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Microbial Biosurfactant Screening: Diversity in Assessment Methods



Surface active metabolites such as biosurfactants are generally produced extracellularly since these molecules possess variable amphiphilic structures that reduce surface and interfacial tension (Twigg et al. 2021). These amphiphilic structures act with diverse polarities since they are comprised of different hydrophilic and hydrophobic moieties, that classify biosurfactants based on structure and function. The hydrophobic moieties of these amphiphilic structures are derived from fatty acid or their derivates while the hydrophilic moieties are derived from peptides, carbohydrates, alcohols, carboxylic acids, carbohydrates, amino acids or phosphate (Eldin et al. 2019). Bacterial biosurfactants have remarkable emulsification and surface properties making them particularly significant in various environmental applications. The reduced toxicity and biodegradability of bacterial biosurfactants makes them even more advantageous over synthetic surfactants (Purwasena et al. 2019). Such surface-active compounds produced by microorganisms also have the ability to form stable emulsions. This strong emulsification of hydrophobic compounds enhances their bioavailability especially in case of bioremediation (Nayarisseri et al. 2018). The applications of biosurfactant are wide and varied in the fields of medicine, industry as well as environment (Tayeb et al. 2022). Due to the non-toxicity of biosurfactant, these are highly specific compared to their synthetic counterparts. In cellular systems, biosurfactants are used in desorption, flocculation, aggregation, adhesion or dispersion etc. They are highly useful in bioremediation of heavy metal, forming stable emulsions as well as reducing surface tension (Eldin et al. 2019). The interest biosurfactants have garnered in numerous fields owing to their applications is only made possible if large scale production of biosurfactants can be achieved economically. Highest hurdle in such cases is the cost and time effective screening of biosurfactant producers. In this review, various conventional and novel biosurfactant screening methodologies have been discussed in detail. Parameters such as use of supernatant, biomass, media, metagenome, imaging and spectroscopic techniques have been used for classification of screening methodologies.

Biosurfactant screening methods

Effective screening of biosurfactant producing bacteria is based on well-chosen experimental design, efficient analytical and assessment methods. Physical and chemical properties can be used to devise screening methodologies specifically for biosurfactant producing bacteria. Surface tension, emulsification, hydrocarbon adherence etc. are all properties whose application can be extrapolated in formulation of biosurfactant screening methods (Koim-Puchowska et al. 2019). Thus far, numerous methods have been developed for this purpose such as oil displacement, drop collapse, surface tension measurement, hydrocarbon adherence, emulsification assay etc.

Furthermore, there are various methods to screen for biosurfactant producing bacteria according to their selective physiological as well as physicochemical properties. For example, physicochemical properties such as salinity, temperature and pH etc. greatly effect biosurfactant activities (Purwasena et al. 2019). Biosurfactants are accumulated between fluid phases either on microbial cell surfaces or extracellularly. Factors such as pH, temperature, operation mode and agitation are used for quantification as well as identification of biosurfactant quality (Nayarisseri et al. 2018).

Most commonly, biosurfactant production ability of bacterial species is evaluated by detecting their presence or absence in supernatant. The effect of biosurfactants – present in supernatant – is studied in such screening methods particularly on surface tension. Some of these biosurfactant screening methods include emulsion test, parafilm-M test, surface tension test etc. The glycolipid nature of biosurfactants can be detected by using either the CTAB methylene blue test in culture for anionic biosurfactants such as rhamnolipids or by phenol sulfuric test in supernatant. The lipolytic activity of biosurfactants can also be detected using tests as Tween 80 substrate test and Phenol red test. Thus, based on the type of methodology used, biosurfactant screening can be supernatant based, biomass (bacterial cell pellet) based, as well as culture based. Some advanced techniques have also been devised for biosurfactant screening in bacterial samples. In screening for potential biosurfactant producers, it is imperative that more than one screening method be used for accu rate identification of biosurfactant producing bacterial strains. Due to the limitations of various test methods, an apparent and strong correlation between results confirms the potency of effective biosurfactant production (Eldin et al. 2019). Most commonly used screening methods are discussed below (Table I).

