Biotechnological potential of microbial bio-surfactants, their significance, and diverse applications

Abstract Globally, there is a huge demand for chemically available surfactants in many industries, irrespective of their detrimental impact on the environment. Naturally occurring green sustainable substances have been proven to be the best alternative for reducing reliance on chemical surfactants and promoting long-lasting sustainable development. The most frequently utilized green active biosurfactants, which are made by bacteria, yeast, and fungi, are discussed in this review. These biosurfactants are commonly originated from contaminated sites, the marine ecosystem, and the natural environment, and it holds great potential for environmental sustainability. In this review, we described the importance of biosurfactants for the environment, including their biodegradability, low toxicity, environmental compatibility, and stability at a wide pH range. In this review, we have also described the various techniques that have been utilized to characterize and screen the generation of microbial biosurfactants. Also, we reviewed the potential of biosurfactants and its emerging applications in the foods, cosmetics, pharmaceuticals, and agricultural industries. In addition, we also discussed the ways to overcome problems with expensive costs such as low-cost substrate media formulation, gravitational techniques, and solvent-free foam fractionation for extraction that could be employed during biosurfactant production on a larger scale.


Introduction
Biosurfactants are compounds that play diverse and notable roles in different industries including soap and detergent industries , petroleum industries , and food and be v er a ge industries and are also involved in environmental bioremediation processes (Cameotra et al. 2010 ). Typically, surfactants have polar heads (hydrophilic) and non-polar tails (hydr ophobic), whic h ar e commonl y r eferr ed to as amphipathic molecules . T his property allows them to form micelles between fluids with different polarities. As an illustration, the inclusion of a surfactant causes an increase in the miscibility of oil and water, which lowers the surface tension between the two liquids (Roy 2017 ). The biosurfactants are surfactants that are released by microorganisms and hav e attr acted the inter est of se v er al r esearc hers due to their ecofriendliness and biodegradability (Shekhar et al. 2015 ). Chemicall y av ailable surfactants hav e negativ e envir onmental consequences and continue to be a pollutant that causes pollution since they are difficult to bioremediate . T he biosurfactants can play a vital role in augmenting the efficacy of bioremediation as they expand the surface area of substrates, form their microenvir onment, and stim ulate em ulsification by the r elease of certain molecules through a variety of processes, such as quorum sensing (Gharaei-Fathabad 2011 ). Because of their beneficial properties, such as increased foaming potential, greater selectivity, lo w er toxicity and biodegr adability, thermo-r esistance, pH and salinity, r ene w able and w aste-material origin, impr ov ed potency, and lac k of carcinogenicity and teratogenicity, biosurfactants have proven to be far superior to oil-based surfactants (Moutinho et al. 2021 ). Globall y, tonnes of oil y waste has incr eased the carbon footprint and caused serious en vironmental issues . T he oily waste is high in carbon, so using it as a base for the creation of products with added value can support the idea of carbon neutrality. Oils may be used dir ectl y as substr ates and can be catabolized by micr oorganisms to create biosurfactants . T he molecules of the twentyfirst century are biodegradable and less hazardous than synthetic surfactants (Gautam et al. 2023 ).
Biosurfactant-pr oducing micr oor ganisms can hav e high molecular weights (pr oteins, lipopol ysacc harides, lipopr oteins, and pol ysacc harides) and low molecular weights (glycolipids and lipopeptides). Biosurfactants of high molecular weight have greater efficacy at stabilizing oil-water emulsions, while those of lo w molecular w eight are good at lo w ering surface tension (Eras-Muñoz et al. 2022 ). The biological functions and uses of various biosurfactants are intimately correlated with the chemical changes in their molecular structures (Morita et al. 2015 ). The molecular weights of the biosurfactants produced by microorganisms r ange fr om low to high. These micr oor ganisms include Pseudomonas cepaci a CCT6659 (40.5 g L −1), Pseudomonas aeruginosa M408 (12.6 g L −1), Bacillus subtilis HSO121 (47.58 g L −1), Rhodococcus erythropolis ATCC 4277 (0.285 g L −1), Candida bombicola (61 g L −1), Candida tropicalis UCP0996 (7.36 g L −1) (Ambaye et al. 2021 ). Numerous studies on Pseudomonas sp , a kind of bacteria recognized for producing rhamnolipids, show that many w ell-kno wn biosurfactants are produced by bacteria. Because of their strong em ulsification ca pabilities, they ar e often r eferr ed to as bio em ulsifiers. Additionall y, r esearc h confirms that fungi and yeast are also involved in the production of biosurfactants (Sáenz-Marta et al. 2015 ).
Biosurfactants are comprised of hydrophilic domains i.e. amphoteric or non-ionic, positiv el y or negativ el y c har ged and hydrophobic domains which is made up of hydrocarbon chains, ther efor e, ar e amphipathic (Otzen 2017 ). These molecules, recognized for lo w ering the surface tension, produce micelles betw een the liquid phases of various polarities (Lombardo et al. 2015 ). The Most widely used surfactants are fatty acids , ethylene , ammonium salt, ethoxylate, pr opylene oxide copol ymers, and sorbitan ester (Satpute et al. 2018 ). The concentration at which these surfactant domains form a supramolecular structure (micelles) calledcritical micelle concentration (CMC) and has lo w er CMC values than synthetic surfactants, making them more potent at low concentrations (Campos et al. 2013 ). In general, biosurfactants could grow progressively in harsh conditions such as pH, salinity, and temper atur e in industrial bypr oducts and waste . T his capability would enable the manufacturing of biosurfactants at a reasonable cost while enabling the utilization of residual substrates to reduce environmental pollution (Carolin et al. 2021 ). Due to their ability to combat viruses, bacteria, and fungi, lactic acid bacteria-derived biosurfactants (LAB) have an advantage over conv entional micr obial surfactants. A lar ge number of LAB strains are being linked to the production of biosurfactants, an important chemical used in the treatment of several kinds of diseases. They are also useful as anti-adhesive coating agents on healthcare insertional elements due to their efficiency as anti-adhesive a gents ov er a wide range of bacteria, which lo w ers hospital infections without the usage of synthetic medications and chemicals (Thakur et al. 2023 ). Many researchers have been successful in isolating biosurfactants from a variety of en vironments , including the marine ecosystem, the natural environment, contaminated areas, and industrial wastes. Due to their unique functional characteristics , they ha v e a wide r ange of uses in industries including a gricultur e , biomedicine , metal, construction, textiles , pulp and paper, pharmaceuticals, and cosmetics (Moutinho et al. 2021 ).
Recently, the demand for biosurfactants has increased manifold because of their profitable properties in different industries, including food, pharma, and a gricultur e. Recombinant or m utant str ains with higher yields may be used as a str ategy to lo w er substrate costs and boost productivity levels to the point where commercial production of biosurfactants eventually becomes economically viable . T he use of biosurfactants as greener amphiphiles might soon be effective on a large scale (Sharma and Sharma 2021 ). This r e vie w article gives an insight into the technologies used in the isolation of biosurfactants and the microorganisms producing biosurfactants, as well as their significance and applications.

Biosurfactants production and their types
Biosurfactants ar e mainl y synthesized by micr oor ganisms-bacteria, yeast, and filamentous fungi, but they can also be synthesized by animals (e.g. bile salts, phospholipids) and plants (e.g. saponin). Micr obe-deriv ed biosurfactants are known to have a strong emulsifying ability and lo w er surface tension. Because biosurfactants' composition includes biomolecules like lipids , proteins , and car-bohydr ates, they ar e mor e complicated structur all y than synthetic surfactants (Nitschke and P astor e 2002 ). Fr om an ecological perspectiv e, biosurfactants ar e crucial in lo w ering environmental pollutants like carbon dioxide and greenhouse gas emissions. Micr obes r elease biosurfactants during the biodegr adation of hydr ocarbons, whic h pr ovide adv anta ges for pr eserving envir onmental sustainability. Ther eby r educing dependency on c hemical degr adation methods (Rahman and Gakpe 2008 ).

