Enrichment of halophilic organisms from contaminated saline lake water and soil samples
To obtain halophilic biosurfactant producing organisms, first halophilic organisms were enriched and isolated, followed by enrichment and screening of biosurfactant producers. The emphasis was on contaminated saline lake ecosystems. Sterile containers were used to collect saline lake water samples and soil samples in its vicinity. Samples of water were collected in sterile glassware and greasy sticky soil samples near the track of salt trollies were collected in sterile bottles and petri-dishes. A total of 20 samples (including water and soil) were taken from diverse locations of Sambhar Lake, near Sambhar salt works, Rajasthan, India.¹²

The following broth media with slight modifications were amended with 3% NaCl (3-5% NaCl for moderate halophile).¹³ A pH meter monitored the medium’s pH (Contech instruments Ltd. Mumbai). Broth was subjected to agitation using a shaker (Classic Scientific) 100 (rpm) at 37 °C for a duration of 7 days.

The pH value at the beginning was set at 7.2 for the following media, incubated for a duration of 1 week at a temperature of 37 °C.

1. Sterile Nutrient broth (NB)
2. Sterile Sehgal-Gibbons (SG) broth^14,15
3. Sterile Halophilic broth^14-16
4. Sterile Zobell broth pH 7.6¹⁷

A 1 mL volume of saline lake water sample was aseptically inoculated into 100 mL of above-mentioned media and kept on a shaker (100 rpm) at 37 °C for 7 days. Similarly, a suspension consisting of 1 g of soil and 10 mL saline was subjected to vortex for a minute, and 1 mL of the supernatant was inoculated into 100 mL of above-mentioned media. After 7 days of incubation, second round of enrichment was undertaken with 10 mL inoculum aseptically transferred to the fresh sterile broth respectively.^14,15 Procedure was repeated until satisfactory growth was obtained.

After the second round of enrichment in above mentioned media the broth sample was subjected to serial 10-fold dilution up to 10^-10 using sterile saline pH (7.4) and 0.1 mL was plated on respective solid media with the similar composition. Along with serial dilution, direct isolation by T-streak method was also performed to get isolated colonies. The incubation of plates was undertaken at a temperature value of 37 °C, for a duration of 7 days for bacteria and number of isolates were obtained.

These different types of halophilic organisms may or may not synthesize biosurfactants, so now they were subjected to enrichment in inorganic mineral salt media seeded with NaCl and an inducer. Various types of mineral salt media were evaluated.¹⁸ N-MSM¹⁹ was found to be relatively better and had relatively lesser precipitate formation.

Enrichment and isolation of the biosurfactant producing halophilic microorganisms
From enrichment experiments for moderate halophiles, bacterial isolates were obtained. Halophiles were enriched in sterile Bushnell-Haas N-MSM which was amended with 3% NaCl, and a hydrocarbon inducer in the form of 1 % (v/v) Kerosene.¹⁹ With an objective to supply minimum carbon in the initial stages for survival 0.1% glucose was added.¹⁷

Different inducers like Kerosene, Coconut oil, Olive oil and Diesel were also evaluated. The different types of oils were initially sterilized by dry-heat method at 140 °C for 1 h for three consecutive days. After 7 days of incubation, second round of enrichment was undertaken with 10 mL inoculum aseptically transferred to the new sterile broth respectively. Broth was observed for turbidity and foam formation, which served as primary indicators for biosurfactant production. After the second round of enrichment, the bacterial broth sample was subjected to serial 10-fold dilution up to 10^-10 using sterile saline pH-7.4 and 0.1 mL was streaked on sterile solid media with similar conditions. Direct isolation by T-streak approach was also undertaken to obtain isolated colonies. The duration of incubation of plates was of 7 days at a temperature value of 37 °C and number of prospective producers of biosurfactant isolates were counted. Those isolates that were able to grow on inorganic mineral salt media amended with 3% NaCl and 1% Kerosene inducer, were selected for further screening.

Initial screening of the capability of the isolate for utilization of a hydrocarbon inducer
Preparation of inoculum for the screening tests
From the initial moderately halophilic isolates, bacterial isolates were able to grow on N-MSM.¹⁹ They were prospective biosurfactant producing moderately halophilic organisms. Different types of colonies exhibiting different morphological features were selected for further investigation. For preservation of cultures, sterile Nutrient Agar (Himedia, Mumbai) slants with 3% NaCl were used and stored at 4 °C.