Biosurfactant screening methods: advantages and disadvantages

Screening methods Advantages Disadvantages References
Emulsification Index

Simple to use

Gives indication of biosurfactant presence

Low stability of emulsion

Surface activity and emulsification capacity do not always correlate

(5, 7)
Surface tension test




Concurrent measurements present difficulties

Variation prone

Oil displacement/Oil spreading test

High precision

Small sample volume

Low quantity of biosurfactant detected

No need for specialized equipment


Amount of oil used influences detection

Drop collapse assay



No need for specialized equipment

Small sample volume

Low sensitivity

(9, 17)
Penetration assay

Used for screening large number of samples


Optical distortion grid assay




Small sample volume

Suitable for automated high throughput screening


Only qualitative

BATH assay




Only qualitative

Tilted glass slide test




If negligible amount of surfactant is present, false results are given

Hydrocarbon overlay agar test



Cannot be used if microbe does not degrade hydrocarbons

Atomized oil assay

Surface enhanced biosurfactant production is shown

Many strains only produce biosurfactant in liquid media

Blood hemolysis test

Preliminary screening method

Also predicts surface activity of producer

Dubious results (lytic enzymes can also cause hemolysis)

Hydrophobic substrates cannot be used as sole carbon source

Diffusion restriction can inhibit zone formation

Blue agar plate test


Allows various culture conditions

Specific for anionic biosurfactants

Inhibits growth of some microbes

(7, 11)
Supernatant based screening methods

Among the wide array of tests used to screen the ability of a bacterial strain for biosurfactant production, most are based on supernatant. Since, biosurfactants are extracellular, their presence or absence in the supernatant can vouch for bacteria's ability to produce biosurfactants. These supernatant-based screening techniques can in turn be based on physical as well as chemical parameters (Fig. 1).

Fig. 1.

Supernatant based biosurfactant screening methods.

(Figure depicts the isolation of microbes from environment and screening for biosurfactant)

Physical parameters as basis for biosurfactant screening
Emulsification index test

Emulsification activity is measured in terms of emulsification index (E24%). Emulsification index is generally based on the ability of biosurfactant to emulsify hydrocarbons, thus, making them more accessible for uptake by the cell. If biosurfactants are present in a sample, they will emulsify the hydrocarbons and from emulsions. Whereas no emulsion will be formed in the absence of biosurfactants (Guerra et al. 2018). In this test, a droplet of oil is added to supernatant in a screw cap test tube. This test tube is vortexed at high speed and then allowed to stand in an incubator for 24 hours at 37°C (Eldin et al. 2019). The emulsification index is calculated by measuring total height of liquid column and the height of emulsified layer using the formula: E24%=Emulsifiedlayerheight/Totalliquidcolumnheight×100 {\rm{E}}24\% = \left( {{\rm{Emulsified}}\;{\rm{layer}}\;{\rm{height}}/{\rm{Total}}\;{\rm{liquid}}\;{\rm{column}}\;{\rm{height}}} \right) \times 100

It should be noted that the emulsification activity of biosurfactant is not corelated with surface tension reduction and is only the indicative of biosurfactant presence (Devi et al. 2020).

Surface tension test

Reduction of surface tension at water and air interface while reduction of interfacial tension at water and oil interface is an ability specifying biosurfactant presence. Such surface-active properties are the result of amphiphilic moieties of biosurfactant and favor aggregation by formation of amphipathic micelles. Surfactant concentration that is needed for micelle formation is known as critical micellization concentration (CMC) and corresponds to the maximum surface tension reduction achieved by the minimum biosurfactant concentration. Efficiency of surfactant as well as its effectivity in terms of surface and interfacial tension measurement are indicated by CMC value (Koim-Puchowska et al. 2019). Surface tension measurement can be done by various methods such as capillary rise method etc. In capillary rise method, supernatant is transferred to glass tubes and immersed in a water bath. Standardized capillary tube is immersed in each glass tube and the height reached by supernatant is calculated as an action of free ascending force to calculate surface ten sion. The equation; γ =r × h × d × g2 can be used to calculate surface tension, where surface tension (mNm−1) is denoted by γ, capillary radius (cm) by r, height of the liquid column (cm) by h, density (g/ml) by d and gravity by g (Eldin et al. 2019). Tensiometers can be also used to determine surface tension of cell free culture broth using various different methods such as Du Nouy Ring method (Nayarisseri et al. 2018).