Microbial synthesis of biosurfactant
According to r eports, micr oor ganisms employ separ ate paths to cr eate the hydr ophobic and hydr ophilic parts of biosurfactants, whic h ar e then combined (T héatre et al. 2021 ). T he biosynthetic pathw ay used b y micr oor ganisms for gr owth depends on the carbon source. As an example, in the case of glycolipid biosynthesis with carbohydrates serving as the only carbon source, both the lipogenic and gl ycol ytic r outes utilize carbon flow to synthesize the lipid moiety and hydrophilic part, respectively (Sánchez 2022 ). Man y substr ates ar e involv ed in the synthesis of biosurfactants ( Fig. 1 ). Their production is influenced by changes in pH, stress, low nitrogen concentrations, and agitation rates, and can be induced by the presence of lipophilic substances . T he biosurfactants rhamnolipid and surfactin, which are produced by the bacteria Bacillus subtilis and Pseudomonas aeruginosa , have been thoroughly r esearc hed by r esearc hers (Sanc hes et al. 2021 ). According to se ver al inv estigations, Candida r eleases lipid-deriv ed biosurfactants made by a fungus called mannosylethitritol (MEL) (Kitamoto et al. 1993 ). Ther e ar e differ ent biosynthetic pr ocesses for the hydr ophilic and hydr ophobic domains of biosurfactants, whic h combine afterw ar d (Kuo and Gardner 2002 ). The metabolic pathway for the production of biosurfactants mostly depends on the carbon source, which may be obtained from lipids and carbohydrates ( Fig. 1 ). when the formation of glycolipids has utilized carbohydrates as the primary source of carbon. The energy source will then switch to the pathways for lipolysis and gluconeogenesis (Fontes et al. 2008 ). As an example, gl ycer ol is used as a medium in the production of rhamnolipid, a biosurfactant produced by Pseudomonas (Gogoi et al. 2016 ). Trichoderma reesei , a filamentous fungus, pr oduces hydr ophobins, a surface-activ e globular pr otein, through the biosynthesis of two genes called hfb1 and hfb2. (Das et al. 2008 ).
The production of rhamnolipids by P. aeruginosa is likely the most w ell-kno wn example of a bacterial glycolipid biosynthetic pathway that has been documented for both non-marine and marine strains (Tiso et al. 2017a ). Two distinct gl ycosyltr ansfer ase units-rhamnosyltr ansfer ase I and II-catal yze the pr oduction of mono-and di-rhamnolipids . T he bicistr onic oper on containing the genes rhlA and rhlB produces the rhamnosyltransferase I protein, although recent research has shown that both RhlA and RhlB proteins also have distinct functions (Wittgens et al. 2017 ). RhlB, a glycosyltr ansfer ase, catal yzes the condensation of dTDP-l-rhamnose and the 3-(3-hydroxy alkano yloxy) alkanoic acids (HAA) to generate mono-rhamnolipids. RhlA produces HAA fr om activ ated hydroxy fatty acids. It should be noted that HAA is already surfaceactive metabolites that are produced from cells as biosurfactants (Tiso et al. 2017a ). Rhamnosyltr ansfer ase II is encoded by the gene rhlC, which is located at a differ ent c hr omosomal location in P. aeruginosa than rhlAB. The synthesis of di-rhamnolipid from mono-rhamnolipid and dTDP-l-rhamnose is catalyzed by this protein (Tiso et al. 2017a ).
Surfactin is produced by a unique mechanism known as nonribosomal peptide synthase (NRPS), unlike the majority of cyclic Figur e 1. T he pathwa y of biosurfactant synthesis. A. Rhamnolipid biosynthesis in the genus Pseudomonas through the metabolic process of gluconeogenesis and fatty acid synthesis. dTDP (deoxythymidine diphospahtate) L rhamnose and 3-(3-hydroxy alkonoyloxy) alkanoateare the ultimate precursor for the synthesis of mono-rhamnolipid follo w ed b y di-rhamnolipidin case of bacteria express the gene RhlC. B. Surfactin biosynthesis occurs in the Bacillus sp. using sucrose as the main carbon source which results in the production of different amino acids-L-Valine , L-Leucine , L-Glutamate , and L-Aspartate, and Fatty acid. These components are assembled into surfactin in the presence of group of genes Sfp and srfABCD in Bacillus .
lipopeptides . T he four modules that together make up NRPS are SrfAA, SrfAB, SrfAC , and SrfAD, which form a linear arrangement of se v en modules. Eac h module is r esponsible for incor por ating one amino acid (Youssef et al. 2005 ). Each module comprises of thr ee catal ytic structur al domains: an aden ylation structur al domain (A), which selects and activates substrates; a small pe ptid yl carrier protein (PCP), which transfers aminoacyladenosine substrates as an enzyme-bound thioester; and a condensation structur al domain (C), whic h forms a peptide bond within acyl-S-PCP intermediates (Ongena and Jacques 2008 ). Four enzyme components , SrfA, SrfB , SrfC , and SrfD, make up the surfactin synthetase complex, in which surfactin synthase is made by multi-enzymatic thio-templates and is responsible for producing surfactin. The beginning stage of surfactin production is lar gel y contr olled by Srf D . The surfactin synthetase operon SrfA is an inducible operon that promotes competence development and sporulation. The sequence of the peptide synthetase modules is consistent with the sequence of the final peptide pr oduct, and eac h module has a variety of domains that add or modify a particular amino acid into the growing peptide chain (Kashif et al. 2022 ).

Types of biosurfactants
Biosurfactants are categorized according to their chemical composition and microbiological origin (Table 1 ). Mono, di, or polysaccharides , anions , or cations comprise the hydrophilic moiety of biosurfactants. In contrast, the hydrophobic moiety is made up of both saturated and unsaturated fatty acids (Abo Elsoud and Ahmed 2021 ). Glycolipids, the most prevalent form of biosurfactant, are composed of fatty acids and carbohydrates connected by either an ester or an ether group (Mnif et al. 2018 ).

Glycolipids
One of the most common types of biosurfactant is glycolipid. They originate from lipids and are composed of lengthy chains of hydroxyaliphatic or aliphatic acid-containing sugars. Well-known subtypes of glycolipids include rhamnolipids , sophorolipids , and trehalolipids (Roy 2017 ). Pseudomonas aeruginosa is primarily responsible for producing rhamnolipids, which have a wide range of uses in bioremediation (Costa et al. 2010 ). It consists of one or two rhamnose sugar groups resulting in the formation of monoor di-rhamnolipid molecules (Raza et al. 2010 ). Another subclass of glycolipids that function as biosurfactants on extracellular surfaces is called sophorolipids . T he two carbohydrate sophorose units that makeup sophor olipids ar e joined to long-chain fatty acids via a glycosidic bond. It has been discovered to be quite helpful in oil bior emediation pr ocedur es (Elshafie et al. 2015 ). Although sophorolipids can lo w er interfacial and surface tension, they are ineffectiv e em ulsifiers (SajadiBami et al. 2022 ). The most pr e v alent sophor olipids ar e those made by yeast such Torulopsis bombicola , T . apicola , and T . petrophilum (Shekhar et al. 2015 ). Trehalose units (a disacc haride unit), whic h ar e discov er ed to be connected to m ycolic acids, mak e up the glycolipids known as trehalolipids. T he size , mycolic acid structure , level of unsaturation, and number of carbon atoms of trehalolipids might vary depending on the organism that produced them (Banat et al. 2010 ). There are se v er al species, including Nocardia , Mycobacterium , and Corynebacterium , that are involved in the production of trehalolipids (Kuyukina and Ivshina 2019 ).