To prepare an inoculum to seed the production flask, each isolate was inoculated in 50 mL of Luria Bertani medium incubated for 18 h until a cell density of 0.5 O.D._{600 nm} was obtained using the McFarland standard. A 2 mL of seed inoculum was aseptically inoculated into 100 mL of a modified N-MSM.

After 7 days-time period, the Erlenmeyer flask was observed for initial changes like turbidity (indicating growth) and foam formation (indicating emulsification of inducer) both changes served as primary indicators of biosurfactant formation. In this initial enrichment, the purpose was to enrich hydrocarbon degraders, with this intention no other carbon source was additionally added and hydrocarbon served as sole source of carbon as well as an inducer. Enriched broth was centrifuged at 10,000 g for a duration of 20 min and further screening procedures were employed to investigate the biosurfactant producing ability using the supernatant.

The shortlisted bacterial isolates were also sub-cultured on sterile NA slants with 3% NaCl and stored at 4 °C.

Methods adopted for screening of biosurfactant producers
Inoculum for screening tests
In this research, bacterial isolates maintained on NA slants were sub-cultured and a homogenous suspension was used for various screening tests.

Initial validation of the screening methods
With an objective to validate the screening results, initially all the primary level tests for stage I screening were performed using an abiotic positive control in the form of 0.1% standard synthetic anionic detergent Sodium Dodecyl Sulphate (SDS). Distilled water served as a negative control. All observations were taken in triplicates. The experimental values were calculated as an average with standard deviation (SD). For data analysis and validation, analysis of variance (ANOVA) test for a single factor was used to calculate the p-value. Different tests parameters were assessed as the dependent variables and the bacterial isolates were independent variables.

Also in the initial screening, after the validation, along with the isolates two biosurfactant producing standard strains (SS1) Bacillus sp. MTCC 2473 (SS2) and Pseudomonas aeruginosa MTCC 10636 (SS2) were procured from CSIR Institute of Microbial Technology, Chandigarh, India, assessed simultaneously and served as biotic positive controls.

Stage I screening
Tilted glass slide (TGS) test
Each isolate was initially subjected to a tilted glass slide test where a colony of an isolate was brought in contact with saline sample at one border of a sterile slide, and the saline drop was observed, if it begins dipping down it could be considered as a positive result.²⁰

Haemolytic test
Each isolate was investigated for biosurfactant production by inoculating the suspension of the isolate from NA slant on sterile superimposed blood agar media (SIBA). After incubation for 24 h at 37 °C if the colony had clearance in its vicinity, it indicated haemolysis of the red-blood cell further indicating biosurfactant production.^21,22

Parafilm M test
Each isolate was subjected to an initial rapid primary screening step for the producer of biosurfactant. The supernatant drop was bought in contact with a Parafilm M sheet and compared with a drop of water placed on the same sheet, if its diameter is greater than water, it could be considered as a positive result.²³ The diameter of the drop (cm) could be taken.

Lipolytic test
For this test, sterile Gorodkowa’s Tributyrin media was utilized to assess lipase activity of the isolates. Each isolate was cultivated at a temperature of 30 °C for 24-48 h. Plates were assessed for a clearance in the vicinity of the growth.²⁴

Oil-drop-collapse method
To perform this test, 2 µL of machine oil was loaded in one well on the 96 well microtiter plate and permitted to equilibrate for 1 h at 30 °C. After this step, the well with a 5 µL drop of supernatant was observed for 1 min to know whether the drop remains intact or collapses.²⁵ If oil drop changes to a flat drop it could be considered as an indicative of biosurfactant production. If the drop remains intact as it is i.e. beaded, then it could be considered as a negative result. Collapse of the drop is indicative of a positive test.²⁶

With an objective to tabulate the results, the observations were noted as ‘+’ to ‘+++’ indicating partial to complete oil spreading; where ‘+++’ indicates flat drop after 1 min, ‘++’ indicates relatively less flat drop after 1 min, and ‘+’ indicates relatively negligible flat drop as compared to the negative control.

Oil displacement test
This test was performed in a petri dish containing distilled water (50 mL). For this test, 20 µL Soybean oil mixed with basic fuchsin (just to make the observation clear and visible) was added, followed by adding the culture suspension (10 µL) on oil layer. Diameter of oil displacement zone was evaluated.²⁷

Stage II screening
CTAB agar method
In order to perform the test, the initial media N-MSM was amended with Cetyl trimethyl ammonium bromide CTAB (0.2 g/L), to this Methylene blue stain was added (0.005 g/L).¹⁹ Plates were incubated and observed for blue concentric rings in the vicinity of a colony after 48-72 h at 37 °C. The bluish halo could be considered as an indicator of an anionic surfactant.²⁸