Parafilm-M test

Parafilm-M test is based on the principle that biosurfactant can destabilize a drop of polar liquid by reducing the surface tension and the interfacial tension present between the drop and hydrophobic surface of the parafilm strip. This reduction in surface and interfacial tension leads to flattening of the drop. The absence of biosurfactant results in maintenance of drop shape, since no change in either surface or interfacial tension takes place (Eldin et al. 2019). Supernatant is dropped on a strip of parafilm at room temperature. Drop is allowed to rest for one minute and then shape and diameter of the drop are evaluated. A flat drop confirms the presence of biosurfactant. A scoring system ranging from partial to complete spreading of drop on the parafilm surface corresponds from + to ++++ (El-Shahed et al. 2020).

Oil displacement/Oil spreading test

Oil displacement test is based on the principle that effectiveness of biosurfactant activity shows positive linear relation to the amount of surfactant present in the sample (Eldin et al. 2019). Biosurfactant, if present in the supernatant, has the ability to display oil and create an oil free clearing zone (Nayarisseri et al. 2018). Oil displacement test distilled water is taken in a Petri dish and oil is dispensed onto the surface of distilled water. The oil forms a thin film on the center of which supernatant is dispensed. If biosurfactant is present in the supernatant it results in displacement of oil and creates an oil free zone due to reduction of surface and interfacial tension. The diameter of oil free zone is compared to the zones created in positive and negative controls. The oil displacement is calculated in cm2 and expressed as biosurfactant activity unit. Biosurfactant activity unit is defined as the surfactant amount that creates oil displaced area of 1 centimeter square. This method is suitable only for qualitative testing during primary screening of biosurfactant producers (Pardhi et al. 2020).

Drop collapse assay

The ability of biosurfactant to modify shape, based on the principle of reduced surface and interfacial tension, according to its surroundings is employed for drop collapse tests. Biosurfactants reduce the interfacial tension between drop and hydrophobic surface and thus the drop collapses (Ghasemi et al. 2019). This method, being highly specific, is reliable for the detection of large unknown consortia, however low levels of sur factant cannot be detected due to low sensitivity. Furthermore, false positives can be shown due to hydrophobicity of bacterial cells, in which case bacterial cells themselves mimic biosurfactant behavior. Oil is applied to the wells of a 96-well microplates and left to rest for 24 hours in order to reach equilibration. A single drop of 48-hour culture free broth is transferred to each well. If biosurfactant is present in the media, drop collapses and bacterial culture giving flat drop is considered to be positive for biosurfactant produced. While, if the drop maintains its shape and a round shaped drop is observed, those cultures are considered as negative for biosurfactant production (Nayarisseri et al. 2018). For quantification of the test, drop diameter can be measured and is larger for experimental group compared to control group in case of positive test results. Software such as Image J can be used for the measurement of drop (Mishra et al. 2021).

Penetration assay

Penetration assay depends upon the hydrophobic and hydrophilic behaviors of biosurfactants i.e., in the presence of biosurfactants, a hydrophobic phase will be exited much quickly by silica gel. Silica gel will enter hydrophilic phase at a more rapid rate than in the absence of biosurfactants. In this test, a microtiter plate filled with hydrophobic paste (usually made of oil and silica gel) is covered with oil. Supernatant of culture broth and uncultured media (mixed with 1% safranin added as indicator dye) are dispensed at the surface of oil covered wells. In the presence of biosurfactant, the color of upper phase turns white from the clear red of safranin after 10 to 15 minutes. However, biosurfactant free supernatant turns cloudy (since there is a modicum of silica gel transfer) but still remains red (Touseef and Ahmad 2018).

Foaming activity

Foam formation due to biosurfactant activity can be studied by shaking supernatant taken is test tube. In case of form formation, biosurfactant presence in the supernatant is verified. Foaming activity can be quantitatively measured using the equation: Foaming=heightoffoam/totalheightofsupernatant×100 {\rm{Foaming}} = \left( {{\rm{height}}\;{\rm{of}}\;{\rm{foam}}/{\rm{total}}\;{\rm{height}}\;{\rm{of}}\;{\rm{supernatant}}} \right) \times 100

This activity also serves as indicator of surface tension reduction by biosurfactants (Bader et al. 2021).

Optical distortion grid assay

Pure water dispensed in a well has a flat surface which becomes concave in the presence of surfactant and causes wetting of the well edges. The fluid takes on the shape of single diverging lens causing image distortion in a grid when viewed from above. A microwell titer plate is used to study the optical distortion of biosurfactant containing supernatant. Black and white grids present on a backing sheet of paper are used for this purpose. Supernatant is placed in the wells and plate is viewed through the grid paper. Optical distortion, in the presence of biosurfactant serves a positive test result (Touseef and Ahmad 2018).