Lipopeptides
Lipopeptides are surface-active biosurfactants with antimicrobial properties ( Surfactinis is mostly produced by the bacteria Bacillus subtilis and has a lengthy carbon chain with less hazardous amino acids (Fei et al. 2020 ). Due to its antibacterial, antiviral, and antimicrobial qualities. It has been emplo y ed in a variety of sectors, such as the cosmetic and oil bior emediation pr ocesses(Dr ak ontis and Amin 2020 ). Arthrofactin is also a type of lipopeptide biosurfactant produced b y Actinomyces , Arthrobacter , and Streptomyces (Sari et al. 2020 ). It is effective at pr e v enting the gr o wth of biofilms b y reducing the surface tension of water from 72 to 24 mNm-1 (Lange et al. 2012 ).

Polymeric biosurfactants
Polymeric biosurfactants such as extracellular polymeric substances (e.g. emulsan) can have deleterious and pr oductiv e effects during microbial biofilm and floc formation (Sajadi Bami et al. 2022 ). They are known to alter surface characteristics during biofouling, including hydrophobicity, color, roughness, and frictional resistance. In the process of biocorrosion of metals, the EPSs can bind with the metal (Vimalnath and Subramanian 2018 ).

Fatty acids and phospholipids biosurfactants
When many bacterial and yeast species are grown on n-alkanes or hydr ocarbon substr ates, they pr oduce huge amounts of fatty acids and phospholipids (Sajadi Bami et al. 2022 ). Se v er al lipids ar e pr oduced by them, suc h as lipopeptides, gl ycolipids, and phospholipids (Hausmann and Syldatk 2014 ). Corynebacterium Lepus is responsible for the production of the most popular phospholipid biosurfactants (Busi and Rajkumari 2017 ).

Applications of biosurfactants
Because they are effective wetting and foaming agents, solubilizers , dispersants , and emulsifiers , as well as detergents, biosurfactants' qualities may be used widely on the commercial scale (Banat et al. 2010 ). The market's availability of chemicals-based products has decreased as the demand for bio-based products has increased (Olasanmi and T hring 2018 ). T his has been determined by the expansion of bio-based chemical patent rights (Tiso et al. 2017 ).

Industrial uses of biosurfactants
Biosurfactants are used in se v er al industrial pr ocesses, including biorefinery and cooling. Ice slurry is a homogeneous substance made up of water and tiny pieces of ice. Processes including cooling, air conditioning, and cold stor a ge systems are all necessary for the de v elopment of ice slurries. By stabilizing them in an icewater slurry, di-acetylated MELs inhibit the build-up of tiny ice particles. MELs are added to biodiesel to impr ov e its flow characteristics, whic h impr ov es its performance at low temper atur es (Madihalli et al. 2016 ). Pr ocesses in bior efineries utilize biosurfactants because they potentially speed up the biodegradation of complicated biomass . T heir presence ma y also hasten the pace at which cellulases break down lignocellulose as a result of their strong binding (Liu et al. 2017 ).
In microbial enhanced oil r ecov ery (MEOR), secondary metabolites of micr obial originsuc h as acids , enzymes , solvents , gases , biopolymers, and the most assuring biosurfactants are used to substitute synthetic surfactants in r ecov ering secondary oil from sedimentary r oc k . Ex-situ and in-situ biosurfactant production are effective approaches to MEOR. In-situ biosurfactant production typically begins with the injection of micr oor ganisms whic h pr oduce biosurfactants, follo w ed b y the infu-sion of nutrients into the reservoir. Industrial bioreactors can also be used to produce ex-situ biosurfactants for subsequent infusion of these substances into the reservoir using CO 2 . Micr oor ganisms pr oduce em ulsifiers and surfactants that r educe surface tension and cause the tr a pped oil to escape. By improving the flushing efficiency of the infiltrated fluid and CO 2 , biosurfactants change the water-holding capacity of the CO 2 that's been injected and the behavior of CO 2 -brine-r oc k at the interface, whic h facilitates the r ecov ery of the oil (Selva Filho et al. 2023 ). In the course of MEOR, nutrients are added to the oil reservoir together with micr oor ganisms capable of producing biosurfactants to promote microbial de v elopment (Sun et al. 2018 ). Biosurfactanat are significant at mobilizing immobile hydrocarbons as it can decrease the surface tension across the oil and rock, lo w ering the capillary forces to transport of oil throughout rock pores (Rawat et al. 2020 ). Rhamnolipid extracted from P. aeruginosa helped to recover mediumweight oils at a rate of 50.45%, an improvement of 11.91% made possible by the presence of the micr obe. Her e, this micr obial a pplication has shown higher r ecov ery r ate than than that attained with the tested synthetic surfactants (Câmara et al. 2019 ). Sometimes, the use of biosurfactants in MEOR is a contentious matter because the biosurfactants r equir ed to r emov e oil r esidues that ar e tr a pped in the por ous r oc ks, ar e undue and it may not be cost effective. Yet, it is considered counterintuitive to use substances for oil r ecov ery whose primary benefit is to take the place of synthetic chemicals in the petr oc hemical industry (De Almeida et al. 2016 ).
Ther e ar e numer ous studies conducted on the use of biosurfactant for the cleanup of oil-contaminated soil. Surfactin is one of the surfactants emplo y ed in biotechnological methods of decontamination, with 85% r emov al by utilizing Bacillus licheniformis derived biosurfactant and 88% with biosurfactant derived from Bacillus subtilis (Alv ar ez et al. 2015 , Khademolhosseini et al. 2019 ). In bioremediation, biosurfactants must undergo biodegradation in soils, which makes them a good ecological replacement for synthetic surfactants. Biosurfactants are adept at technology and can be disc har ged in-situ, wher e they may carry out their effects with less subsequent handling effort than their synthetic surfactants (Silva et al. 2020 ).

Medical applications
Lipopeptides are biosurfactants known for their stability over a wide pH range and heating them at high temperature does not result in the loss of their surface-active property. Recent studies show that they also possess antimicrobial properties. For example, rhamnolipid which are produced by the genus Pseudomonas are known to have po w erful antimicrobial properties as monorhamnolipids have a bacteriostatic effect, di-rhamnolipids exhibit a bactericidal effect on P. aeruginosa (Diaz et al. 2016 ). Ther e ar e some biosurfactants which are having a synergic effect on antibiotics by augmenting their uptake efficiency into the cell (Hage-Hulsmann et al. 2018 ).
Due to their antibacterial, anti-adhesive, and enzymeinhibiting qualities, biosurfactants have been emplo y ed in the medical and pharmaceutical sectors for a variety of ther a peutic purposes (Markande et al. 2021 ). Gene-releasing biosurfactants, pharmaceuticals, and also antiviral properties, and anticancer activities are some of the key areas of study involving these biomolecules in the fields of pharmacy and medicine (Dr ak ontis and Amin 2020 ). Recent r esearc h has focused on COVID-19 management tactics and investigated the use of biosurfactants as cleansers , disinfectants , en vir onmentall y friendl y sanitizers, antivir al a gents , and anti-inflammatories . Sophorolipids ma y be used as ther a peutic a gents to combat the SARS-CoV-2 virus, according to a new study (Daverey et al. 2021 ). SARS-CoV-2 is a positive sense single stranded RNA envelope virus which can be pleomorphic or spherical. The four main structural proteins (NC-nucleoca psid pr otein, M-membr ane pr otein, E-enca psulation protein, and S-spike protein) as well as five to eight nonstructural auxiliary proteins are all encoded by the genome . T he ACE2 receptor location on the epithelial cells that line the r espir atory tract of the host is where the spike glycoprotein attaches to the viral particle (Baglivo et al. 2020 ). The anionic c har acter of sophorolipids disturbs the viral en velope , causing the structural elements to disintegrate and consequently interfering with the connections between virus protein surfaces and cell-host receptor sites (Kashif et al. 2022 ). Ad ditionally, the y are found to be suitable for usage in future innovations like nanobiotechnology and effective medication delivery systems . Also, they ha v e dr awn consider able inter est fr om the scientific comm unity due to their str ong ther a peutic qualities, making them useful in the tr eatment of SARS-CoV infection as well as for anti-vir al, imm unomodulatory, anti-cancer, wound healing, and other conditions (Kumari et al. 2023 ).
Biosurfactants are often emplo y ed in antimicrobial and antivir al r esearc h and also in drug deliv ery. The use of biosurfactants as potential substitutes to manage biofilms has been thoroughly inv estigated in r ecent years. Biosurfactants c hange the surface c har acteristics of bacterial cells that are connected with their membr anes and pr e v ent them fr om adhering to other substrates. Additionally, it has been shown that biosurfactants generated by Gr am-negativ e bacteria pr e v ent micr oor ganisms fr om de v eloping biofilms and communicating with one another (Kashif et al. 2022 ). T he biosurfactants ha v e antibacterial action a gainst a v ariety of pathogenic pathogens, including Candida albicans, E. coli, and others. According to a new study conducted by Gupta et al., a glycolipid biosurfactant made from B. licheniformis SV1 sho w ed fast wound-healing action (Gupta et al. 2017 ). Similarly, another study found that the administration of lipopeptide biosurfactants from Acinetobacter junii B6 impr ov ed the r ate of wound healing in rats (Ohadi et al. 2017 ). The antioxidant c har acteristics of the biosurfactants, which lessen o xidati ve stress by reducing the production of r eactiv e oxygen species and incr easing the activities of fr ee r adical scav engers, may be the cause of the r a pid wound healing when provided with lipopeptides. Because the structure includes both a fatty acid chain and a peptide group, lipopeptide biosurfactants also have anticancer action. Due to their potential involvement in apoptosis, cell proliferation, signal suppression, and cell cycle inhibition, biosurfactants hav e latel y been examined for their anticancer c har acteristics (Kashif et al. 2022 ).