BATH test
For this test the prospective isolates were cultivated in modified N-MSM in the presence of an inducer. After the appearance of visible turbidity, broth was centrifuged for 10 min at 10,000 g and the pellet was obtained. After giving saline washings, the cell suspension was utilized for the BATH test. Cell suspension with a volume of 2 mL was treated with n-hexane (100 µL) and the preparation was vortexed for a minute and allowed to settle under static conditions at 30 °C for 1 h. After the duration of 1 h two layers were formed, and the aqueous layer was pooled. Using spectrophotometer in visible range, the factor evaluated was absorbance at a wavelength of 600 nm. In order to estimate the percentage of the hydrophobicity the ratio of the absorbance of suspension after and before incubation was subtracted from 1 and multiplied by 100.²⁹

Measurement of emulsification index
For this test, a volume of 2 mL of Kerosene and 2 mL of filtrate was mixed and vortexed for a minute. It was observed for formation of an emulsion and its maintenance for 24 h. For calculation purpose, emulsion height was divided by the mixture’s complete height and the answer obtained was multiplied by 100.³⁰

Determination of surface tension
Evaluation of surface tension at periodic intervals is crucial in biosurfactant investigation.³¹ Isolates were cultured in N-MSM amended with an appropriate inducer and incubated on the shaker (150 rpm) at 37 °C for 5 days. As a control for comparison, uninoculated N-MSM with oil as abiotic control was maintained for initial measurement. Du-Nouy ring Tensiometer (manufactured by Ganga Scientific, Kolkata), was subjected to calibration using distilled water (72 dynes/cm) and used for measurement.³² The decrease in surface tension was noted and calculated as a percentage decrease.

After the screening steps, the isolate/s showing the highest emulsification index, and reduced tensio-active force of mineral salt medium were chosen for identification studies and production of biosurfactant.

The above tests were conducted for all the isolates, and it was noted that HB-1, HB-26, SH-6, SH-7 along with few others performed relatively better, out of these isolates SH-6 consistently maintained good results so henceforth the promising isolate SH-6 was investigated further.

Identification of the promising biosurfactant producer
Gram staining
The bacterial isolate was subjected to Gram staining and the smear was observed under oil-immersion lens (100X).

Identification of the biosurfactant producing isolate
Sequencing analysis of 16S rRNA gene
Along with morphological and biochemical preliminary analysis, the further steps of the identification of the bacterial isolate SH-6 were done using partial sequencing of the 16S rRNA gene at HiMedia laboratories Pvt. Ltd., Thane.

The uncontaminated QC passed culture plate was used for colony PCR. The genomic DNA was isolated as per the standard protocol and processed further. Molecular Biology grade water (50 µL) was used as suspending medium for 1-2 well-isolated colonies selected from the plate and subjected to proper mixing, followed by incubation for 10 min at 95 °C and a brief centrifugation. The supernatant thus obtained was used as a PCR template using 16S universal primers:

16S universal primers used for bacterial isolate SH6 16S rRNA gene amplification:³³
Primer working from forward: 16S27F (5′-CCAGAGTTTGATCMTGGCTCAG-3′)
Primer working from reverse: 16S1492R (5′-TACGGYTACCTTGTTACGACTT-3′)

PCR products were further purified by salt precipitation and analysed using agarose gel in order to assess PCR amplicons along with 500 bp of DNA ladder Using the approach of BDT v3.1 chemistry, purified amplicons were evaluated by cycle sequencing and then subjected to sequencing on genetic analyzer (ABI 3500 XL). With an objective to generate good quality base reads covering the target, internal primers were also incorporated to obtain near full-length sequence. BLASTN search algorithm was used for assessment of sequences against the microbial nucleotide sequence databases.

BLAST analysis was undertaken. Sequences of 16S rRNA in the form of the .ab1 trace files obtained from the sequencer were manually customized, changed into a readable file based on format of FASTA and then assembled into a sequence in a contiguous manner. After these modifications the files were exported as a FASTA file. Different software’s like Chromaslite and MEGA (Molecular Evolutionary Genetic Analysis) were explored for initial sequence-based data. For bacterial isolate, the SILVA database v138.1 was employed for database search of the assembled consensus sequence³⁴ using the BLAST tool.³⁵

The FASTA format of the sequence was accepted by BLASTN program. Later an appropriate bacterial and archaeal 16S rRNA database was selected in order to search for homology for the given sequence. The steps enabled in the identification of the species. Multiple sequence alignment was undertaken. Along with MEGA other software like CLUSTALW was also explored, since it is always better to validate output using two or more tools. The obtained 16S rRNA gene sequence was considered for the multiple sequence alignment by the CLUSTALW program. For the Phylogenetic analysis from the database, highest similarity 1000 hits from the beginning were obtained. From a source consisting of different taxa, ten neighbour sequences exhibiting close similarity were aligned using MUSCLE aligner.