Chemical parameters as basis for biosurfactant screening
Phenol sulfuric test

Phenol sulfuric test is based on the action of sulfuric acid on carbohydrates to create monosaccharides by removing water molecules to form furfural compounds. These compounds condense with phenol to give a dark yellow color. Unfortunately, this method detects almost all classes of carbohydrates and not only glycolipid based biosurfactants. It is very difficult to distinguish biosurfactant glycolipids from other carbohydrates present in supernatant (Eldin et al. 2019). In the phenol sulfuric test, supernatant is added to the phenol solution and concentrated sulfuric acid is introduced drop by drop until a characteristic orange color develops. The mixture is shaken and left at room temperature in order to allow reaction when biosurfactant is present. The development of color indicates biosurfactant presence.

Bromothymol blue assay

Bromothymol blue assay can be used for high throughput detection of all three classes of biosurfactants specifically lipopeptide based biosurfactants. This quantitative assay is a colorimetric technique based on the principle that lipopeptide biosurfactant changes color in the presence of bromothymol blue that can be quantified as a linear response to concentrations at 410 nm and 616 nm spectrophotometrically. This test can be performed using either cell-free broth for biosurfactant screening or purified biosurfactant for quantitative assays. Such chemical tests for biosurfactant screening, however, have some limitations such as reagent preparation, use of toxic chemicals etc. (Ong and Wu 2018).

Methylene blue active substances (MBAS) assay

MBAS assay or methylene blue active substances assay is an analysis method that uses methylene blue for the detection of surfactant based on their anionic nature. It is a colorimetric analysis since the color satu ration increases did the increase in concentration of anionic surfactants present in the sample. These anionic surfactants include phosphates, sulfonates, carboxylates as well as sulfates.

The basic principle of this method is the color reaction between methylene blue and the anionic surfactant. Since methylene blue is a cationic dye, an ion pair forms due to the reaction between cationic methylene blue and anionic surfactant resulting in color change. Firstly, a sample containing surfactants is acidified. A solution of methylene blue and chloroform are added and reagents are distributed throughout the aqueous and organic phases by agitating the biphasic solution. If an ion pair is created due to the presence of surfactant, it is extracted in the organic phase (Singh et al. 2021). Nonionic surfactants, on the other hand, can be detected using cobalt thiocyanate active substance assay (Muthukumaran 2022).

Surfactant dependent dye solubilization

Victoria Pure Blue BO (VPBO) dye can be used for screening of biosurfactants in culture supernatant. This method is a colorimetric assay used for detection of different anionic and non-ionic biosurfactants and quantification of biosurfactant concentration in a sample. This method is based on solubilization dependent on detergent presence or absence. If biosurfactant is present in the sample/supernatant, the immobilized dye is solubilized as a result of micelle formation and hence the specific absorption of VPBO increases. The amount of dye released as result of solubilization by biosurfactant reflects the amount of biosurfactant in a linear logarithmic trendline and can be used to obtain quantifiable data. Whereas if biosurfactant is absent in the supernatant, VPBO solubilization does not take place and the overall specific absorption of VPBO remains the same. Microtiter plates containing Victoria pure blue BO dye coatings are obtained and supernatant is transferred to the assay plates. Plates are sealed with aluminum and incubated at 23°C for 1 hour with constant shaking at a speed of 750 rpm. Absorbance of solution before and after incubation is measured at 625 nm. Changes in VBPO absorption can be used for quantification of biosurfactant concentration in supernatant by calculating logarithmic trendline equations from plots of standard biosurfactant. In contrast to less specific chemical reactions between reducing sugar and surfactants, this method depends on surface activity and enable quantification compared to current semi-quantitative colorimetric methods. One limitation observed is that the behavior of biosurfactant assembly to macrostructure changes in response to pH. Thus, when using this method for screening of biosurfactant, it is advised to work in a pH range that is suitable for solubilizing biosurfactant micelle formation (Kubicki et al. 2020).

Colony /Biomass based screening methods
Tilted glass slide test

Surface tension is decreased as an action of biosurfactant. In this method, a droplet of 0.9% NaCl is dispensed on the surface of glass slide and a single colony is transferred to the drop. Slides are placed in a tilted position. If surface tension decreases due to biosurfactant action, water flows over the surface whereby the test is considered positive and vice versa (Touseef and Ahmad 2018, Sohail and Jamil 2020).