Cosmetic applications of bio-surfactants
Biosurfactants have vital physiochemical properties for preserving health y skin. F or instance, their molecules' fatty acid ends help moisturize the skin's rough and dehydrated surfaces . T he accessible fatty acids may also function as anti-oxidants, which would pr e v ent the cr eation of fr ee r adicals br ought on by UV r adiation (Thakur et al. 2021 ). Skin infection pathogens including Staph ylococcus aureus , P. aeruginosa, Candida acnes, and Streptococcus pyogenes have all been demonstrated to be effectively suppressed by a variety of biosurfactants. Because of this, biosurfactants are being suggested as a potential substitute for conventional antibiotics, e v en though their bactericidal activity can often be weak (Loeto et al. 2021 ). Their usage in cosmetic industries is major because of their wetting, foaming solubilizing, dispersing, and emulsification properties (Varvaresou and Iakovou 2015 ). The biosurfactants ar e highl y compatible with the skin and also don't cause any irritation. The antimicrobial properties of biosurfactants pr ov ed to be a boon for cosmetic pr oducts (Lourith and Kanla ya vattanakul 2009 ). It has been discovered that the carbohydrates , lipids , and proteins included in biosurfactants are similar to the membrane found on skin cells (proteins and phospholipids). Due to their unique structural property, biosurfactants can cross the skin cell membr ane, whic h can activ ate potential benefits on hair repair, skin protection processes, and the regulation of protein skin barrier functions . T he mo vement of substances across the skin cell membrane is controlled by lipophilicity and interfacial properties (Ferreira et al. 2017 ).
Lar ge cosmetic cor por ations often pr ovide 10 000 distinct cosmetic items, and 25-30% of these goods ar e r eform ulated annually. The use of novel active components for consumers or the industry is a factor in around 10% of these reformulations . T hese businesses add up to 80 new components to their product line per year  ). In the aforementioned context, using biosurfactants is one way to satisfy the need for novel components. RelipidiumTM (a body and face moisturizer made by BASF in Monheim, Germany), SopholianceTMS (a deodorant, face cleanser, and sho w er gel made b y Gi vaudan Acti v e Beauty in P aris, France) and Kanebo skincare, (a moisturizer , cleanser , and UV filter made by Kanebo Cosmetics in Tokyo, Japan) are a few products whic h incor por ate these biomolecules (Adu et al. 2020 ). Additionall y, Evonik, a German c hemical compan y is now set tec hnologies for the production of rhamnolipid as foam promoters in cosmetic pr oducts, assur ance the a pplicability of biosurfactant as one of the safety and active ingredients in formulation of cosmetic products. In 2010, Evonik was able to de v elop biotec hnological tec hniques for producing microbial biosurfactants on a commercial le v el  ). In the current circumstance, using biosurfactants is one way to satisfy the need for novel components as the y re present minimal dangers to humans and the environment due to their biodegr adable, r ene wable, or non-toxic natur e, whic h is in the interests of the developing consumer segment and, as a result, the cosmetic business . In vestment in the practical study of such biomolecules has a good possibility of producing reformulations and the creation of novel, safer cosmetics (Olasanmi and Thring 2018 ). Glycolipid and lipopeptide biosurfactants more br oadl y hav e a low le v el of toxicity, and antimicr obial, and dermatological moisturizing properties that make them a great deal better than chemical surfactants in recent demand for cosmetic and personal skincare products (Adu et al. 2020 ).
Kao Co. Ltd. in Ja pan is a pr ominent pr oducer of sophoolipid substances for use as a humidifying agent in different commercial goods such as hair moisturizers, skin moisturizers, and lipstic ks. Sophor olipids can encour a ge hair r egr owth and pr eserv e the skin (Adu et al. 2023 ). These biosufactant may also reduce fat deposits in the skin by boosting adipocytes' production of the hormone leptin. According to various research, rhamnolipids are also thought to be biocompatible and a pr ospectiv e component for usage in medical formulations of cosmetics as well as personal dermatological products (Karnwal et al. 2023 ). Human keratinocyte cells, which are crucial for skincare applications, were studied by Adu et al. to compare the effects of manufactured surfactants (sodium lauryl ether sulphate) and natur all y pr oduced glycolipid biosurfactants (rhamnolipids and sophorolipids). The results show that the acidic nature of mono-rhamnolipids and sophor olipids v ery modestl y affects cell survival and inflammatory cytokine production, but the impact of various glycolipids on cells varies depending on their chemical makeup. Di-rhamnolipids have been shown to effectively minimize inflammation and increase the anti-inflammatory action of cytokines at noninhibitory concentrations, making them a potential replacement for chemical surfactants in skin care products and helpful for dermatological conditions like psoriasis (Adu et al. 2023 ). According to Etemadzadeh et al. ( 2023 ), salt-tolerant Bacillus halotolerans produces a biosurfactant that shows several therapeutically r ele v ant pr operties and may be used as a r aw ingr edient in the manufacture of food, pharmaceutical, and cosmetic products. In vitro studies on the derived lipopeptide sho w ed that 90.38% effectiveness at 0.8 mg/mL with antibacterial and antioxidant properties . Besides , it has anticancer potential through induction of apoptosis in MCF-7 cells while possing no negative effect on unharmed HEK-293 cells (Etemadzadeh et al.2023).

