Rhamnolipids bio-production and miscellaneous applications towards green technologies: a literature review

Search methodology

This literature review goal is to focus on bioproduction, the potential applications of rhamnolipids in pharmaceuticals, soil bioremediation, agriculture and food industries in addition to future perspectives, especially with their positive economic and environmental impact. To achieve our goal of ensuring comprehensive and unbiased coverage of the literature, we targeted highly peer-reviewed, especially CC-BY-4.0 licensed articles published in English, starting from the year 2000 (except for a few basic articles), with no geographical location limitation including: case studies, research articles, review articles and market analyses employing search engines, mainly Google and Firefox. In order to be precise in our search, some search terms were used such as Rhamnolipids, Bioproduction, Biosurfactants, Bioemulsifier, Functional properties, Biotechnology and Green economy. For further precision, the exclusion criteria included factors or characteristics related to genetics. These factors cannot be well covered within the current data which may confuse the readers and affect the targeted outcomes. Our efforts are primarily motivated by a desire to learn more about different bioproduction, and the potential applications of rhamnolipids toward green economy.

Rationale and intended audience

Biosurfactants are safe, multi-functional, emulsification, micelle production, and oil displacement. From environmental perspective, biosurfactants are biodegradable, environmentally compatible, and non-toxic. The sustainability of science thus requires well-behaved alternates that best suit the demand. Integration with green economy recommends valorization of agro-food wastes as suitable feedstock for added value sustainable biosurfactant production to mitigate global carbon footprint and contribute to the United Nations SDGs goal. The biosurfactants’ growing market size was valued for over USD 8 billion in 2022 and is set to depict around 5% CAGR from 2023 to 2032. Thus, this review may attract the attention of a wide array of audience in fields related to biosurfactants production, industrial applications, environmental sciences, microbiology and marketing.

External environmental factors

PH is always required for the growth of microbial and the fermentation of biosurfactants. In Pseudomonas aeruginosa, surface tension measurements differs due to the carboxylic acid moiety (pKa 5.5 for rhamnolipid aggregates), solution adjusted to pH 4.0 or 8.0 create solutions of the protonated nonionic or deprotonated anionic mono-rhamnolipids, respectively (Palos Pacheco et al., 2017; Cerqueira Dos Santos et al., 2024). In C. antarctica biosurfactants production, the best production was obtained at pH ranged from four to eight (Begum, Saha & Mandal, 2023).

Temperature is one of the most important environmental factors in biosurfactant production. The growth of microorganisms is associated with a particular temperature range. Thus, the fermentation of carbohydrates needs an optimum well-maintained temperature due to its impact on the metabolic process and physical properties of the fermentation broth. In case the temperature exceeds the optimum level, denaturation of enzymes and other proteins may occur (Begum, Saha & Mandal, 2023). The optimal temperature for the synthesis of rhamnolipids by P. aeruginosa 44T1 on glucose was determined to be 37 °C (Robert et al., 1989). Others claimed that when Pseudomonas sp. DSM 2874 employed n-alkanes as a substrate, it was feasible to control the rhamnolipid composition of the surfactant consisting of four compounds by temperature (Syldatk et al., 1985).

As temperature has a broad effect on bacterial metabolism and regulation, different hypothesis may provide an explanation for this behavior. RNA-thermometers are generally reported to display a comparably high degree of “leakiness” and inefficiency when inhibiting translation, which could result in differences in rhamnolipid production rates. In many pathogenic bacteria, expression of virulence genes is induced at 37 °C, and the underlying mechanisms are often based on RNA-thermometers. The presence of an RNA-thermometer in the rhlAB operon coding for the rhamnosyltransferase complex required for rhamnolipid biosynthesis (Noll et al., 2019).

Agitation and aeration are very important factors in the fermentation process as (1) they affect the activities of heat, nutrients and oxygen distribution in the medium, (2) they affect medium viscosity, (3) they provide the oxygen necessary for cell growth and fermentation process, (4) they reduce the exhaust gases generated during the process (Begum, Saha & Mandal, 2023).

Salinity of the halotolerant strain and many marine and petroleum-contaminated soils that have a saline environment is crucial. The effect of salinity on Pseudomonas aeruginosa performance in the production of biosurfactants was measured by the surface tension of media. The reduction of surface tension in media containing Pseudomonas aeruginosa should be maintained over 1.4% NaCl concentration for the best quality (32.1 mN/m) (Ahmadi et al., 2021).

Internal factors

Carbon source can affect the produced biosurfactant amount, composition and quality. Various carbon sources could be applied for the production of biosurfactants. Long chain fatty acids such as in soybean oil are applied as carbon source in the rhamnolipids fermentations. Their de-foaming properties can weaken liquid films by competing with foaming metabolites and destabilize the formed bubbles, thus, suppressing the rhamnolipids fermentation broth foaming behavior (Gong et al., 2021).

