Sample collection and bacteria isolation
Five raw cow milk and five homemade dahi samples were collected from the local market of Gangtok Sikkim, India, and processed for bacterial isolation on De Man, Rogosa and Sharpe (MRS) agar media with 0.1 % CaCO3. Bacteria were isolated by serial dilution method at 10− 4concentration of the pure milk/dahi samples (Ghatani and Tamang 2017).
Screening for biosurfactant (BS)production
Pure isolates were cultured in MRS broth at 37 ˚C for 48 h. The culture broth was centrifuged at 8000 rpm for 20 min to obtain the cell-free supernatant used for screening the BS. A qualitative (Oil displacement test)(Youssef et al. 2004) and quantitative screening techniques. i.e. estimation of Emulsification Index (Satpute et al. 2008) and surface tension measurement by Du Noüy ring method (Gudiña et al. 2010) was used to select efficient biosurfactant producing isolates.
Oil displacement test
A thin layer of 20µl of engine oil (Servo2T Supreme, India) was made on 30 ml distilled water in 100 mm glass petri plate, and 10 µl of cell-free supernatant was added gently to the oil film. The displacement of oil showed the presence of BS in the media(Youssef et al. 2004).
Estimation of Emulsification Index (EI24): 5 ml of each sunflower oil and culture supernatant was taken in a test tube and vortexed for 5 min. The tubes were kept undisturbed overnight. After 24 hours, the total length of liquid in the media and the emulsified solution's length was measured. EI24 was calculated using the formula mentioned by Satpute et al (Satpute et al. 2008).
Measurement of surface tension
DuNoüy platinum ring method is the most accurate method for measuring surface tension. The surface tension of the 48 hours culture supernatant was measured with Kruss GmbH Hamburg tensiometer's help at 25˚C(Gudiña et al. 2010; Jazeh et al 2012).
Production and extraction of biosurfactant: Lactic acid bacteria were cultured in MRS broth for 48 hours at 25˚C in a shaker incubator at 120 RPM. The culture broth was centrifuged at 12,000 rpm at 4˚C, and the supernatant was acidified with 5M HCl by adjusting to pH 2. The supernatant was kept at 4˚C overnight to allow the biosurfactant to settle down at the flask's bottom. After harvesting the precipitate by centrifugation (12,000 rpm for 20 min and 4˚C), it was suspended in CHCl3:CH3OH (2:1) solution and centrifuged again. Then the surfactant was collected from the interphase of CHCl3 and CH3OH. Collected biosurfactant was suspended in distilled water with adjustment of pH to 7. Finally, the biosurfactant was lyophilized for further chemical analysis (Chander et al. 2012).
Phenotypic identification of BS producing bacteria was made by microscopic observations, Gram staining, growth at different conditions and sugar fermentation tests according to the “Bergey’s manual of systematic bacteriology” (Vos et al. 2009). The genotypic characterisation was done by 16S rRNA gene sequencing. Bacterial genomic DNA was isolated by a modified phenol-chloroform method (Sambrook et al. 2006). Full-length 16s rRNA gene region was amplified by 27F and 1492R primers with standard PCR protocol (Sachdev and Cameotra 2013; Bee et al. 2019). PCR product was subjected to Sanger sequencing, and the forward and reverse sequences were aligned by Codon Code Aligner 7.1.2 software. The identity of bacterial isolates was confirmed by the BLAST tool from the NCBI nr/nt database. Partial sequence data were deposited in the GenBank nucleotide sequencing data library(Bento et al. 2005).
Purification of biosurfactant:100µg lyophilized biosurfactant was dissolved in 1ml methanol and applied on analytical Silica gel 60F254 plates (Merck, Germany). The chromatogram was developed in duplicate with mobile phase Chloroform: Methanol: Ammonia water::60:35:5. One plate was treated with ninhydrin solution, and another plate was treated with hydrochloric acid. Biosurfactant was purified by preparative Thin Layer Chromatography (TLC) with the mobile phase chloroform: methanol: ammonia water::65:35:5 on HF254 silica gel plate (Merck, Germany). The bands were observed under UV light at 254 nm (Varadavenkatesan and Murty 2013; Antonious et al. 2015).
