Supplement to this trend, many researchers used other methods in this primary screening such as the cultivation of bacteria on blood agar medium and the drop collapse method as a sensitive and easy method. A total of 38 out of 56 isolates gave a good response to the employed screening tests e. The best results in decreased surface tension production. Results of a pesudomonas agar test, b gsp agar plate and c oil displacemnt with lig01 isolate double diffusion on agar of an anionic biosurfactant.
In vitro cytotoxicity and antimicrobial activity of. The demosponge halichondria halichondria panicea pallas. Pdf a combination of rapid and easy assays of biosurfactant. Mineral agar plates 19 were coated individually with 100. An alternative setup using a fixed underneath light source and image analysis software. Isolation of biosurfactant producing bacteria from garbage soil. To confirm the ability of isolates to produce biosurfactant, agar blue plate, oil displacement test, drop collapsed assay and measurement of surface tension were conducted. Original article production and characterization of. The biosurfactant producing ability of bacteria was investigated by oil displacement agar plate. The endogenous isolate ora9 from contaminated hydrocarbon soil was found to be a good biosurfactant producer. Methods discussed above have both advantages and disadvantages. A spot of culture grown on lb medium was placed on a methylene blue agar plate 0. Isolation of biosurfactant producing bacteria from garbage. The thin layer chromatography revealed a possible glucidic composition of biosurfactants.
Selected colonies were streaked on blue plate agar containing the cationic surfactant cetyltrimethylammonium bromide ctab and the basic dye methylene blue and incubated at 37 0c for 78 days 18. Briefly, a single colony of bacterial strain was spotted onto nutrient agar plates spread with 60. Pdf screening and characterization of biosurfactantproducing. The ctab cethyl trimethyl ammonium bromide agar plate method was developed by siegmund and wagner 1991. Oil spreading assay, emulsification index e24, drop collapse test, blue agar plate method cetyl trimethyl ammonium bromidectab, blood agar haemolysis, reduction in sft and phenol sulfuric acid method were used to. The best isolate was identified as pseudomonas aeruginosa. Ctabmethylene blue agar test the ability of a bacterium to produce anionic biosurfactant was examined on ctabmethylene blue agar plate as described by seigmund et al. Isolation of chlorpyrifos degrading bacteria from garden soil int j zoo animal biol isolation of chlorpyrifos degrading bacteria from garden soil rajesh m, archana b and samundeeswari m. The biosurfactant showed emulsification activity on a wide range of temperature between 20100. Nov 16, 2012 screening for biosurfactant producing strain was performed using methylene blue agar plates containing cetyltrimethyl ammonium bromide ctab siegmund and wagner 1991.
The microbes of interest are cultivated on a light blue mineral salts agar plate containing the cationic surfactant cetyltrimethylammonium bromide ctab and the basic dye methylene blue. Mar 23, 2018 a total of 38 out of 56 isolates gave a good response to the employed screening tests e. Lb agar plates were inoculated with ongrown culture of isolates and incubated at 30 c for 4872 h. Boil agar plate tests mineral salt agar medium coved by crude oil was used to detection of biosurfactant production. Methylene blue agar plates were prepared modifying a mineral salt medium. Thus, productive colonies are surrounded by dark blue halos. These methods can have both advantages and disadvantages. Isolation of biosurfactant producing bacteria from our local.
Bacterial biosurfactant from citrobacter freundii mg812314. Madhura warde published on 20160317 download full article with reference data and citations. These isolates were tested for biosurfactant production under sub. All dilutions were spread on nutrient agar plate in triplicate and then overlaid the agar plate surface with lubricant oil. Production and characterization of rhamnolipid biosurfactant. Many biosurfactant producing microbes were isolated from soils or water samples which are contaminated with hydrophobic organic compounds like e. Msm contained microelements needed for an efficient bacterial and fungal. Isolation and screening of microorganisms from oil contaminated soil samples with biosurfactant producing ability was investigated. A dark blue halo around the culture was considered as positive for biosurfactant production. Ctab agar plate method used in this study determines the ionic charge of biosurfactant. Biosurfactant production trait of bacillus cereus isolated.
Among the 214 fungi, 20 strains displayed a clearly visible dark blue halo around the cultures table 2. Characterization of biosurfactants produced by novel strains of. Blue agar plates were prepared using bhm supplemented with ctab 20mg100ml, cationic surfactant, methylene blue 0. When the anionic surfactant in the culture supernatant combines with the cationic surfactant ctab, it will produce dark blue insoluble ion pair with cetyltrimethylammonium bromide and methylene blue. Different screening methods namely, hemolytic assay ha, modified drop collapse mdc, tilted glass slide tgst, oil spread method osm, blue agar plate bap, hydrocarbon overlaid agar hoa plate, emulsification index ei, emulsification assay ea were assessed for their efficiency to detect biosurfactant producing marine bacteria. According to these physiological roles, biosurfactant producing microbes can be found in different environments. Review on biosurfactant production and its application.
