfactants offers a significant advantage, as they spontaneously segregate into two immiscible phases by both reducing the surface tension and enhancing the solubility of hydrophobic compounds. Their environmentally friendly and non-toxic characteristics, coupled with resilience to elevated temperatures and resistance to varying pH levels, highlight their importance.
Biosurfactants play a crucial role in breaking down hydrocarbon compounds and providing bio-available substrates for microorganisms, thereby enhancing microbial activity—a pivotal factor in soils contaminated with hydrocarbon compounds. For instance, urban cultivation for food production has highlighted the role of biosurfactants in protecting the soil quality through the integration of organic matter and appropriate cultivation techniques, which can prevent the degradation caused by chemical treatments. Additionally, research has shown that soil quality significantly impacts population mobility in agricultural regions, with improved soil conditions leading to reduced migration, thereby underlining the importance of maintaining soil health.
Conclusions and Future Prospects
Biosurfactants represent a promising and versatile solution for enhancing agricultural sustainability and addressing environmental challenges. The integration of biosurfactants into precision agriculture systems, supported by advancements in biotechnology, genomics, and nanotechnology, offers new opportunities for optimizing agricultural efficiency and sustainability. Future research should focus on enhancing the biosurfactant production efficiency, optimizing application strategies, and developing tailored solutions for specific agricultural challenges. The advancement of synthetic biology, microbial engineering, and green chemistry approaches can significantly improve biosurfactant yields and functional diversity, facilitating their large-scale deployment. As research progresses, biosurfactants have the potential to mitigate climate change impacts, improve soil health, and enhance crop resilience.
In agriculture: The surfactant can be used insolubilization, wetting, emulsification, foaming. Dora presents the natural surfactants to meet the standard of organic agriculture, ensure the organic throughout.
For additional insights into agricultural surfactant options, refer to the Dora surfactant series.