Food industries
Biosurfactants play a very important role in food industries because of their non-toxic nature and easy biodegradability. To feed the growing population, it is necessary to increase the pr oductivity of a gricultur e . T hey also contribute further to the pr oduction of food, whic h includes (i) soil impr ov ements, (ii) stimulation of effective foliar fertilizers uptake, (iii) protection from phytopathogens, (iv) amelioration microbe-plant interactions (Sac hde v and Cameotr a 2013 , Liu et al. 2016 ). T hey ha ve se v er al useful applications in the manufacturing of food and also possess antibacterial and anti-biofilm properties that are utilized for sanitization and to pr e v ent food from spoiling (Kiran et al. 2017 ). Silva et al. used biosurfactants to substitute 50%-100% of the plant fat in cupcakes as part of a bakery-related application. Thr ough the r emov al of trans-fatty acids, the nutritional content of the cupcake was somewhat improved by the substitution of plant fat with a biosurfactant (Silva et al. 2020 ). The main reasons why food is wasted each year throughout the world are the deterioration in food quality, microbiological contamination spoilage, and the short shelf life of particular goods. The usage of biodegradable packaging and antimicrobial ad diti ves are only a couple of the many strategies being investigated right now to deal with these problems (Cofelice et al. 2019 ). Kourmentza et al. r ecentl y r e ported the antibacterial potential of the lipope ptide BS against foodborne pathogens such as Bacillus sp . Additionall y, they r eported antibacterial activity against the filamentous fungus Byssoc hlam ys fulv a, Candida krusei, and Paecilomyces variotti (Kourmentza et al. 2021 ). The yeasts Starmerella bombicola, Meyerozyma guilliermondii Candida sphaerica , and Sacc harom yces cerevisiae are among the microorganisms that have recently been reported for the production of biosurfactants . T hese organisms have the potential to produce substances with emulsifying properties and surfactant activities along with antibacterial and antioxidant qualities (Ribeiro et al. 2020 ). Regardless of the wide range of food a pplication possibilities, v arious studies ar e necessary to de v elop a pr actical a pplication that can execute functions in complicated food matrices under diverse processing circumstances. It's crucial to create methods that use these biomolecules at the most modest feasible concentration for optimum performance for an affordable application (Augusto et al. 2020 ).

Bioremediation
Biosurfactants ar e ca pable of withstanding extr emes in temper ature, salinity, and envir onmental abr asion while maintaining their stability. Petroleum and heavy metals are detoxified from the contaminated environment utilising biosurfactant-based remedia-tion a ppr oac hes and micr oor ganisms that pr oduce biosurfactants (SajadiBami et al. 2022 ). Also aids in the dissolution of hydrophobic pollutants in water. T hey ha ve been shown to be the best substitute for c hemicall y manufactur ed surfactants used in bioremediation because of their inherent low toxicity and great biodegradability (Mao et al. 2015 ). The em ulsification pr operty and higher solubility of biosurfactants promote cellular utilizations of contaminants (Shah et al. 2016 ). Organic and metallic pollutants are unavailable to microorganisms for breakdown, which is one of the reasons why they persist in soil for such a long period without being eliminated. Additionally, the pollutants' interactions with the environment and the microbe may be insufficient, which prevents the micr oor ganism fr om carrying out the r equir ed catabolic pr ocesses . T her efor e, biosurfactants pr oduced by bacteria and fungi play a crucial role in solubilizing hydrophobic contaminants, allowing for their direct elimination (Abbot et al. 2022 ). The biosurfactants produced by Stenotrophomonas sp. S1VKR-26 can be utilized to bioremediate w astew ater contaminated with petrolatum (Patel and Patel 2020 ). The lipopeptide-type biosurfactant Bacillus cereus UCP 1615 can clean up oil spills (Durval et al. 2020 ). The hydrophilic compound's solubility is increased by the biosurfactant that was isolated from Rhodococcus erythropolis HX-2, which also speeds up the biodegradation of petroleum (Hu et al. 2020 ). The release of biosurfactant in the soil helps to enhance the biodegradation process in bacteria, as schematically represented in Fig. 2 .

Significance of biosurfactants
Micr obes ar e essential in the pr oduction of biosurfactants. Due to their microbial origin, they offer se v er al beneficial c har acteristics, including low toxicity, high stability, eco-friendliness, simple biodegradability, the ability to function at severe temperatures, and the capacity to withstand a wide range of pH.

En vironmental compa tibility
Environmental pollution is a major concern no w adays because of the increasing population. To control pollution, we have to spread awareness among people that will contribute to w ar ds cleaning up environmental pollution. About two decades ago, it was found that ther e ar e a pplications of biosurfactants that can be used for environmental sustainability. Also play a very crucial role in the r emov al of major greenhouse gas i.e. carbon dioxide from the atmosphere (Rahman and Gakpe 2008 ). Chemicall y pr oduced surfactants found to deplete non-r ene wable petr oc hemical r esources (Henkel et al. 2012 ). Accum ulation of their counter parts puts the environment in danger because they are non-biodegradable (Rahman and Gakpe 2008 ). Hydrophobic contaminants produced by the oil and gas sector can collect in soil. Soil contamination can be caused by a variety of things, including storage tank leaks, spills, and pipeline leaks from accidents involving oil exploration, refinement, and shipment. In addition to being poisonous, these contaminants are also obstinate , intractable , and hazardous (da Silva Faccioli et al. 2022 ). Surfactants enable contaminants to desorb from soil particles and favor their mineralization and microbial breakdown by lowering their surface and interfacial tensions. Another method of contamination r emov al is phytoremediation, whic h involv es plants absorbing contaminants with the aid of biosurfactants (Fenibo et al. 2019 ). The P. cepacia CCT6659 biosurfactant sho w ed potential for use in the biological remediation of soils. In trials with soil polluted with hydrophobic organic matter, an indigenous consortium and biosurfactant treatment resulted in the breakdown of 95% of the contaminants within 35 to 60 days (Silva et al. 2014a ). Additionally, biosurfactants are frequently used to clean the soil, increase the amount of nutrients in the soil, and function as biocides, focusing on bacteria. Pesticides are made mor e bioav ailable by biosurfactants, whic h speeds up the pr ocess by which these sediment and soil contaminants degrade (Rawat et al. 2020 ). They are also involved in the remediation of heavy metals . Hea vy metal r emov al r ates wer e ac hie v ed with the use of biosurfactants (41% for Ni, 30% for Cr, 29% for Pb, and 20% for Zn), and r emov al r ates of F e , Zn, and Pb from soil using an anionic biosurfactant made by C. sphaerica UCP0995 in various combinations with NaOH and HCl were 95, 90, and 79%, respectively , Liduino et al. 2018. According to many reports, using biosurfactants in these formulations is a sustainable substitute for synthetic surfactants. P. aeruginosa rhamnolipids can be utilized to stabilize water-in-diesel emulsions to be utilized as fuel. This mixtur e is r equir ed to lessen viscosity during the tr ansportation of diesel, the primary energy source, as well as to lessen emissions of fine particulates and gases containing hydrocarbons (Fenibo et al. 2019 ).

Low toxicity
Synthetic surfactants after use are released into w astew ater str eams whic h imposes a thr eat to the ecosystem (Iv ank ovi ć and Hreno vi ć 2010 ). T he damage caused by synthetic surfactants to the water body depends on their concentrations. As increased concentration leads to the growth of algae by which other micr oor ganisms' cell membranes become more permeable and result in its disintegration (Yuan et al. 2014 ). Glycolipid and lipopeptide biosurfactants br oadl y hav e low toxicity, antimicr obial, and skin surface moisturizing properties that portrayed as suitable substance in replace of chemical surfactants in cosmetics and skincar e pr oducts (Adu et al. 2020 ). The toxicity imposed by surfactants depends on their hydrophobicity. Due to the increase in toxicity le v els, ther e is a high c hance that it will be consumed by animals and disturb the animal food chain. Aquatic animals such as fishes absorb these surfactants through their body surface and gills and then pass it to the blood circulation. When consumed by humans through food, it will lead to damage to enzyme activity (Iv ank ovi ć and Hr enovi ć 2010 ). Anal yzing the usa ge of surfactin pr oduced fr om Bacillus subtilis HSO121 indicated that this compound is a non-toxic, non-irritating substance, making it a safer one to be emplo y ed in detergent formulations (Fei et al. 2020 ). A comparison of Marlon A-350, a synthetic surfactant, and a biosurfactant produced by Pseudomonas aeruginosa r e v ealed that the biosurfactant was non-toxic while the synthetic surfactant was extr emel y harmful in all experiments (Muthusamy et al. 2008 ).
Santos et al. discov er ed that biosurfactant samples obtained at 0.02 and 0.06% did not significantl y incr ease the fatal rates for Artemia salina , whereas 0.08% resulted in the death of 100% of all the larvae (Santos et al. 2017 ). In a study by Santos et al. biosurfactants produced by Streptomyces sp . DPUA1559 in concentrations of 50, 100, and 150 mg/mL with CMC (10 mg/mL) w as sho wn to have no detectable fatal rates (Santos 2013 ). Because of this reason, biosurfactant discov ery pr ov ed to be a boon against synthetic surfactants.

