Screening, Isolation and Molecular Characterization …
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2.5 Extraction of Biosurfactant
The extraction of the biosurfactant was done with the initial step of bacterial cell
removal by centrifugation at 12,000 rpm for 20 min, the cultured supernatant adjusted
to pH 2.0 with the aid of 0.1 M HCl. The biosurfactants extracted with a solvent
mixture containing chloroform and methanol in the ratio 2:1v/v in a separation funnel.
After shaken vigorously, and allowed for phase separation, the lower organic phase
concentrated, and anhydrous sodium sulfate added to remove water, and the residue
was dried to obtain biosurfactants (Pacwa-Płociniczak et al. 2011).
2.6 Molecular Characterization of Biosurfactant Producing
Bacteria–16S RRNA Gene Sequencing
The DNA was isolated from the bacterial culture and was used in PCR to amplify
the bacterial 16 s rRNA using 16 s rRNA PCR kit (800). Using the primers from
the kit, amplification of 800 bp amplicon done, and no amplicon was visible in the
negative control. The expected sized amplicon of 800 bp observed in the positive
control. The test amplicon of 800 bp was purified using magnetic beads, and the
product sequenced by Sanger’s method of DNA sequencing. The sequencing results
were assembled and compared with the NCBI database.
2.7 Characterization of Biosurfactants from Serratia
Marcescens Using Thin Layer Chromatography
Silica gel plate prepared on the ratio 1:2, with 1 mm thickness, i.e., 10 g in 20 ml
distilled water which was then allowed it to stay for 30 min. The silica gel plate
was kept in the hot-air oven for 1 h at 100 °C to activate the absorption. Chloroformmethanol of ratio 85:15 used as the developing solvent. 20 μl of biosurfactant spotted
on the silica gel plate (Sivagamasundari and Jeyakumar 2016), and Ninhydrin reagent
sprayed. The retention factor Rf (eq. 2), which defined as the distance travelled by
the compound to the distance travelled by the solvent calculated.
Rf = (Distance travelled by the compound/Distance moved by the solvent front)
(2)
247
2.5 Extraction of Biosurfactant
The extraction of the biosurfactant was done with the initial step of bacterial cell
removal by centrifugation at 12,000 rpm for 20 min, the cultured supernatant adjusted
to pH 2.0 with the aid of 0.1 M HCl. The biosurfactants extracted with a solvent
mixture containing chloroform and methanol in the ratio 2:1v/v in a separation funnel.
After shaken vigorously, and allowed for phase separation, the lower organic phase
concentrated, and anhydrous sodium sulfate added to remove water, and the residue
was dried to obtain biosurfactants (Pacwa-Płociniczak et al. 2011).
2.6 Molecular Characterization of Biosurfactant Producing
Bacteria–16S RRNA Gene Sequencing
The DNA was isolated from the bacterial culture and was used in PCR to amplify
the bacterial 16 s rRNA using 16 s rRNA PCR kit (800). Using the primers from
the kit, amplification of 800 bp amplicon done, and no amplicon was visible in the
negative control. The expected sized amplicon of 800 bp observed in the positive
control. The test amplicon of 800 bp was purified using magnetic beads, and the
product sequenced by Sanger’s method of DNA sequencing. The sequencing results
were assembled and compared with the NCBI database.
2.7 Characterization of Biosurfactants from Serratia
Marcescens Using Thin Layer Chromatography
Silica gel plate prepared on the ratio 1:2, with 1 mm thickness, i.e., 10 g in 20 ml
distilled water which was then allowed it to stay for 30 min. The silica gel plate
was kept in the hot-air oven for 1 h at 100 °C to activate the absorption. Chloroformmethanol of ratio 85:15 used as the developing solvent. 20 μl of biosurfactant spotted
on the silica gel plate (Sivagamasundari and Jeyakumar 2016), and Ninhydrin reagent
sprayed. The retention factor Rf (eq. 2), which defined as the distance travelled by
the compound to the distance travelled by the solvent calculated.
Rf = (Distance travelled by the compound/Distance moved by the solvent front)
(2)