Table 2.

Methods for screening of biosurfactant and its significance.

Methods	Protocol	Significance	References
Drop collapse method	Drop collapse activity was observed by adding a drop of crude oil and culture supernatant onto the glass slide	A straight forward 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 quantitative level0	(Jain et al. 1991) (Plaza et al. 2006) (Morais et al. 2017)
Oil displacement method	50 mL of distilled water was poured into the Petri dish and 2 mL of crude oil was added in such a way it distributes uniformly on water surface. Afterwards, culture supernatant of 500 µL was added and biosurfactant activity can be seen by clear zone on the oil surface	Most widely used technique for quick and simple preliminary detection of bacteria that produce biosurfactants and it can be used when the biosurfactant activity and quantity are limited	(Plaza et al. 2006) (Walter et al. 2010) (Satpute et al. 2018)
Hemolytic activity	Culture broth were incubated overnight, 10 µL of culture inoculated onto 5% sheep blood agar and then incubated the plates at 37°C for 48 h. The zone of lysis was observed around the colonies	Used as a primarily screen test for biosurfactant production. However, it is not a very accurate approach for biosurfactant production test in bacteria since many good biosurfactant producersare left out due to negative haemolytic zone on blood agar and also non biosurfactant producers also showed hemolytic activity.	(Thavasi and Jayalakshmi 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 followed by addition of 2 mL of crude oil to it. Then whole assembly was vortexed for 15 min at room temperature and allowed 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 accelerate substrate breakdown and bioavailability will be boosted. Emulsifying hydrocarbon molecules consequently 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 lower the CMC value, the process will be more efficient	(Sriram et al. 2011) (Sambanthamoorthy et al. 2014)
Penetration method	96 well ELISA microplates were taken. Hydrophobic paste containing oil and silica gel was prepared and its 200 µL were added to the wells. Subsequently, biosurfactant activity was measured by taking 10 µL of crude oil followed by addition of 90 µL culture supernatant with 10 µL of safranin solution	The presence of biosurfactants significantly speeds up silica gel's transition from the hydrophobic paste to the hydrophilic 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. Followed by streaking of given isolated strain and left the plates for incubation for 7 days at 37°C. The presence of emulsification halo around culture growth plate shows the activity of biosurfactants	A straightforward photometrical assay for determining hydrophobicity of bacteria showing bacteria's capacity to cling to hydrocarbons is a property shared by microorganisms that produce biosurfactants	(Rosenberg et al. 1980) (Burd and Ward 1996) (Shoeb et al. 2015)
Victoria Pure Blue BO	Microtitre plates coated with VPBO was prepared 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. The 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.	This assay is the direct quantification of biosurfactant and it doesn't require any extraction steps. This technique offers wide aspects for comparative determination of different culture conditions for biosurfactantproduction and represents high throughput screening of biosurfactant producing microbial strains.	(Kubicki et al. 2020)