Biodegradability
Biosurfactants derived from microbes can easily undergo the process of bioremediation or biosorption in comparison to syntheticall y av ailable surfactants (Desai and Banat 1997 ). Marine micr oor ganisms pr oducing biosurfactants ar e best for biosor ption of solvent polycyclic sweet-smelling hydrocarbons and contamination caused by phenanthrene over aquatic surfaces (Gharaei-Fathabad 2011 ). Microbial surfactants are susceptible to biodegradation because of their natural origin and do not accumulate in soil and water. The enzymatic activities of certain micr oor ganisms break down these surface-active substances by first cleaving and then inactivating the surfactant monomers. Surfactant monomers are known to be broken down by a number of enzymes (Kashif et al. 2022 ). The pol ysacc haride bac kbone of em ulsions , for instance , is broken by the enzyme emulsan polymerase, rendering the molecule inactive (Santos et al. 2016 ). According to r esearc h that looked at the biodegradability of sophorolipids made by a non-pathogenic strain of Candida bombicola, biosurfactants degr aded instantl y in comparison to synthetic surfactants, which continued to function even after eight days (Ahn et al. 2016 ). Rhamnolipids wer e discov er ed to disintegr ate in both aerobic and anaerobic en vironments; whereas , the synthetic surfactant (Triton X-100) only decayed partially in aerobic environments and failed to degrade anaerobically (Kashif et al. 2022 ). There is a study conducted by Moldes et al. for bioremediation of soil contaminated with octane . T he biosurfactants produced by Lactobacillus pentosus results in a reduction of octane concentration in soil nearly to 60% after 15 days of treatment. The biodegradation rate of octane increased to 76% after 30 days of treatment. While the r emov al r ate of octane is thr ee times slo w er in the absence of biosurfactants (Moldes et al. 2011 ).

Temper a ture and pH tolerance
Biosurfactants produced by microbes that live in extreme conditions can tolerate a wide temper atur e and pH range. Surface tension can be r eadil y r educed by biosurfactants at higher temperatures, but it is more difficult to do so in low-temperature conditions . To o v ercome the temper atur e barrier and reduce surface tension, it is, ther efor e, r easonable to deduce that micr oor ganisms from cold environments will have a longer, less-branched hydr ophobic c hain (Antonioli Júnior et al. 2022 ). Depending on the envir onment's temper atur e, pr essur e, pH, structur e, and le v el of solution stability, lipopeptides' emulsification capabilities might vary (Kumar and Ngueagni 2021 ). For instance, pH can affect the emulsifying action of surfactin formed by B. subtilis . It produces an emulsion that remains stable with kerosene at pH le v els ov er 7, ho w e v er, if the pH falls below 3, the emulsion is not formed (Long et al. 2017 ). The isolated Planococcus sp. XW-1 sho w ed exceptional ability in the production of surfactants at low temperatures and the degradation of petr oleum. Intr oducing Planococcus sp. XW-1 at 4 • C caused the degradation of 54% of crude oil. These results indicate that Planococcus sp. XW-1 is a good option for in-situ biore-mediation of marine ecosystems contaminated with petroleum in the north Yellow Sea during the winter (Guo et al. 2022 ). A study conducted on lic hen ysin pr oduced by Bacillus licheniformis found it to be resistant up to a temperature of 50 • C and having a pH range of 4.5 to 9 and can tolerate NaCl and Ca concentrations up to 25 to 50 gL −1 (Purwasena et al. 2019 ). The biosurfactant produced by Bacillus subtilis strain JA-1 is found to have surface activity and emulsification capability that are stable even at pH le v els of 7-8. Pseudomonas aeruginosa RS29 de v eloped a biosurfactant that demonstrated pH, saline and temperature stability. Even under these harsh circumstances, it was said to have significant foaming and emulsifying capabilities (Rufino et al. 2008 ). Since industrial applications require a wide pH and temperature range for their pr ocedur es, it becomes mor e important to focus on nov el microbes that will produce under extreme conditions (Das and Mukherjee 2007 ).

Techniques used for screening of biosurfactants
Biosurfactants can be isolated from different locations such as oil fields, petroleum and hydrocarbon-contaminated sites, garbage soil, thermophilic and halophilic en vironments , marine en vironments, and many more other sites yet to be explored. As for example, Pseudomonas aeruginosa SP4 has been isolated from petroleumcontaminated soil (P ornsunthornta wee et al. 2008 ), Pseudomonas and Bacillus sp found to be isolated from soil contaminated with domestic w astew ater (Femi-Ola et al. 2015 ), V ibrio sp . LQ2 was isolated from cold-seep sediment (Zhou et al. 2021 ) and Azotobacter chroococcum was isolated from the marine environment (T ha vasi et al. 2009 ).
Screening of biosurfactants involves many methods (Table 2 ). Morais et al. performed the drop collapse method to observe drop collapse activity of crude oil shown by culture supernatant using glass slide (Morais et al. 2017 ). The oil displacement technique reported by Satpute involved adding 2 mL of crude oil and 50 mL of distilled water to a Petri dish in such a way that the oil is distributed e v enl y acr oss the w ater's surface. Afterw ar ds, a culture supernatant of 500 μL was added and the presence of biosurfactant shows clear zones on the oil surface (Satpute et al. 2018 ). Lipase assay was performed by Kumar and his colleagues with modifications ,10 μL of o v ernight cultur e br oth was added to tributyrin agar medium plates, which were then incubated at 37 • C for 48 hours. Around colonies producing biosurfactants, the zone of lysis was seen (Kumar et al. 2017 ). The penetration method can be used as a primary screening method for biosurfactants, 96 well ELISA micr oplates wer e taken. A hydr ophobic paste made with oil-200 μL and silica gel was poured into each well, the activity of biosrurfactant was observed by the addition of crude oil (10 μL), of culture supernatant (90 μL), and 10 μL of safranin solution (Kumar et al. 2017 ). The hemolytic activity method pr ov ed to be a clear indicative test for biosurfactant production by many authors (Tabatabaee et al. 2005 ). This pr ocedur e involv ed incubating cultur e br oth for an ov ernight period, inoculating 10 L of cultur e ov er 5% sheep blood agar, and then incubating the plates at 37 • C for 48 h. A lysis zone was seen surrounding the colonies (Roy 2017 ). The presence of biosurfactants can be measured by emulsification index (EI%) and emulsification assay. To calculate the emulsification index (EI%), a 48-h-grown culture was centrifuged at 10 000 rpm for 10 min to collect supernatant. The equal amount of culture supernatant and crude oil are mixed and vortexed for 15 minutes at r oom temper atur e, allo w ed to stand for 24 h at dark chamber Table 2. Methods for screening of biosurfactant and its significance.