Nitrogen source is an important factor that affects the quality and production rate of biosurfactants including; ammonium chloride, urea and ammonium sulfate. The nitrogen addition showed to effect on cell growth and reduced the surface tension. Furthermore, the nitrogen source in the environment is very important for metabolism especially cellular synthesis and bacterial functions. For production of biosurfactant by Pseudomonas fluorescens, olive oil and ammonium nitrate were used, as the best carbon and nitrogen sources, respectively.

Micro- and macronutrients are essential factors affecting RLs production. Broth medium supplemented with metal ions such as MgCO₃ and Fe₂+ can enhance microbial rhamnolipids production Cassava wastewater represents a low-cost culture medium rich in macro-nutrients (proteins, starch, sugars) and micro-nutrients (iron, magnesium) (Begum, Saha & Mandal, 2023; De Oliveira Schmidt et al., 2023).

In conclusion, for rhamnolipid production process, all of these issues should be taken into account, demonstrating the need for a multidisciplinary strategy integrating several disciplines and technologies.

Analytical methods

Chromatographic techniques such as thin-layer chromatography (TLC) and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) are available analytical methods for determining rhamnolipids (Heyd et al., 2008). TLC is generally used to analyze culture broth extracts of rhamnolipid composition. Based on either the number of sugars in the molecule (mono-rhamnolipids or di-rhamnolipids) or the length of the fatty acid alkyl chain, normal or reversed phase TLC has produced effective separation of rhamnolipid combinations (Long et al., 2013). Rhamnolipids are not accurately quantified by TLC, and considerable sample amounts are required (in comparison to those needed for other tests). However, HPLC-MS not only distinguishes between various rhamnolipid species but also offers precise structural details and quantification of the rhamnolipids present in the sample. For optimal results, reversed-phase silica columns and negative electrospray ionization are typically utilized in this approach (Déziel et al., 2000; Pinzon-Gamez, 2009). Although this technique is quite accurate at analyzing rhamnolipids, the equipment is very expensive, and prior purification procedures are necessary to get rid of salts and other impurities that can interfere with the ionization of rhamnolipids in the MS. Losses of analyte due to manipulation during the purification process might also affect the quantitation (Heyd et al., 2008; Pinzon-Gamez, 2009). Rhamnolipids in aqueous solutions can be identified and measured using an alternate technique such as ATR-FTIR (Leitermann et al., 2010). Although this method similarly depends on the availability of expensive equipment, it is suitable for the quick and repeatable examination of rhamnolipids. The procedure still needs to be standardized and its accuracy needs to be increased.

Indirect methods

Colorimetric methods

Anthrone, phenol-sulfuric acid, and orcinol are among the colorimetric techniques that are frequently utilized (Raza et al., 2007). In these procedures, rhamnolipids are extracted from the aqueous sample into an organic solvent, and the rhamnose and lipid portions of the molecule are then separated by acid hydrolysis. After the sugar has reacted with phenol, orcinol, or anthrone, it is then measured spectrophotometrically. These techniques are widely used for rhamnolipid quantitation, but because of the powerful acid required, they are time-consuming, difficult, and dangerous to use (Zhu & Zhang, 2008). They are also indirect as rhamnose is detected rather than rhamnolipids, and further steps must be taken to establish the ratio of the sugar to lipid moieties in the combination (which may alter throughout manufacture) to accurately determine rhamnolipid concentration. Additionally, several solvents, inorganic salts (such as NaCl), carbonyl or oxidizing substances, and proteins may also affect the measurement (Heyd et al., 2008; Pinzon-Gamez, 2009). The effectiveness of strain selection is increased by a quick and simple method for rhamnolipid analysis, development of processes and metabolic engineering for increased output. Subsequently, Siegmund & Wagner (1991) used agar plates with methylene blue and cetyl trimethyl ammonium bromide (CTAB) (SW plates), and they suggested a semi-quantitative technique. The technique depends on cationic chemicals like methylene blue or CTAB and the ability of anionic surfactants to produce insoluble ion pairs. Rhamnolipids, an anionic biosurfactant, can interact with these cationic molecules to form a complex, which can be seen by the development of dark blue patches on the agar plates (Pinzon-Gamez, 2009). Therefore, colonies established around rhamnolipid-producing strains can be identified by the dark blue halo, and the regions of the halo could be connected with the quantification of rhamnolipids produced. This technique is applied for strain selection and screening research which is unique to glycolipids (Pinzon-Gamez, 2009), especially for distinguishing between rhamnolipid producers and non-producers. This approach is frequently insufficiently quantitative to identify the strain that produces the most among many others, particularly when many of them generate other pigments that obstruct the creation or detection of the blue halo. Additionally, variables like various bacterial cell growths, cultivation times, and agar plate filling levels can all readily affect the halo diameter (Pinzon-Gamez, 2009).