BATH assay

Bacterial cells able to produce biosurfactant often exhibit cell hydrophobicity by adhering to hydrocarbon surface. Generally, the adhesion of bacterial cell to crude oil as well as cell surface hydrophobicity increases with time. There are, however, some factors such as temperature, pH, organic phases and ionic strength that can influence adhesion (Khanpour-Alikelayeh et al. 2020). In this test, culture pellet is made via centrifugation, washed twice and suspended in a buffer salt solution of K2HPO4 and KH2PO4 (16.9 g/L and 7.3 g/L respectively). The pellet is diluted to an optical density of 0.5 at 610 nm, using the same buffer salt solution. Crude oil is added to the cell suspension and vortexed. The cell and crude oil mixture is allowed to rest for 1 hour in order to separate the aqueous and crude oil phases. Optical density of aqueous phased at 610 nm is measured and the percentage of cells that adhere to oil is calculated using the formula: 1%ofbacterialcelladherence=1ODshakenwithoil/ODoriginal×100 1\%\;{\rm{ of}}\;{\rm{bacterial}}\;{\rm{cell}}\;{\rm{adherence}}\; = \;\left[ {1 - \left( {{\rm{OD}_{{\rm{shaken}}\;{\rm{with}}\;{\rm{oil}}}}/{\rm{OD}_{{\rm{original}}}}} \right)} \right] \times 100 where the ODshaken with oil is the optical density of the mixture containing cells and crude oil while the ODoriginal is the initial optical density of the cell suspension e.g., 0.5.

In some cases, bacterial strains give positive results for BATH test but if drop collapse test is performed, negative test results are observed. The ability some highly hydrophobic, bacterial cells to act as biosurfactants themselves influences these test results. Since no extracellular biosurfactant is produced, negative results are observed for drop collapse test. Further confirmation of bacterial cell adherence to hydrocarbon (BATH assay) can be made using a chemical technique i.e., by adding INT (iodophenyl nitrophenyl-phenyl tetrazolium-chloride) solution and observing under a light microscope. INT is an indicator that changes color when reduced. INT is taken up by bacterial cells and reduced in the presence of biosurfactant. In BATH assay, if bacterial cells actively adhere to the oil, INT is reduced inside the cells and turns red. This change in color observed under microscope also indicates the viability of bacterial cells under experimentation (Nayarisseri et al. 2018).

Screening methods on the basis of incubation media
Hydrocarbon overlay agar (HOA) test

HOA test is used for the identification of hydrocarbon-clastic bacteria and gives a measure of bacteria's hydrocarbon degrading activity (Nayarisseri et al. 2018). It has been suggested that many microorganisms such as bacteria produce biosurfactants in order to enhance the solubilization and emulsification of nonpolar substrates such as hydrocarbons. Hydrocarbons are the sole source of carbon in a media and their emulsification by the action of biosurfactants facilitates the overall process of growth and maintenance (Trudgeon et al. 2020). This ability of biosurfactants is exploited in hydrocarbon overlay plate test by measuring the overall emulsification of hydrocarbon that is observed in form of emulsification halo zone around bacterial colonies (Eldin et al. 2019).

Hydrocarbon overlay agar (HOA) plate test is conducted using a minimal salt agar plate whose surface has been coated in oil and serves as indicator of tolerance against hydrocarbon (Touseef and Ahmad 2018). The bacterial culture in which presence of biosurfactant is suspected is cultured onto the surface of oil coated agar plate and incubated at 30°C for seven to 10 days. The growth of colonies is studied for the presence of emulsified halo zone around the colonies that is indicative of biosurfactant presence.

Atomized oil assay

Oil/hydrocarbon is displaced by the activity of biosurfactant and bright zone or halos are created in the presence of biosurfactant. In this method, a droplet of oil or a mist of liquid paraffin is sprayed over bacterial culture plates in a fine spray. The formation of halos around bacterial colonies is indicative of biosurfactant production. Atomized oil assay is highly sensitive even for very low concentrations of biosurfactant, however is limitable only to microbes culturable on a solid medium (Touseef and Ahmad 2018).