References
Dr op colla pse method Dr op colla pse activity was observ ed by adding a dr op of crude oil and culture supernatant onto the glass slide A straight forw ar d and practical approach that provides a delicate yet quick manner of producing biosurfactant and evaluation of the presence of biosurfactants can be done on a qualitative and quantitativ e le v el0 h. The zone of lysis was observed around the colonies Used as a primarily screen test for biosurfactant production. Ho w ever, it is not a very accurate approach for biosurfactant production test in bacteria since many good biosurfactant producersare left out due to negativ e haemol ytic zone on blood a gar and also non biosurfactant producers also sho w ed hemolytic activity.
(T ha vasi and J ay alakshmi 2003 ) (Roy 2017 ) Emulsification index (EI%) 48 h grown culture was subjected to centrifugation at 10,000 rpm for 10 min. After centrifugation, 2 mL of culture supernatant were taken and follo w ed b y addition of 2 mL of crude oil to it. Then whole assembly was vortexed for 15 min at room temperature and allo w ed it to stand for 24 h. Emulsification index were calculated after 24 h E24 can display the proportion of biosurfactants produced throughout the degradation process and the capacity of stable biosurfactants to acceler ate substr ate br eakdown and bioav ailability will be boosted.
Em ulsifying hydr ocarbon molecules consequentl y enhances their bioavailability (Dusane et al. 2011 ) (Leite et al. 2016 ) Critical micelle concentration (CMC) The ring method was used to measure surface tension using a DuNouytensiometer at room temperature. Micelle formation occur at particular concentration of biosurfactants known as CMC. With the help of distilled water various concentration (0-500 mg/L) of extracted biosurfactant was formulated and the surface tension was recorded Biosurfactant's effectiveness is determined by how well it dissolves in aqueous solutions. A specific quantity of biosurfactant is necessary to reduce surface tension to a minimum level, and this amount is related to the CMC value; the lo w er the CMC value, the process will be more efficient The presence of biosurfactants significantly speeds up silica gel's tr ansition fr om the hydr ophobic paste to the hydr ophilic phase, resulting in a change in colour (Walter et al. 2010 ) (Kumar et al. 2017 ) Oil coated agar plate method Oil coating was applied over the surface of nutrient agar media plate.
Follo w ed b y str eaking of giv en isolated str ain and left the plates for incubation for 7 days at 37 • C. The presence of emulsification halo ar ound cultur e gr o wth plate sho ws the activity of biosurfactants A straightforw ar d photometrical assay for determining hydrophobicity of bacteria showing bacteria's capacity to cling to hydrocarbons is a pr operty shar ed by micr oor ganisms that pr oduce biosurfactants (Rosenberg et al. 1980 ) (Burd and Ward 1996 ) (Shoeb et al. 2015 ) Victoria Pure Blue BO Micr otitr e plates coated with VPBO was pr epar ed using VPBO (0.1mg/ml) in isopropanol solution. The isopropanol gets evaporated and NaOH solution is added to each well and incubated for 10 minutes at room temperature . T he plate was dried after the aspiration of NaOH. The culture supernatantswere loaded in assay plate, sealed and incubated for 1 h. In a fresh clean 96 well microplate, VPBO dependent absorbance were measured at 625 nm. Based on reference, the value of the biosurfactant are determined.
T his assa y is the direct quantification of biosurfactant and it doesn't r equir e an y extr action steps . T his technique offers wide aspects for compar ativ e determination of different culture conditions for biosurfactantproduction and represents high throughput screening of biosurfactant producing microbial strains. (Kubicki et al. 2020 ) and determined the emulsification index (Dusane et al. 2011 ). In the emulsification assay, 3 mL of culture supernatant and 0.5 mL of oil were placed in a test tube, vortexed for 5 min, and then left at r oom temper atur e for 1 h. After that, the mixtur e's aqueous phase was collected, and an absorbance measurement at 400 nm was taken (Campos et al. 2015 ). A DuNouy tensiometer was used to measure surface tension using the ring technique at room temperature . T he critical micelle concentration (CMC) method was utilized to determine the surface tension property of biosurfactants. A certain concentration of biosurfactants known as CMC results in the formation of micelles. Different concentrations of the extracted biosurfactant (0-500 mg/L) were created with the use of distilled water, and the surface tension was measured (Sambanthamoorthy et al. 2014 ). In the oil-coated agar plate method, oil coating was applied over the surface of the nutrient agar media plate. Follo w ed b y streaking of a giv en isolated str ain and leaving the plates for incubation for 7 days at 37 • C. The presence of an emulsification halo around the culture growth plate shows the activity of biosurfactants (Burd and Ward 1996 ). The chemical composition and component analysis of the isolated biosurfactant is done by FTIR spectroscopic analysis (Shimadzu, Japan). Using this tec hnique, samples wer e cr eated by mixing potassium br omide pellets with 1 mg of biosurfactant in a homogeneous suspension. The infr ar ed spectrum was determined by using an integrated plotter. With a resolution of 4 cm -1 , the IR spectrum's range was 450-4500 cm -1 (Ferr eir a et al. 2017 ).
Combining se v er al scr eening tec hniques is necessary for the effective and simultaneous identification of microbial biosurfactants (Satpute et al. 2008 ). For the primary scr eening dr op colla pse method, the oil displacement method and hemolytic method can be used. The dr op colla pse method is a straightforw ar d and practical a ppr oac h that pr ovides a delicate yet quick manner of producing biosurfactants (Jain et al. 1991 ). Additionall y, it enables e v aluation of the presence of biosurfactants on a qualitative and quantitativ e le v el (Płaza et al. 2006 ). The oil displacement test is the most widely used technique for quick and simple preliminary detection of bacteria that produce biosurfactants (Płaza et al. 2006 ). Additionally, it can be used when the biosurfactant activity and quantity are limited (Walter et al. 2010 ). Previous investigations have shown the efficacy and dependability of this a ppr oac h (Huy et al. 1999, Youssef et al. 2004. The hemolytic test is not a very accur ate a ppr oac h for biosurfactant pr oduction since some r esearc h demonstr ates that it eliminated man y good biosurfactant producers and, in some reports, strains with positive hemolytic activity were found to be ineffective for the production of biosurfactants (T ha vasi and Ja yalakshmi 2003 ). T he most widely used technique for quantifying biosurfactant production is the emulsification index. Theor eticall y, a thic ker em ulsion layer will signify a gr eater pr oduction of biosurfactants . Moreo ver, the E24 can display the proportion of biosurfactants produced throughout the degr adation pr ocess . T he capacity of stable biosurfactants to acceler ate substr ate br eakdown and bioav ailability will be boosted. The deterior ation r ate can thus be incr eased (Leite et al. 2016 ). According to CMC, a biosurfactant's effectiveness is determined b y ho w w ell it dissolves in aqueous solutions. Surface tension reduction to a minimal le v el r equir es a certain amount of biosurfactant that is proportional to the CMC value, and the lo w er the CMC v alue, the mor e effectiv e the pr ocess will be (Srir am et al. 2011 ). Man y r esearc hers performed lipase assay for microbial biosurfactant production, the isolate confirmed lipase production on the Tributyrin Agar (TBA) plate by displaying the zone of clearing (Patel et al. 2021b, Zarinviarsagh et al. 2017 ). The penetration method is based on the observation that the presence of biosur-factants for determining the hydrophobicity of bacteria and is also known as the bacterial adherence to hydrocarbons significantly speeds up silica gel's transition from the hydrophobic paste to the hydr ophilic phase, r esulting in a color change (Walter et al. 2010 ). Unlike other isolates, the biosurfactant-free supernatant becomes cloudy while still being red (Hussain et al. 2021 ). In the oil-coated a gar plate a ppr oac h, bacteria's ca pacity to cling to hydr ocarbons is a property shared by microorganisms that produce biosurfactants (Shoeb et al. 2015 ).