Vegetable oils and oil wastes several studies

Rapeseed oil, babassu oil, and maize oil are examples of plant-derived oils that have demonstrated their ability to serve as efficient and affordable raw materials for the manufacturing of biosurfactants. Similarly, different bacteria produced rhamnolipid, sophorolipid, and mannosylerythritol lipid biosurfactants using vegetable oils such as sunflower and soybean oils (Mukherjee, Das & Sen, 2006). Growing P. aeruginosa 47T2 c on olive oil mill effluent (OOME) as the only carbon source (a significant waste issue in Spain) will provide rhamnolipids (Mercad et al., 1993), in addition to different lipophilic wastes that might be used in rhamnolipids production (Makkar, Cameotra & Banat, 2011).

Canola oil, soybean oil, and glucose were used as a combination for P. aeruginosa UW-1 to produce rhamnolipids, and the results showed a 10–12 times increase in rhamnolipid production in vegetable oils compared to glucose (Sim, Ward & Li, 1997). Additionally, P. aeruginosa UG2 cultures cultured on maize oil as the only carbon source were reported to produce a variety of rhamnoli (Mata-Sandoval, Karns & Torrents, 2000). P. aeruginosa isolate Bs20 cultured on a medium containing soybean oil also produced rhamnolipid (Abdel-Mawgoud, Aboulwafa & Hassouna, 2009). The rhamnolipid was generated as a thick, sticky, oily, yellowish-brown liquid with a fruity smell. It also showed very high surface activity, emulsifying ability, and qualities that allowed it to withstand heat and light. These features suggested that rhamnolipids are suitable for application in the bioremediation of hydrocarbon-contaminated locations or the petroleum industry, and this conclusion was later verified (Perfumo et al., 2010). Additionally, Bacillus velezensis S2 is expected to play a significant role in oil remediation from the environment as well as serve as a potential source of non-toxic and eco-friendly biosurfactants for various industrial applications (Sultana et al., 2024).

Mixed substrates of vegetable industries

Some researchers adopted a mixed substrate strategy to make processes more cost-effective (Haba et al., 2000). To create stable emulsions with kerosene oil, nine Pseudomonas and two Bacillus strains were able to reduce the surface tension (to about 32–36 mN/m). P. aeruginosa 47T2 used waste frying cooking oil (sunflower and olive oil) as substrates and generated 2.7 g/l of rhamnolipid with a production yield of 0.34 g/g (Makkar, Cameotra & Banat, 2011). Using low-cost raw materials, researchers were able to produce larger quantities of biosurfactants, with yields of 4.31, 2.98, and 1.77 g/l of rhamnolipid biosurfactants using, respectively, soybean oil, safflower oil, and glycerol. DS10-129 P. aeruginosa (Rahman et al., 2002). Rapeseed oil was used as the substrate in a biotechnological process that produced 45 g/l of combinations of mono and di-rhamnolipids by Pseudomonas sp. DSM 2874 (Trummler, Effenberger & Syldatk, 2003; Makkar, Cameotra & Banat, 2011). The formation of rhamnolipid (1–4), L-(+) rhamnose, and (R, R)-3- (3-hydroxydecanoyloxy) decanoic acid was caused by the direct addition of Naringinase to resting cells. This was one of the first attempts to produce pure rhamnolipid using an integrated microbial/enzymatic approach (Makkar, Cameotra & Banat, 2011).

Another Brazilian research team isolated P. aeruginosa LBM10 from a southern coastal region of the country. This strain was able to grow on various inexpensive carbon sources, including fish oil, soybean oil, soybean oil soap stock, and glycerol, producing a biosurfactant of the rhamnolipid type (Prieto et al., 2008).

Restaurant frying oil wastes

Remaining cooking or frying oil is a further raw material utilized in the vegetable sector. This large source of inexpensive fermentative waste is rich in nutrients. The globe over, a lot of cooking oil is produced in restaurants. According to estimates, the United States alone produces 100 billion L of oil waste per week (Shah, Jurjevic & Badia, 2007). Few studies have taken advantage of these frying oils’ enormous potential for the generation of biosurfactants (Zhu et al., 2007). P. aeruginosa zju.u1 M. was used to produce 20 g/l rhamnolipid in a 50 L bioreactor (De Lima et al., 2009). Using various waste-frying soybean oils, the effectiveness and scope of biosurfactant production by the P. aeruginosa PACL strain were examined; a maximum rhamnose concentration of 3.3 g/l, an emulsification index of 100%, and a minimum surface tension of 26.0 mN/m was attained (Makkar, Cameotra & Banat, 2011).

Lactic whey and sugar industry wastes

Lactic whey from dairy products makes a cheap and practical substrate for the manufacture of biosurfactants, which can represent a good substrate for commercial biosurfactant synthesis than synthetic media (Mukherjee, Das & Sen, 2006). However, research showed that molasses, with high sugar content, is a promising substrate for the generation of biosurfactants. P. aeruginosa GS3 produces rhamnolipid biosurfactants while growing on molasses and maize steep liquor as the main carbon and nitrogen sources (Patel & Desai, 1997). The product might be used in oil recovery due to strong surface activity and emulsification properties. Producing rhamnolipids from soy molasses is a resource that is readily available and reasonably priced (Rashedi et al., 2005), such as growing P. aeruginosa EBN-8 on molasses as the only source of energy and carbon to produce a microbial surfactant. Maximum rhamnolipid yields (1.45 g/l) were attained (Makkar, Cameotra & Banat, 2011).