Characterisation of biosurfactant
Chemical characteristics of isolated BS were determined by mass-spectroscopy (MS), infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectrometry.
The various biosurfactant fractions were isolated from the purified BS by LC-MS (Thermo Finnigan LCQ Advantage Max Ion Trap Mass Spectrometer Hyphenated with Thermo Finnigan Surveyor HPLC system). BS samples were dissolved in HPLC grade methanol (Merck, Germany) at the concentration of 2mg/ml, and 2µl was injected into the C18 column(5µl, 4.6 X 250mm) with the flow rate of 0.20 ml/min. The mobile phase was acetonitrile/water (with 0.1% TFA) gradient (10–90%). ESI-MS was obtained in positive mode with the scanning range of 50 to 2000Th(Antonious et al. 2015).
FT-IR spectroscopy was used to elucidate the chemical bonds and functional groups present in the BS structure (Pornsunthorntawee et al. 2008).BS sample was analysed in Alpha FTIR spectrophotometer(Bruker, Germany), equipped with Opus graph plotter(Pornsunthorntawee et al. 2008). The lyophilized dry biosurfactant sample was mixed with potassium bromide (KBr) and compressed into a tablet form (Yalcin E 2010), scanned under 700 to 4000cm− 1 with the resolution of 4cm− 1.
TLC purified BS sample was dissolved in deuterated water (D2O) in the concentration of 50µg/ml, and 1H-NMR spectroscopy was done to determine the structural characteristics of the BSwith the instrument ‘Bruker AFCEND-Germany spectrophotometer’. The scanning was done at the frequency of 400mHz (Makkar and Cameotra 1999). The spectrograph was analysed with ‘Mesternova’ software.
Effect of Biosurfactant on seedlings germination
First, the seeds were washed with 3% sodium hypochlorite to make them free from undesired fungal spores. Five different sets of three petri plates were prepared with 10 ml of 0, 150, 300, 450 and 600µg/ mlBS solutions soaked in filter papers and incubated in the dark. In each plate, five healthy maize seeds were incubated, and the growth of the seedlings was observed for one week. The observation was taken at a regular interval of 24 h.
In-vitro effect of biosurfactant on plant growth
The soil samples were collected from different agriculture sites of Sumbuk village, situated in South Sikkim, India. The soil was sterilised by autoclaving at 121oC for 20 min twice. Two sets of five pots were prepared and marked as lactic acid bacteria, and control treatment, respectively and each pot was filled with 1.25 kg of soil. In parallel, broth culture of E. faecium LM5.2 was prepared with the cell density of 108 cells/ml by adjusting the optical density 0.08 at the wavelength of 600 nm with UV-Visible spectrophotometer (PerkinElmer Lambda 25) (Bhuvaneswari et al. 1980; Bai et al. 2003). A set of 5 pots was inoculated with 20 ml of culture broth with the initial population of LAB in each pot was about 8x 105cells per kg soil. An equal amount of sterilised MRS broth was added to the control pots. The pots were incubated for one week at room temperature in undisturbed condition. For the first week, the observation for the seedlings development was made every 24 hours. Later, the plant length, number of leaves, length of leaves, and the plant's width were measured after every week for one month. After one month, the plants were removed from the soil carefully without damaging the roots, and fresh weight, dry weight and moisture content were recorded (Bhuyan-pawar et al. 2015).
Effect of Biosurfactant on rhizosphere microflora
After harvesting the plants, each potting soil's microbial loads were analysed using the serial dilution method. Mycorrhizal spore was extracted from each set of 100g soil by the sieving method. Mycorrhiza spores were counted manually with the help of a zoom stereomicroscope.
All the data were replicated at least thrice to obtain the mean and the standard deviation. The initial screening of biosurfactant was analysed by One way ANOVA followed by Bonferroni post hoc analysis to determine the significant differences (p < 0.05) among the means of different isolates. Student's two-sample t-test analysed differences in the means of the effect of plant growth. All the analysis were performed in RStudio 3.6.3.