Production of biosurfactant
Inoculum preparation
For bacterial isolate seed inoculum, 18-20 h saline culture suspension of Pseudomonas aeruginosa SH-6 was prepared. Density of the inoculum was set at 0.5 O.D.₆₀₀ nm using McFarland standard. From the culture 2% v/v inoculum was used to inoculate 100 mL sterile modified N-MSM¹⁹ amended with 3% NaCl and 1% Kerosene.

Extraction and estimation of biomass
The bacterial isolate was cultivated for 6 days in the respective Mineral basal media i.e. N-MSM at 37 °C for Pseudomonas aeruginosa SH-6 to facilitate the production of biosurfactant. The flasks were incubated on shaker at 100 rpm. After incubation and centrifugation of production broth at 10,000 g for 15 min, cell pellets and crude biosurfactant supernatant were obtained. The supernatant was used to extract the biosurfactant by the technique with the use of an acid to facilitate precipitation and use of solvent to facilitate extraction. The suspended pellets were further separated by centrifugation. A temperature of 50 °C for a duration of 24-48 h was employed for drying of cell pellet. This process was continued till a stable reading of the dry weight was obtained.³⁶

Method for extraction of biosurfactant
The bacterial biosurfactant was synthesized using N-MSM and different solvents in combination and different proportions were evaluated to obtain the best possible extraction. Taking cognizance of green chemicals and with a purpose to investigate alternative solvents, we evaluated different solvents such as non-polar Chloroform and polar Methanol in the ratio of 2:1,³⁷ solvents such as Chloroform and Ethanol in the ratio of 2:1,^38,39 solvent system comprising of Chloroform, Methanol and Water in the ratio of 10:05:07,⁴⁰ Acetone-based precipitation at a low temperature,⁴¹ chilled Ethyl acetate^42,43 and Dichloromethane.⁴⁴

Recovery of the biosurfactant
After the respective incubation in the mineral media, the production media was centrifuged at a high speed 10,000 g for 15 min. As per the situation, the culture broth was processed either in fat tubes or Eppendorf tubes. For Eppendorf tubes cooling microfuge was operated at 10,000 g for 15 min. Initial weight of the fat tube or the Eppendorf tube was determined and used in biomass and biosurfactant dry weight calculations. Wherever applicable sterility was maintained throughout the biosurfactant extraction process. Broth was made cell-free, and it was acidified (pH 2.0). For the purpose of lowering the pH 6N HCl was used, the acidified broth was refrigerated for 18 h at 4 °C. Post cold treatment, broth was centrifuged for 15 min at 10,000 g, 0.1 NaHCO₃ was used as a dissolving media for the precipitate and subjected to solvent extraction using organic solvents i.e. Chloroform:Methanol (2:1) and subjected to thorough mixing using a separatory funnel and the phases separated were stabilized. Multiple cycles of extraction were undertaken and at each step the pooled fractions from the organic phase were dried for evaporation of traces of solvents at 50 °C, and contents were subjected to treatment with water and again re-extracted using a solvent mixture. The objective behind this step was to purify the residue to a considerable degree. The organic layer had concentrated biosurfactant. With an objective to concentrate biosurfactant and recover solvents, rotary evaporator device was employed. Biosurfactant mass obtained was treated with n-Hexane in order to eliminate the unused oil.⁴⁵ Crude viscous biosurfactant was treated at 50 °C for a duration of 24 h to obtain a dried mass of biosurfactant.⁴⁶

After the downstream processing of the biosurfactant from the broth, the residue obtained was subjected to Column chromatography to obtain different fractions, which were then subjected to Thin-layer chromatography (TLC). Presence of biosurfactant in the sample on comparing with the standard reference Rhamnolipid R-90 (90% pure, AGAE) helped in the confirmation of the biosurfactant.

Characterization of biosurfactant
Initial detection of biosurfactant components using chromatography
Biosurfactant residue was dissolved in organic solvent and centrifuged at 10000 g for 5 min to collect the organic phase to retrieve solid crude residue for column chromatography. As per the literature^47,48 rhamnolipids obtained from Pseudomonas species consists of congeners, and there could be different homologues present. Hence, column chromatography helped in separating mono-rhamnolipids, di-rhamnolipids present, if any. Later the fractions obtained were combined for further analysis as per the requirement.