Other unconventional substrate sources

P. aeruginosa MTCC 2297 produced rhamnolipids exclusively from orange fruit peeling (George & Jayachandran, 2009). Fewer yields were recorded when glycerol was used by P. aeruginosa as the only carbon source for the synthesis of rhamnolipid as opposed to conventional hydrophobic low-cost substrates (Makkar, Cameotra & Banat, 2011). Glycerol was used as a substrate for P. aeruginosa DS10-129 to produce rhamnolipid biosurfactants with a yield of 1.77 g/L (Rahman et al., 2002), while P. aeruginosa cultured on a basal mineral medium with glycerol as the only carbon source produced 15.4 g/L rhamnolipids (Guo-Liang et al., 2005). Using glycerol as a carbon source for microbial growth and biosurfactant synthesis is feasible (Lee et al., 2004). The generation of biosurfactants using fish oil for improved culture medium for P. aeruginosa BYK-2 KCTC 18012P resulted in an increased rhamnolipid yield of 17 g/l. The utilization of fish oil as a substrate showed good potential for the production of biosurfactants as inexpensive substrate that will aid in the cost-effective manufacturing (Makkar, Cameotra & Banat, 2011).

Biosurfactant mechanism

When a single amphipathic biosurfactant molecule is added to liquid water, the hydrophobic sections of the molecule will orient above the water surface, minimizing energy interactions with polar water molecules. Naturally, polar aqueous environments will not be preferred by hydrophilic regions over the somewhat hydrophobic gas phase above the surface. This partitioning will continue until the liquid surface is saturated as additional biosurfactant molecules are added to the mixture. These additions will break the hydrogen bonds that are in charge of the water’s high surface tension, resulting in a drop in surface tension. At the point of surface saturation, further surface tension reductions will be restricted, and as biosurfactant is added, molecules will be driven into the aquatic milieu. Once there are multiple biosurfactant molecules in the aqueous environment, micelle formation, in which hydrophobic biosurfactant areas cluster together and are shielded from water interactions by a hydrophilic shell, will result in the lowest energy state between the water and biosurfactant molecules. Micelles are constantly forming, dispersing, and reforming by enlisting fresh biosurfactant molecules (Singh, Kuhad & Ward, 2009).

Biosurfactants versus synthetic surfactants

Concern for environmental preservation has boosted interest in the creation of and characteristics of biosurfactants since the last decade of the previous century (Cohen & Exerowa, 2007). Aside from their potential for cost-effective manufacture, biosurfactants also have the distinct advantage of being biodegradable, which makes them “green” chemicals that are good for the environment (Whang et al., 2008; Salihu, Abdulkadir & Almustapha, 2009; Abdel-Mawgoud, Lépine & Déziel, 2010). Even under settings of high salinity, temperature, and pH, biosurfactants can maintain their characteristics (Ron & Rosenberg, 2002; Salihu, Abdulkadir & Almustapha, 2009), and are compatible with human skin, minimal irritant (Pornsunthorntawee et al., 2008). Among the special qualities of these agents are their ability to increase the bioavailability of poorly soluble organic compounds, like polycyclic aromatics, as well as other significant benefits like bioavailability, activity under a variety of conditions, ecological acceptability, low toxicity, and their capacity to be modified by biotechnology and genetic engineering (Tugrul & Cansunar, 2005; Salihu, Abdulkadir & Almustapha, 2009). Therefore, using them could provide some answers for the bioremediation of polluted soil and subsurface habitats (Salihu, Abdulkadir & Almustapha, 2009; Lai et al., 2009).

Water’s surface tension can be reduced by a good surfactant from 72 to 35 mN/m, while the interfacial tension between water and hexadecane can be reduced from 40 to 1 mN/m (Mulligan, 2005). B. subtilis surfactin may lower the interfacial tension of water and hexadecane to less than 1 mN/m and the surface tension of water to 25 mN/m. The surface tension of water is reduced by rhamnolipids from P. aeruginosa to 26 mN/m, while the interfacial tension of water and hexadecane is reduced to 1 mN/m (Muthusamy et al., 2008). The surface tension and interfacial tension of T. bombicola have been reported to be reduced to 33 mN/m and 5 mN/m, respectively (Cavalero & Cooper, 2003; Muthusamy et al., 2008). In general, biosurfactants are more effective and efficient than chemical surfactants, and their CMC is 10–40 times lower, meaning that a smaller amount of surfactant is required to achieve the greatest reduction in surface tension. The surface activities of many biosurfactants are not impacted by environmental factors like pH and temperature. Temperature (up to 50 °C), pH (4.5−9.0), and NaCl and Ca concentrations up to 50 and 25 g/l, respectively, did not affect the lichenysin of B. licheniformis JF-2 (Muthusamy et al., 2008). The surface activity of a lipopeptide from B. subtilis LB5a remained constant from pH 5 to 11 and up to 20% NaCl concentrations after autoclaving (121 °C/20 min) and six months at −18 °C.