Original research biosurfactant production by a newly. The diameter of the clear zone is a qualitative method used as an indicator of biosurfactant production. Blue agar plate bap method anionic biosurfactant, specifically rhamnolipids were detected by this technique. Rhamnolipids, produced predominantly by pseudomonas aeruginosa, are biosurfactants with important applications. Blue agar plate method bushnell hass agar medium bha supplemented with glucose as a carbon source 2% and. Screening and characterization of biosurfactantproducing. Isolation and characterization of potent biosurfactant.
A clear zone around culture was considered positive. Isolation and screening of biosurfactant producing bacteria. The plates were visually inspected for halo zone of oil displacement around the colonies, indicative of biosurfactant production morikawa et al. Blue agar plates con taining cetyltrimethylammonium. Emulsification activity was measured according to the method of cooper et al. Estimation of biosurfactant activity of an alkaline. Methylene blue agar plates containing cetyl trimethylammonium bromide ctab 0. Natural manipueira medium showed minimum surface tension of 28 mnm lb5a isolate whereas for decanted manipueira the lowest value was 26 mnm isolate lb2a. Isolated microorganism was plated on mineral salt medium containing cationic surfactant cetyltrimethyl ammonium bromide ctab and the basic dye methylene blue. Anionic bs producing colonies on blue agar plate were identified following the formation of dark blue halos around the colonies on a light blue plate background. Average diameter of growth on manipueira agar was 7. Pure water in a hydrophobic well has a flat surface but when surfactant is present, it appears concave, taking the shape of the wells vaux and cottingham, 2001. Characterization of hydrocarbondegrading and biosurfactant. Ctab agar plate method blue agar plates containing cetyltrimethylammonium bromide ctab 0.
Estimation of biosurfactant activity of an alkaline protease. Screening of anionic biosurfactants production among fungi. Isolation and selection of biosurfactantproducing bacteria article pdf available in world journal of microbiology and biotechnology 121. Ten micro liters of the appropriate culture was placed into each well. Production of biosurfactant by nocardia otitidiscaviarum. Screening of biosurfactant producing bacteria blue plate agar method this method was developed by the wegnar et. Mineral salt medium, used for blue agar plate as assay medium along with supplementation of feso 4, 7h 2o 10 mgliter with ph 6.
Screening of surfactant producing bacterial strains isolated from. Mineral salts agar medium msa 9 supplemented with carbon sources 2% and cetyltrimethylammonium bromide ctab. The strains were isolated on blue agar plates containing ctab and methylene blue as proposed and developed by siegmund and wagner for. The best results in decreased surface tension production biosurfactant were observed in three isolates ksat 30 mnm followed by ksam 35 mnm and then ksa9 39 mnm comparing with.
Isolation and characterization of biosurfactant production. The biosurfactant producing ability of the microorganisms isolated from the soil samples was investigated by hemolytic assay, drop collapse test, emulsification index and methylene blue agar plate method. Pdf isolation and selection of biosurfactantproducing. The mediterranean sponge halichondria halichondria panicea was explored as a novel matrix for the isolation of biosurfactant. Oil spreading assay, emulsification index e24, drop collapse test, blue agar plate method cetyl trimethyl ammonium bromidectab, blood agar haemolysis, reduction in sft and phenol sulfuric acid method were used to screen microbial biosurfactant producers. The cultures were isolated and maintained on nutrient agar slants and were stored at 4c. Six biosurfactant producing bacterial strains were isolated from soil sample collected from the premises of a coconut oil mill. Hydrocarbon overlay agar method 18 was performed with some modifications. The results from all methods indicate that the biosurfactant can designated as glycolipid. Thirty microlitre of cell free supernatant of each test isolates grown from msm 10 ml was loaded into the each well prepared in methylene blue agar plate using cork borer 4. Characterization of biosurfactant produced by the endophyte. Isolation, characterization and identification of bacterial. Biosurfactants were observed by the formation of dark blue halos around the colonies. Thirty microlitre of cell free supernatant of each test isolates grown from msm 10 ml was loaded into the each well prepared in methylene blue agar plate using cork borer 4 mm.
