Microbial Biosurfactant Screening: Diversity in Assessment Methods

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).

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% of bacterial cell adherence = 1−ODshaken with oil/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 OD_{shaken with oil} is the optical density of the mixture containing cells and crude oil while the OD_original 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).

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.

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 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).

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).

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 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 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.

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).

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.

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 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).

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).

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) 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).

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 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).