Contrary to manufactured surfactants, molecules made by microbes are quickly destroyed (Mohan, Nakhla & Yanful, 2006; Muthusamy et al., 2008), especially those appropriate for use in environmental applications like bioremediation (Mulligan, 2005), and the cleanup of oil spills. The literature contains very little information on the toxicity of microbial surfactants. They are typically regarded as low- or non-toxic products, making them suitable for usage in food, cosmetics, and pharmaceuticals. According to one report, a synthetic anionic surfactant called Corexit had an LC₅₀ (concentration fatal to 50% of test species) against the bacteria Photobacterium phosphoreum that was ten times lower than that of rhamnolipids, indicating that the chemical-derived surfactant was more hazardous. Regarding toxicity and mutagenesis potential, a biosurfactant from P. aeruginosa was compared to a synthetic surfactant (Marlon A-350) that is often used in the industry. The biosurfactant was shown to be just marginally less hazardous and non-mutagenic than the chemical-derived surfactant, according to both assays (Muthusamy et al., 2008).

Emulsions that are stable and have a long shelf life can be created. Biosurfactants can emulsify or de-emulsify an emulsion. In general, high-molecular-mass biosurfactants outperform low-molecular-mass biosurfactants as emulsifiers. T. bombicola sophorolipids have been found to lower surface and interfacial tension, however they are poor emulsifiers (Cavalero & Cooper, 2003). Liposan, in contrast, has been successfully utilized to emulsify edible oils even though it does not diminish surface tension (Muthusamy et al., 2008). The fact that polymeric surfactants cover oil droplets to create stable emulsions gives them additional benefits. Making oil/water emulsions for food and cosmetics is made easier thanks to this feature. Rhamnolipids are now utilized in housekeeping and cleaning products as surface-active agents. According to OECD-tests 301F, 201, and 202, these items have little influence on aquatic life, are entirely biodegradable, and are environmentally benign (Leitermann et al., 2010).

Bioremediation and enhanced oil recovery

Alkanes, cycloalkanes, aromatics, polycyclic aromatic hydrocarbons, asphaltenes, and resins are some of the many hydrocarbons found in petroleum. The best surfactants for breaking down industrially produced oil-in-water and oil-in-oil emulsions, cleaning up oil spills, and emulsifying oil (Wang & Mulligan, 2004; Pinzon-Gamez, 2009). In addition to hexadecane, tetradecane, virgin, creosote, and hydrocarbon mixes in soils, rhamnolipid addition demonstrated to promote the biodegradation of hexadecane, octadecane, n-paraffin, and phenanthrene in liquid systems (Maier & Soberón-Chávez, 2000). Both improved substrate solubility for the microbial cells and interaction with the cell surface, which increases the surface’s hydrophobicity and makes it easier for hydrophobic substrates to associate, are potential pathways for accelerated biodegradation. The mineralization of octadecane increased to 20% from 5% for the controls at a concentration of 300 mg/L rhamnolipids (Zhang & Miller, 1992; Wang & Mulligan, 2004). During the development of hexadecane, the biosurfactant-producing strain produced more of an increase in cell surface hydrophobicity than a non-producing strain did (Beal & Betts, 2000). Hexadecane’s solubility was elevated by rhamnolipids from 1.8 to 22.8 mg/L. There are signs that inhibition can also happen. Rhamnolipids and the fertilizer Inipol EAp-22, according to other investigations, improved the biodegradation of aromatic and aliphatic chemicals in the aqueous phase and soil reactors (Churchill et al., 1995). When cleaning up Exxon Valdez oil spills at Alaskan gravel, rhamnolipids and pressurized water were compared. Rhamnolipids were discovered to greatly outperform water alone in successfully releasing oil from polluted gravel (two to three times Pinzon-Gamez, 2009). Rhamnolipids have been examined for their ability to biodegrade as well as for their efficacy in cleaning soils tainted with petrol and other hydrocarbons in numerous research articles. An aqueous rhamnolipid solution was shown by Neto et al. (2009) to be 3.2 times more effective than water at removing organic matter from the soil. Rhamnolipids and various bacterial consortia have been used to demonstrate the bioremediation of n-alkanes in petroleum sludge: C8–C11 alkanes were destroyed, and alkanes in the range of C12–C21 were degraded up to 83–98% (Pinzon-Gamez, 2009). Primary pumping techniques only retrieve about 30% of the crude oil from the field in the petroleum industry. During water, steam, or fire flooding recovery operations, the injection of surfactants lowers the surface and interfacial tensions of the oil in the reservoir, enabling oil flow and penetration through reservoir pores (Norman et al., 2004; Pinzon-Gamez, 2009). The many processes involved in the deterioration of organic substrates are stimulated by rhamnolipids. Depending on how the substrate is given, the biodegradation process’ effectiveness and the precise mechanism of action of rhamnolipid may change. This group demonstrated that as opposed to matrices with smaller pore sizes or in sea sand, matrices with pore sizes higher than 300 nm accelerated the breakdown of hexadecane to a greater extent (Calvo et al., 2009).