Blood hemolysis test

Blood agar plate test is conducted to evaluate the hemolytic activity of surfactant. Blood agar media is inoculated with bacteria culture and incubated at 37°C for 48 to 72 hours. After incubation the formation of clear zone indicating hemolysis is observed (Pardhi et al. 2020). The hemolytic zones are categorized as white for incomplete hemolysis (α), greenish for complete hemolysis (β), and no change in color in case of no hemolysis (γ). Hemolytic activity of biosurfactant however well documented – as ideal method for detection of rhamnolipids and surfactins – has some limitations pictures such as formation of clear zones around bacterial colonies by action of other lytic enzymes, minimum to no support of hydrophobic substrates, and hindrance in biosurfactant production due to diffusion restriction which consequently leads to inhibition of clear zone formation. Since this particular method creates a lot of false negative or false positive results, hemolysis is not considered a reliable criterion for the detection of biosurfactant activity (Eldin et al. 2019).

Blue agar plate (BAP) test

Extracellular anionic biosurfactants can be detected by CTAB-methylene blue agar plate test. Biological surface-active compounds such as biosurfactants form insoluble ion pairs in the presence of cationic surfactant CTAB (cetyl trimethyl ammonium bromide). These ion pairs are indicated by formation of dark blue halos due to the action of methylene blue dye base in the media. This method, however only applicable in case of anionic biosurfactants, is ideal for the detection of extracellular glycolipids (Islam and Sarma 2021). A minimal salt agar media supplemented with glucose, CTAB and methylene blue is used in this test. Petri plates containing media are inoculated with bacterial culture and incubated at 37°C for 7 to 8 days. After incubation, plates are stored at 4°C to develop color for an additional 24 hours. Formation of halo zone with dark blue coloration around the bacterial colonies serves as indicative of biosurfactant presence. The diameter of dark blue zone created around bacterial colonies, is usually considered as proportional to biosurfactant concentration, albeit semiquantitively. However, CTAB also forms complexes with polysaccharides. Hence, if polysaccharides are present in the supernatural, the reliability of this test is considered to be significantly minimized (Eldin et al. 2019).

Agar double diffusion test

Cell bound biosurfactant have a characteristic ionic behavior that can be used as a screening strategy. In the presence of two charge bearing compounds, of either the same or opposite types, precipitation lines are formed due to the action of biosurfactant. In this test, uniformly spaced wells are made in agar surface and filled with 24-hour culture of test strain in Nutrient broth media. On either side of wells anionic and cationic compounds are introduced. Generally, sodium dodecyl sulfate (SDS) and Cetyl trimethyl ammonium bromide (CTAB) are used in this test, as anionic and cationic compounds respectively. Plates are incubated for 24 hours at 25°C. After incubation, presence or absence of precipitation lines is observed (Touseef and Ahmad 2018).

Tween 80 substrate test

Tween 80 induces lipase gene expression and thereby stimulates lipase secretion. It also serves as substrate for the enzymatic action of lipases and estrases. When lipases act on Tween 80 substrate, oleic fatty acids are released. Calcium ions present in the growth medium react with these fatty acids and transform them to calcium oleate that is water insoluble and appears in form of white precipitate (Eldin et al. 2019). For Tween 80 agar plate test, petri plates are inoculated with bacterial culture and incubated at 30°C for three to five days. Due to the action of lipolytic biosurfactant, the presence of white precipitate is detected in the medium is indicative of biosurfactant presence.

Phenol red test

Phenol red is a pH indicator dye that is used in order to detect pH changes in the occurring due to acidity. When extracellular lipases act on triglycerides, fatty acids are released. These fatty acids cause a change in the acidity of medium (Eldin et al. 2019). For lipase assay by phenol red agar plate test, the plates are inoculated with bacterial culture and supplemented with oil. Bacterial plates are incubated at 30°C for three to five days. After incubation formation of hydrolysis zone around the bacterial colonies as well change in color of phenol red to a bright yellow is indicative of biosurfactant presence.

Antifungal activity

Bacterial biosurfactants such as surfactin have the ability to inhibit fungal growth by various defensive mechanisms especially against cellular membranes. Since antifungal activity has been linked to strong biosurfactant production, it can be used to screen for the presence or absence of biosurfactant. Agar spot test can be used to determine antifungal activity of bacterial strain. In this test, fungal plug is inoculated in the center of bacterial culture plate and each bacterial strain is spotted at a distance from the fungus (approximately 2.5 cm). Growth inhibition of fungus is studied at day 7 of incubation at 25°C. Zone of inhibition are created around bacterial colonies that produce biosurfactant. Diameter of inhibition zones is measured in mm (Kaboré et al. 2018).