Large scale production of biosurfactants
Surfactants hold a huge market demand worldwide. But the use of synthetic surfactants imposes harmful effects on both human life as well as imbalance the ecosystem because of their nonbiodegr adable natur e. So, it becomes mor e important to focus on natur all y occurring gr een substances to ov ercome this existing pr oblem. Biosurfactants deriv ed fr om micr obes hav e solv ed this problem. Because they are eco-friendly and biodegradable, biosurfactants show many properties such as heterogeneity, substrate specificity, and biodegr adability whic h has gotten a lot of attention fr om r esearc hers . Abo v e mentioned pr operties motiv ated to w ar ds their large-scale production globally (Gaur and Manickam 2021 ). The k e y factors that influence their production on a large scale are the growing organism, the substrate utilized, downstream processing, and financial inputs. Many fermentation processes are used to produce biosurfactants on a larger scale (Kr onember ger et al. 2010 ). The fermentation pr ocedur e was carried out in a benchsize bioreactor by Joshi and Desai et al. for the manufacture of biosurfactants by Bacillus sp. Within 10-12 h of fermentation, the maxim um concentr ation of biosurfactants was gener ated at 70-100 CMD (Critical micelle dilution) when given parameters such as initial dissolved oxygen (DO) that was set to 100% saturation and aeration rate k e pt at 1.0 vvm airflow (Joshi and Desai 2013 ). Amani et al. ( 2010 ) provided the volumetric oxygen transfer coefficient (kLa) for scaling up the production of biosurfactants, which aided in assessing increased productivity at the commercial level. In contrast to shaking flasks, they were able to boost biosurfactant production from Bacillus subtilis by 28% (Amani et al. 2010 ). Cav alcanti et al. addr essed the lar ge-scale pr oduction of biosurfactants by Bacillus invictae UCP 1617 (Cavalcanti et al. 2020 ). To demonstr ate the inter action between v arious pr oduction circumstances and the r esponse v ariable in a 5 L bioreactor, they used a complete 23-factorial design. By using this a ppr oac h, the pr oduction of biosurfactants becomes 1 g/L in 72 hours, while their introduction into a 50 L bioreactor resulted in a 72-h yield of 2.42 + 1.1 g/L. Upgrading the culture medium using a response surface a ppr oac h will enable str ain Wic kerhamom yces anomalus CCMA03558 to produce biosurfactants at a larger scale. By creating a pre-optimized medium that was tested in flasks and bioreactors, batc h cultur e was employed to produce biosurfactants . T he results of this experiment sho w ed that the surface tension of the biosurfactants generated in the bioreactor and flask, respectively, decreased to 29.3 and 31.4 mN/m from 49.0 mN/m. After 24 h of fermentation, the combination of the largest microbial load and the lo w est surface tension value w as ac hie v ed in a 5 L bior eactor. The production of biosurfactants associated with growth is influenced by both of the pr e viousl y mentioned factors (Souza et al. 2018 ). For the large-scale manufacture of rhamnolipid from the bacterium Pseudomonas aeruginosa , a bench-scale bioreactor has shown to be highly helpful. This study examined the rate of production and dependence of micr obial gr owth on oxygen by de v eloping a non-dispersive oxygenation a ppar atus under controlled conditions. When fermentation was carried out using a 1 mg/L oxygen set point, it was noted that the synthesis of gl ycer ol and biosurfactants was consumed less (de Kr onember ger et al. 2007 ). This a ppr oac h pr oduced 15.0 mg/L/h of rhamnolipids, whic h increased to 2.0 g/L/h following 7 days of fermentation. Remarkably, it was discov er ed that the alter ation in oxygen concentration did not affect the rate of consumption, supporting the idea that the oxygenation process can enhance the efficiency of biosurfactants. The specific oxygen absorption rate increases along with the exponential microbiological growth. This proves that oxygen is crucial for the de v elopment of microbes (de Kronemberger et al. 2007 ).
In another study, response surface methodology (RSM) and genetic algorithms (GA) were used to build a numerical model by mathematicall y anal yzing the v ariables and their inter actions with the production statistics (Liyana-Pathirana andShahidi 2005 , Patel et al. 2021a ). It has been reported that scaling up the production of biosurfactants from Bacillus am yloliquef aciens SK27 is ac hie v ed by combining the method of employing RSM and GA under optimal fermentation conditions. According to a report, the v alue r ecor ded b y RSM and the optimal activity of the oil displacement zone by the GA analyzer were determined to be extremely similar. The output of biosurfactants is boosted by 1.2 times using this amalgamation technique (Malik and Kerkar 2019 ). A costlimiting stage makes up 60% of the entire manufacturing cost in addition to downstream processing (such as production, separ ation, and extr action pr ocedur es) (Chen et al. 2021 ). To obtain biosurfactants of high purity combinations of techniques can be used among different methods such as acid precipitation, salt precipitation, chromatography, ultra-filtration, gravity separ ation, and solv ent extr action (Jimoh and Lin 2019 ). Furthermore, it was studied that the choice of extraction method plays a v ery important r ole to obtain biosurfactants of high purity as differ ent extr action methods giv e differ ent purity r ates. Other fac-tors that should be optimized while considering large-scale production of biosurfactants ar e pH, temper atur e, carbon-nitr ogen r atio, and aer ation (Gaur and Manic kam 2021 ). Singh et al. ( 2019 ) described some of the important biosurfactant-producing strains with the maximum yield at a specific amount of substrates under optimum temperature that have been depicted in Fig. 3 .

Challenges of biosurfactant production
Although biosurfactants are superior to synthetic surfactants in many wa ys , they are still unable to compete on a commercial le v el because of highproduction costs and low yields (Olasanmi and Thring 2018 ). According to the r eport pr esented by Hazra, the production cost of biosurfactants is 20%-30% higher than that of synthetic surfactants, causing issues with large-scale production (Hazra et al. 2011 ). The high production cost of biosurfactants is mainly due to two main reasons . T he first is related to the high cost of the substrate used, which varies from 10-30% (Sobrinho et al. 2013 ) to 50% (Luna et al. 2015 ) of the final cost of production. The second is related to the production process , which in volves the purification step and accounts for the high pr oduction cost, whic h r anges fr om 60% (Fr eitas et al. 2016 ) to 70%-80% (Santos et al. 2016 ). Because of these r easons, r esearchers need to focus on reducing the production cost, which can be done by using gravity separation methods that involve biosurfactant r ecov ery by separ ating the surfactant-ric h phase from the fermentation broth (Makkar and Cameotra 1999 ) and foam fractionation, in which a solvent-free method is used for the separation of biosurfactants from the culture medium (Santos et al. 2016 ). On the other hand, low substrate materials, such as waste from agro-industries, which are rich in carbohydrates and have high lipid content, make substr ates extr emel y useful and r easonabl y priced for the synthesis of biosurfactants (Joshi et al. 2008 ). This will lead to lo w er ov er all manufacturing costs and mor e cost-effectiv e scaling up (Banat et al. 2010 ). There is a statement by Br oc khaus , i.e . 'sustainability is only sustainable when it is pr ofitable,' whic h helps in understanding the need for economic feasibility in biosurfactant production (Brockhaus et al. 2017 ).

Conclusion and Future perspectives
Surfactants play a very important role in various industries worldwide, but their chemical origin imposes hazardous consequences for sustainable de v elopment. Biosurfactants ar e gr een compounds that are derived from microbes and have proven to be the best alternative to c hemicall y pr oduced surfactants. T hese ha v e man y beneficial pr operties, including foaming potential, thermos-resistance, low toxicity , biodegradability , and the ability to withstand extreme pH and temper atur e . T hese properties, when combined, ar e extr emel y beneficial in their widespr ead a pplications, attr acting the attention of man y r esearc hers. One of the major applications is in the cleaning of the environment via hydr ocarbon bior emediation, as they gr ow on hydr ophobic surfaces, thereby augmenting the nutrient uptake of the hydrophobic substrate . T here are different techniques emplo y ed for screening biosurfactants, such as the drop collapse method, emulsification index, hemolytic activity, and man y mor e. Understanding the biosynthesis pathway of microbes associated with biosurfactant production can help optimize techniques used for screening and extr action pur poses.
The production cost of biosurfactants is m uc h higher than that of chemical surfactants. which is a major limitation associated with biosurfactants. So, there is a need to explor e ne w tec hniques or optimize old ones. Further attention is needed so that resear chers can w ork on reducing the downstr eam pr oduction costs and increasing their yield. Several methods, such as salt precipitation, acid precipitation, chromatography, gravity separation, ultr afiltr ation, and solv ent extr action, can be used to pr oduce biosurfactants of high purity. For large scales of biosurfactants, optimization of other factors like carbon-nitr ogen r atio, pH, temper atur e, and aer ation is r equir ed. Mor eov er, r esearc hers need to focus on the discovery of nov el str ains that can grow on cost effective substrates.

Consent to participate
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