Remediation of a heavy metal-contaminated soil by rhamnolipid

Arsenic and heavy metal exposure is linked globally to negative health impacts, according to reports (Wang & Mulligan, 2004; Rodrigues et al., 2006). Natural processes like weathering and erosion of enrichments as well as human activities like mining and smelting operations may release these harmful elements into the environment. For polluted soils or solid wastes, several remediation strategies are proposed, including excavation and landfilling, thermal treatment, stabilization, and solidification (Wang & Mulligan, 2004; Wang & Mulligan, 2009). Due to the prohibitive costs or land space requirements, however, the adoption of these methods has been constrained (Wang & Mulligan, 2009). They can be combined with other substances, including salts like sodium chloride and/or additions like alcohol. By reducing interfacial tension and stimulating the formation of aqueous micelles, these chemicals would be most efficient in encouraging the mobilization of organic molecules with high lipid and relatively low water solubility. Ion exchange, precipitation-dissolution, and counter-ion binding are three potential processes for the extraction of heavy metals by surfactants. To limit the mobility of the pollutants, polymers or foams might also be added. Heavy metal-contaminated soil can be successfully treated with rhamnolipids (Wang & Mulligan, 2009). According to several studies, rhamnolipids work better than tap water and other synthetic surfactants in removing of metals, including cadmium, nickel, lead, and chromium. The stability constants of rhamnolipid-metal complexes are in the following order: Cu2+ > Pb2+ > Zn2+ > Fe3+. Metal mobilization from sediment and soil requires rhamnolipid adsorption (Mulligan et al., 1999; Ochoa-Loza, Artiola & Maier, 2001; Wang et al., 2008; Wang & Mulligan, 2009). The negatively charged rhamnolipids may compete with arsenic for adsorption sites, squelching it and mobilizing it as a result. Arsenic mobilization by rhamnolipids may be explained by an anion exchange between the arsenic anions and the rhamnolipids (Wang & Mulligan, 2009).

Application in cosmetics and personal care products

Rhamnolipids are utilized in a variety of formulations in health care products, including toothpaste, antacids, acne pads, anti-dandruff treatments, contact lens solutions, deodorants, and nail care items (Kanlayavattanakul & Lourith, 2010). Surfactants with high surface and emulsifying activity are necessary for these products (Vasileva-Tonkova et al., 2001), especially emulsifying activity, which is essential for these products’ textural consistency (Haba et al., 2003). Due to their skin compatibility and extremely minimal skin irritation, cosmetics containing rhamnolipids have been trademarked and utilized as anti-wrinkle and anti-aging products. As a result, one business launched these products in a variety of dosage forms (Aurora Advanced Beauty Labs, Inc., St. Petersburg, FL, USA) and announced a variety of rhamnolipid-containing cosmetic items that would be sold (Desanto, 2008). Although, in the skincare industry, sophorolipid and rhamnolipid biosurfactants have demonstrated the potential to offer a natural, sustainable, and skin-compatible alternative to synthetically compounds, there are three main barriers for market adoption of glycolipid technology and utilization in academic research and skincare applications include: (1) low rhamnolipid yield, (2) impure preparations and/or poorly characterized congeners, (3) lack of the safety/bioactivity assessment of sophorolipids and potential pathogenicity of some native glycolipid-producing microorganisms. Consequently, more rhamnolipids testing and experimental techniques/methodologies are recommended for increasing the acceptance of glycolipid biosurfactants for use in skincare applications (Adu et al., 2023).