Antimicrobial and antiadhesive activity

Biosurfactants serve as therapeutic antimicrobial agents due to their potential for self-association and pore creation in cell membrane. Antimicrobial activity of biosurfactants against various microorganisms such as Enterobacter aerogenes, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, and Bacillus cereus can be determined by various tests such as agar well diffusion method etc. In case of agar well diffusion method, biosurfactant solution i.e., supernatant as well as liquid culture can be added to wells made in solid agar media plates and plates are incubated at 37°C. Antimicrobial activity of biosurfactant against microbial culture can be observed in form of clear inhibition zones around the wells (Ghasemi et al. 2019). Studies show that adsorption of biosurfactant against solid surfaces reduces microbial adhesion. This ability also aids in inhibition of pathogenic microbial colonization. Antiadhesive activity of biosurfactants against microbial strains therefore can be exploited as screening test for biosurfactant producing bacteria and serves as a reliable criterion.

Metagenomic screening methods

Metagenomics provide a bypass from traditional cultivation methods and make it efficient to handle diverse microbial populations. Metagenomic approaches have been devised by taxonomic and functional analysis using databases and computational tools such as NCBI BLAST (27), COG (clusters of orthologous groups of proteins), Biosurfactants and Biodegradation Database (BioSurfDB) (22), KEGG (Kyoto Encyclopedia of Genes and Genomes), antiSMASH (antibiotic and secondary metabolite analysis shell) etc. (Islam and Sarma 2021). Knowledge of microorganisms and genes involved in biosurfactant production, their identification and characterization help in determination of specific microbial diversity in a consortium. Collective microbial genome analysis aided by bioinformatics tools can be aimed at identification and screening of taxa as well as genes involved in biosurfactant production (Gaur et al. 2022). A few such approaches are listed as follows:

Genome sequencing of unknown consortia

The extraction of DNA from environmental pool aids in screening for biosurfactant producing genes. A stable isotope probe (SIP) can be used for effective and efficient discovery and identification of biosurfactant producing strains. Since biosurfactants have significance in hydrocarbon degradation, targeting the gene specific for polyaromatic hydrocarbon (PAHs) degradation can aid in screening for biosurfactant producers. Exploration of crude oil fields have led to the discovery of various key genera of hydrocarbon degrading bacteria, many of whom are biosurfactant producers (Gaur et al. 2022). Genomic DNA is obtained from microbial consortia and sequenced to obtain metagenomic data. Metagenomic data is analyzed using different computational analysis such as alignment, taxonomic hierarchy mapping and dendrogram drawing etc. in order to map out metabolic pathways specific for biosurfactant production. Bacterial strains in which such biosurfactant specific genomic sequences are identified are screened from the consortia (Guerra et al. 2018).

PCR detection of known biosurfactant genes

PCR detection of biosurfactant specific genes can also be done for screening of biosurfactant production bacteria. Many genes involved in biosurfactant biosynthetic pathways have been identified. Rhamnolipid biosurfactants are produced by rhlAB genes (Alyousif et al. 2020) while srfA operon containing srfA, srfB and srfC genes is responsible for production of surfactin biosurfactant. In case of hydrocarbon degradation, gene alkB mediates petroleum hydrocarbon degradation while nah gene mediates alkane and aromatic hydrocarbon degradation (Islam and Sarma 2021). Gene sfp has been reported to be involved in the biosynthetic pathway of surfactin, a lipopeptide antibiotic. Surfactin also aids in biofilm formation owing to its specific surface-active properties. Due to its detergent like action against biological membranes, as a defense mechanism, surfactin is considered to be a powerful biosurfactant (Kaboré et al. 2018).

Vector construction from metagenomic library

Metagenome libraries are being used in for vector construction derived biosurfactant screening. Such vectors – including but not limited to lipids and proteins – have proven efficient tools in diverse biosurfactant identification from non-cultivable microorganisms. Metagenome derived ornithine lipid identification has been proposed as a novel functional screening methodology for biosurfactants, in a recent study. A metagenomic library using environmental DNA from Escherichia coli, Streptomyces lividans and Pseudomonas putida shuttle vector was constructed. This library was used for sequence analysis to confirm ornithine acyl-ACP N-acyltransferase as being responsible for biosurfactant activity. Transposon mutagenesis was also explored. This vector was used to study and identify ornithine lipid in unknown microbial strains as indicative of biosurfactant activity. Use of multiple hosts, in case of biosurfactants, also increases substrate diversity during biosurfactant synthesis (Williams et al. 2019). Another study proposed the use of a protein having oil degradative activity, namely MBSP1, without substrate dependence. Additionally, MBSP1 transformed cells showed increased aliphatic hydrocarbon degradation (Araújo et al. 2020).