Screening concepts for the isolation of biosurfactant. The plate was viewed using a backing sheet of paper with a grid. Methods like hemolytic assays are not reliable and sensitive, because this. The plates were visually inspected for clear zone hemolysis around the colony. Ctabmethylene blue agar plate assay pseudomonas desmolyticum ncim 2112 pd 2112 was initially assayed for. Himedia, india and methylene blue 5 g ml were used to detect extracellular anionic glycolipid production 11. Higher emulsification index indicated a higher emulsification activity of the tested biosurfactant vandana and peter, 2014a. Isolation and characterization of potent biosurfactant producing bacteria from petroleum contaminated soil and sea water written by dr. Isolation of biosurfactant producing bacteria from. Evaluation of different screening methods for biosurfactant. Blue agar plates containing cetyltrimethylammonium. The ctab agar plate method is a semiquantitative assay for the detection of extracellular glycolipids or other anionic surfactants 18. Biosurfactant production was screened by determining hemolysis on blood agar, blue halo on ctab agar and emulsification activity.
Apr 24, 2019 supplement to this trend, many researchers used other methods in this primary screening such as the cultivation of bacteria on blood agar medium and the drop collapse method as a sensitive and easy method. A colony surrounded by emulsified halos was considered positive for biosurfactant production. In the present study, for the first time, the blue agar plate method was also applied to fungi. Thirty microliters of cellfree supernatant was added into the hole. Paper open access detection of rhamnolipid production in. The blue agar plates were prepared from mineral salts agar medium supplemented with 0. Characterization of biosurfactant produced by pseudomonas.
Paper open access detection of rhamnolipid production. It is also possible to detect biosurfactant producing and hydrocarbon degrading activity simultaneously on agar plate by overlaying with hydrocarbon kokare et al. Assessment of different screening methods for selecting. Isolation, characterization and identification of bacterial biosurfactant mahalingam p. A study on biosurfactant production from marine bacteria. Dark blue halo zone in the methylene blue agar plate supplemented with ctab confirmed the presence of an anionic biosurfactant siegmund and wagner, 1991. Holes on the agars were created using 4 mm cork borer. Mineral salt agar medium supplemented with glucose as carbon source 2% and cetyltrimethylammonium bromide ctab.
Instead of a rin g, a platinum plate, a so called wilhelmy plate, can be applied in the same manner. Isolation of biosurfactant producing bacteria from petroleum. The ctab agar plate method is a semiquantitative assay for the detection of extra cellular glycolipids or other anionic surfactants. Anionic biosurfactantproducing colonies on blue agar plate are identified following the formation of dark blue halos around the colonies on a light blue plate background. Noteworthy, to the best of our knowledge this is the first report of a wide screening aimed to identify anionicbiosurfactants producing fungi. The synthesis of extracellular glycolipid biosurfactant was detected using the siegmund and wagner 1991 technique. For efficient culture screening according to rhamnolipid productivity, the method using agar plates containing methylene blue mb and cetyl trimethylammonium bromide ctab was reexamined. The microbes of interest were cultivated on a light blue mineralsalts agar plate, containing the cationic surfactant cetyltrimethylammonium bromide and the basic dye. Mineral salt agar medium supplemented with glucose as carbon source 2% and. Screening and molecular analyses for biosurfactant. Evaluation of screening methods for the isolation of. Ctabmethylene blue agar plate assay pseudomonas desmolyticum ncim 2112 pd 2112 was initially assayed for rhamnolipid production using mineral salt cetyltrimethylammoniumbromide ctabmethylene blue agar plate method ctab 0. Evaluation of screening methods for the isolation of biosurfactant producing marine bacteria. Thirty microlitre of cell free supernatant was loaded into.
Blue agar plate containing cetyl trimethyl ammonium bromide ctab 0. For tested pseudomonas strains the emulsification index e24 values ranged between 1858% nhexadecane and respectively 1540% for flukaoil. Improved detection of rhamnolipid production using agar. Results were recorded based on the type of clear zone observed sarvanan 2012. Isolation and screening of biosurfactant producing. Ctab plates were prepared with wells for injection of the samples. Pure bacterial culture was plated onto blue agar 17. Shallow wells were cut into the surface of the indicator plates.
Biosurfactant production with various optimization parameters deepti somayajula, akhilesh shenai, sheryl paul, sharmila savio, tara poduval. Positive colonies dark blue colonies were selected. The biosurfactant producing ability of the microorganisms isolated from the soil samples was. By oil agar plate method supernatant 1oil, 3h, and b23 isolates were add to the wells in mineral salt medium agar olate covered with 5ml of crude oil, 1oil, 3h, and 23000 showing a clear zone around the wells in mineral salts agar medium as showed in figure 5. The isolates were screened for the production of biosurfactant using a range of qualitative and quantitative tests like oil spreading technique, blood agar haemolysis test, drop collapse test, ctab agar plate test, tilted glass slide test, emulsification index and determination of surface activity section 3. Isolation and screening of biosurfactant producing microorganisms. Isolation and characterization of biosurfactant producing. In this study biosurfactant producing bacteria were isolated from 50 terrestrial samples collected in areas contaminated with petroleum in several districts in bangkok, thailand. Pdf isolation and selection of biosurfactantproducing bacteria.