Applications in pharmaceuticals

RLs have a variety of antibacterial characteristics (Itoh et al., 1971). In particular, they are proven to have an inhibition effect against a wide range of bacteria, including both Gram-negative and Gram-positive species, such as Bacillus subtilis. Bacillus cereus and Rhodococcus erythropolis. The cell envelope is the target of synthetic surfactants. Similarly, the biological membrane is intercalated into and destroyed by RLs’ permeabilizing effect in their suggested mechanism of action (Sotirova et al., 2008). Three different strains of P. aeruginosa cultivated on various types of vegetable oil waste have been the subject of extensive research by the Manresa group on the antibacterial effects of mixes of RL congeners (Benincasa, 2007; Abdel-Mawgoud, Lépine & Déziel, 2010). Surfactin, rhamnolipids, and mannosileritritol lipids show functional properties including; antimicrobial, biodegradability, demulsifying and emulsifying capacity (Begum, Saha & Mandal, 2023; De Oliveira Schmidt et al., 2023). Various RL combinations showed significant antimicrobial activity against several Gram-negative species, with Serratia marcescens, Enterobacter aerogenes, and Klebsiella pneumoniae being the most susceptible ones, and nearly all tested Gram-positive species, including Staphylococcus, Mycobacterium, and Bacillus. They also discovered strong inhibitory efficacy against a variety of fungal species, including the phytopathogens Botrytis cinerea and Rhizoctonia solanii, the filamentous fungus Chaetomium globosum, Aureobacidium pullulans, and Gliocladium virens, but no discernible impact on yeasts (Abalos et al., 2001). Investigations were made into the algicidal potential of the rhamnolipid biosurfactants (a combination of Rha-Rha-C10-C10 and Rha-C10C10) generated by Pseudomonas aeruginosa. According to the findings, Heterosigma akashiwo, a species of harmful algal bloom (HAB), could be affected by the biosurfactants’ algicidal properties. In addition, the rhamnolipids showed substantial lytic activity towards H. akashiwo at higher concentrations (4.0 mg/L), which significantly hindered the development of H. akashiwo in a medium containing them (0.4–3.0 mg/L). Rhamnolipids’ effects on the growth of Gymnodinium sp. and Prorocentrum dentatum, two more HAB species, were also investigated. The cells of P. dentatum were inhibited or lysed at higher concentrations (1.0–10.0 mg/L) compared to the significant algicidal action on H. akashiwo, while the cells of Gymnodinium sp., were not suppressed by the same therapy, demonstrating the ability of rhamnolipids to control HABs only (Wang, 2004). The extent of the alga’s ultrastructural damage was severe at high rhamnolipid concentrations and for long periods of contact, according to morphometric examination at the ultrastructural level using transmission electron micrographs. The plasma membrane experienced the initial reaction, which led to some disintegration. The absence of a membrane made it easier for rhamnolipid biosurfactants to enter the cells and allowed other organelles to be damaged. This led to the injury of chloroplast, vacuolization of mitochondria, deformation of the cristae, disruption of the nuclear membrane, and condensation of chromatin in the nucleus, suggesting that the lytic activity of rhamnolipids was primarily caused by their strong surface activity and their propensity to cohere on the surface afterward, the ultrastructure suffers irreparable damage and organelles lose their functionality, causing the cells to lyse (Wang & Mulligan, 2004).

Applications in agricultural and plant protection

RLs were found to have strong zoosporicidal activity against a variety of zoosporic phytopathogens, including species from the Pythium, Phytophthora, and Plasmopara genera, likely by zoospore lysis (Abdel-Mawgoud, Lépine & Déziel, 2010). P. aeruginosa strain PNA1’s biocontrol ability depended on the generation of RLs since rhlA mutant was substantially less effective at preventing Pythium sp.-caused plant disease (Abdel-Mawgoud, Lépine & Déziel, 2010). The FDA has authorized the use of a biofungicide made using rhamnolipids to stop plant pathogenic fungi in fruit, vegetable, and legume crops (Nitschke & Costa, 2007). Additionally, these formulations are thought to be low-toxic and nonmutagenic (Pinzon-Gamez, 2009). ZonixTM is another rhamnolipid biofungicide (Jeneil Biosurfactant Co, Saukville, WI, USA), used to protect horticulture crops from harmful fungi and to regulate them (United States Environmental Protection Agency).

Plant diseases and pests decrease agricultural yield and production quality (Savary et al., 2019). Despite that chemical techniques, such as pesticides and fungicides, have assisted in lowering the impact of these plant infections, their long-term usage is impractical due to their negative impacts on the biodiversity of helpful soil microorganisms and public health (Faloye & Alatise, 2017). Because of their lower toxicity, higher efficacy, and stability under extreme environmental conditions, biosurfactants have recently received increased attention as an alternative eco-friendly strategy for managing plant diseases. They can be made from inexpensive substrates and have a wide range of structural variations. Most significantly, they are environmentally friendly because they decompose (Abdel-Mawgoud, Lépine & Déziel, 2010). Rhamnolipids offer a wide range of antibacterial and antiviral activities. They were shown to be effective against Gram-negative bacteria, such as Xanthomonas campestris, which causes black rot in crucifers and bacterial spot disease in pepper and tomato, and Ralstonia solanacearum, which causes bacterial wilt in many Solanaceous plants (Kim et al., 2000). It has also been demonstrated that rhamnolipids have antifungal properties. Colletotrichum falcatum, which causes red rot in sugarcane, has its spore germination reduced by Pseudomonas aeruginosa’s extracted rhamnolipid (Borah et al., 2016). It has been shown that rhamnolipid plant treatments can both prevent Colletotrichum orbiculare anthracnose in cucurbits and protect pepper plants from blight disease (Vatsa et al., 2010). Rhamnolipids showed to exhibit antifungal activities against the molds Cercospora kikuchii, Fusarium verticillioides, Cladosporium cucumerinum, Colletotrichum orbiculare, Botrytis cinerea, Cylindrocarpon destructans, Magnaporthe grisea, and Phytophthora capsici in greenhouse experiments (Borah et al., 2016; Monnier et al., 2018). Rhamnolipids were found to have lytic action against Phytophthora sp., Pythium sp., and Plasmopara sp. zoospores in in vitro tests (Perneel et al., 2008).