Imaging and spectroscopic techniques used for biosurfactant screening

The properties of biosurfactants are being explored in depth to devise innovative screening methodologies such as biofilm disruption assay, etc. Recent advancement in the field of biosurfactant screening are as follows.

Biofilm disruption detected by ERT

Microbial biofilms are surface associated complex aggregates of microbial cells in an extracellular matrix. These hydrophobic aggregates are highly resistant against antimicrobial agents and have high interfacial tension. Surface active molecules remove biofilms either by destroying the extracellular matrix or altering surface adhesion properties by emulsification, leading to overall decrease in interfacial tension. Due to their low toxicity, amphipathic biosurfactant molecules are preferred in biofilm removal as well as hydrocarbon and xenobiotic remediation (Bhadra et al. 2022). In recent studies, electrical resistance tomography (ERT) has been reported as a screening method by detection of biosurfactant mediated biofilm disruption. Sophorolipids and rhamnolipids have been reported to disrupt bacterial biofilms leading to their eventual removal. Electric resistance tomography can be used to detect either biofilm disruption or removal by action of biosurfactants. Biofilms are nonconductive and their electrical properties are governed by electrolyte diffusion and porosity of biofilm layer. Pre-formed biofilms with well-known electrical conductivities can be used to check the effect of biosurfactant presence or absence. ERT, a non-intrusive imaging technique, can be used to monitor the temporal and spatial resolution of biofilms. This tomographic technique also provides detection independent of biofilm growth location. In this method, the electrical conductivities of biofilms before and after biosurfactant aided disruption are measured. The deviation of control from experimental group can be represented as zonal boundary averages for biofilm removal in terms of conductivity values (De Rienzo et al. 2018).

Matrix assisted laser desorption/time of flight mass spectrometry (MALDI-TOF/MS)

Matrix assisted laser desorption/time of flight mass spectrometry (MALDI-TOF/MS) was used in a recent study for the screening of glycolipid biosurfactant producing bacteria. Crude extract of well-known biosurfactant producer initially used MALDI-TOF/MS and then environmental samples were screened using broth cultures. Results were compared to evaluate biosurfactant production. This method can be used reliably for the rapid screening of specific biosurfactant producing strains such as glycolipid type biosurfactant (Sato et al. 2019).

Desorption Electrospray Ionization Mass Spectrometry (DESI-MS)

Biofouling results from an excessive buildup of microbes whose extracellular bioactive metabolites can clog purification membranes and reduce efficiency. At this air-water interface, microbial biosurfactants influence the water-membrane interface during bacterial adherence mechanisms. This biosurfactant production, as a result of quorum sensing, is strictly maintained at transcriptional level and allows rapid screening of biofilms at membrane surface. Thus, biosurfactants have a particular molecular signal, that can be detected in form of DESI-MS specific signatures, indicative of biofilm initiation. In Pseudomonas aeruginosa, biofilm formation is mediated by rhamnolipids that forewarn the process of biofouling. Mass spectrometry specifically ambient ionization techniques can also be used for the detection of surfactant-metal complexes, in case of heavy metal toxicity (Jakka Ravindran et al. 2018).

Laser ablation electrospray ionization mass spectrometry

Laser ablation electrospray ionization mass spectrometry can be used for the study of biofilm production in situ. This biofilm production is accompanied by the production of various competitive biological control factors within a chemically complex matrix. Such factors including biosurfactants mostly of lipopeptide classes e.g., fengycin and surfactin can be detected by laser ablation electrospray ionization mass spectrometry as screening criteria for biosurfactants producers (Bacon et al. 2018).


Various concurrent methods and selection criteria of biosurfactant producers are discussed in this review. Specific methods like MBAS assay for anionic biosurfactants can be used for targeted microbial isolation. Expertise in imaging and spectroscopic techniques permits explorations of large populations. Metagenomic libraries are being employed for characterization of novel microbes and surface-active metabolites using techniques such as vector construction.

Angielski, Polski
Częstotliwość wydawania:
4 razy w roku
Dziedziny czasopisma:
Life Sciences, Microbiology and Virology