Rhamnolipids’ capacity to promote membrane permeability, which results in cell rupture and lysis, may be a factor in their antibacterial activity. They interact with the surface lipids and proteins, removing them from the cell membrane and altering the osmolality and the structure of the cell wall. Important membrane functions, such as transportation and energy production, are affected by this alteration in protein conformations (Banat et al., 2010; Sotirova et al., 2008).

Rhamnolipids have been shown to have the potential to boost plant immunity to diseases that are both biotrophic and necrotrophic. To protect grapevine against Botrytis cinerea, rhamnolipids from Pseudomonas aeruginosa and Bacillus plantarii produce reactive oxygen species (ROS) and activate MAP kinase (Varnier et al., 2009). Rhamnolipid-exposed plants have stimulated the Arabidopsis defense marker genes PR1, PDF1.2, and PR4 (Sanchez et al., 2012). They can also activate defense genes in cells of rapeseed, wheat, and tobacco (Varnier et al., 2009; Monnier et al., 2018) if rhamnolipids enter inside the lipid bilayer of the plant membrane and are situated close to the lipid phosphate group of the phospholipid bilayers, or if they require a specialized receptor in the plasma membrane of the plant cell (Monnier et al., 2018).

Applications in the food industry

Due to their non-toxic properties, biodegradability, high surface and interfacial activity, high thermal and chemical stability, and sustainability through production from renewable resources, rhamnolipids have the potential to be used as emulsifiers and stabilizers in many applications of food processing, bioremediation, and food additives (Banat, Makkar & Cameotra, 2000). Because they can bind protein with a high affinity and improve the solid surface’s wettability, they are effective for cleaning ultrafiltration (UF) membranes of protein (Sinumvayo & Ishimwe, 2015).

In addition to having antifungal activity against Candida albicans, the biosurfactants (BSs) also have an antibacterial impact against several pathogenic bacteria, including Neisseria gonorrhoeae, Escherichia coli, Staphylococcus saprophyticus, Enterobacter aerogenes, and Klebsiella pneumonia (Giani, Zampolli & Gennaro, 2021). The quality of packed food is improved by rhamnolipid coatings in functional food packaging because of its antibacterial, antifungal, bacteriostatic, and physicochemical qualities.

The use of rhamnolipids enhances the quality of bakery products such as bread, hamburger buns, soft rolls, Chinese steam bread, croissants, argentine bread, schnittbotchen, cake, and sponge cake in terms of dough or batter stability, volume, shape, structure, dough texture, hardness, the width of the cut, and microbiological preservation (Magario, 2009). The qualities of buttercream, decorating cream, and non-dairy cream filling for croissants, Danish pastries, and other fresh or frozen fine confectionery products are also improved by using them. Rhamnolipids can be used in ice cream formulations to control consistency, delay staling, solubilize flavor oils, stabilize fats, and reduce spattering in addition to preventing separation, preventing the agglomeration of fat globules, stabilizing aerated systems, and modifying rheological properties (Giani, Zampolli & Gennaro, 2021).

When compared to dangerous synthetic preservatives, their antimicrobial capabilities can recommend rhamnolipid for extending shelf life either directly by preventing food contamination as a food preservative or indirectly by acting as a detergent to clean surfaces in touch with the food. They increased shelf life and reduced hemophilic spores in UHT soymilk when combined with niacin. The shelf life of salad was increased and the formation of mold was reduced by adding niacin and rhamnolipids. The combination of natamycin, nisin, and rhamnolipids in cottage cheese increases shelf life by preventing the formation of mold and bacteria, particularly gram-positive and spore-forming bacteria (Sinumvayo & Ishimwe, 2015).

It is simple to hydrolyze rhamnolipids to create rhamnose sugar (Pinzon-Gamez, 2009). It is employed in the production of various chemical compounds and as a premium flavoring agent on the market (Maier & Soberón-Chávez, 2000; Pinzon-Gamez, 2009). Currently, the production of rhamnose involves extracting quercitrin from oak bark, naringin from citrus peels, or rutin from oak bark or other plants. This technique uses caustic ingredients and produces a significant amount of toxic waste (Chayabutra, Wu & Ju, 2001).

Applications as anti-biofilm agents in industrial water systems

Biofilms are bacterial communities on the surfaces of man-made and natural structures. Biofilm formation in industrial water systems such as cooling towers results in biofouling and biocorrosion represents a major health concern and economic burden. Traditionally, they were treated with alternating doses of oxidizing and non-oxidizing biocides, chemical surfactants or combination of both. Biosurfactants were shown to be powerful anti-biofilm agents and can act as biocides and biodispersants. Long-chain rhamnolipids isolated from Burkholderia thailandensis inhibited biofilm formation between 50% and 90%, while a lipopeptide biosurfactant from Bacillus amyloliquefaciens inhibited up to 96% and 99%. Additionally, the efficacy of antibiotics can be increased up to 50% when combined with biosurfactants, Thus, co-formulation with biosurfactants represents an ecological, cost-effective, and renewable solution with diminished impact when water is released into the environment. Industrial water users can involve biosurfactants combined with other molecules, such as polymers and bio-based surfactants, to proffer novel and safe alternatives (Jimoh et al., 2023).