Column chromatography
For silica gel-based column chromatography, chloroform was used as the dissolving medium for the residue. Loading of sample was undertaken from the column top. Washings were given to the column with 100% chloroform with an objective to eliminate any impurities, neutral lipids present if any. An attempt was made to gradually proceed from non-polar to polar in terms of the polarity of the mobile phases, hence initially a mixture of non-polar chloroform and polar methanol was adopted, and the ratio of these solvents was changed in the sequential order from 2:1 to 1:1 and finally to 1:2 (v/v) respectively. Fractions of 10 mL were collected in dilution tubes and were subjected to TLC to check the presence of congeners of rhamnolipids. A solvent system comprising of chloroform, methanol and water (65:7:2) was used for TLC.⁴⁹ The first ones to elute from the column could be mono-rhamnolipids followed by di-rhamnolipids. The fractions were then combined and validated by TLC, HPLC and HPTLC analysis.

Thin-layer chromatography
Aluminium plates that were pre-coated with Silica gel (G60, F254, Merck Germany 6×3 cm) were utilized. Sample preparation was done by dissolving 0.1 g of the partially purified biosurfactant in 1 mL of analytical grade methanol. A 10 µL volume of the sample was spotted twice with intermittent drying, at a point which was on the reference line at a distance of 2 cm away from the plate’s bottom. In a similar manner, different plates were prepared to detect lipids, carbohydrates and amino acids. With an objective to detect carbohydrates, another plate was prepared for specific detection of sugars using Rhamnose (1%) prepared in distilled water, as a standard. For mobile phase, solvents i.e. Chloroform:Methanol:Acetic acid [65:10:4 (v/v/v)] were used.⁵⁰ Proper precautions were taken to maintain the saturation of the vapour phases. Mobile phase was permitted to run till 75% of the plate and the developed plates were air-dried before detecting various components. Lipids were detected with the help of iodine vapour.⁵¹ A 1% Ninhydrin reagent prepared in acetone was sprayed to detect amino acid groups. Different reagents relevant to carbohydrates were used. Molish’s reagent (consisted of 5-8 drops of concentrated H₂SO₄ added to 10% a-Naphthol solution made in 96% ethanol)⁵² and Anthrone reagent (obtained by adding 1 g of Anthrone prepared in 5 mL of H₂S0₄ to 95 mL Ethanol) was used to detect carbohydrates.⁵³ Plates were then subjected to a temperature of 100 °C and observed until the appearance of definite spots. With an objective to detect carbohydrates and sugars respectively, the plates were assessed for pale violet and yellow spots. Brownish yellow spots for lipids and purple spots for amino acids were checked. The R_f value of sugars was calculated for further studies.

HPLC analysis of biosurfactants
HPLC analysis of the biosurfactant was undertaken at National Facility of Biopharmaceuticals (NFB) at G. N. Khalsa College, Matunga Mumbai. The biosurfactant sample was subjected to derivatization so to generate phenacyl esters. The biosurfactant obtained after processing and purification was dissolved in 100% Acetonitrile having 2-bromoacetophenone and triethylamine in the molar ratio (1:4:2). The mixture was then kept in a Sonicator for 1 h at 60 °C. In this system p-bromophenacyl esters were then injected into the column of HPLC for the qualitative detection of Rhamnolipid. As a standard, Rhamnolipid R-90 was used, and the test samples were diluted 1:20 in 10% Acetonitrile. The mobile phase consisted of a gradient having phase A: Water and phase B: Acetonitrile, 10% to 60% of mobile phase B. A reverse phase C18 column having a dimension 150 x 4.6 mm at 40 °C was used. A sample volume of 20 µL was injected with a flowrate was 0.8 mL/min, a runtime of 30 min wavelength and was evaluated at 272 nm.³

Separation of components of biosurfactant and confirmation of results of TLC by HPTLC
Biosurfactant obtained after acid precipitation and solvent extraction was dried and used for chromatographic analysis. An amount of 0.1 g was dissolved in HPTLC grade Methanol (1 mL) used as a solvent and its components were used for analysis in automated HPTLC system (CAMAG, Switzerland) at Anchrom Laboratory, Mulund (East) Mumbai. Earlier attempts were also made by dissolving test sample in HPTLC grade Chloroform. A 200.0 x 100.0 mm plate (1.05554.0001) (Merck Darmstadt, Germany) with silica gel 60 F₂₅₄ was a stationary phase. The test samples (5,10,15 µL) were spotted on this plate using an automated sampler (LINOMAT V A, S/N: 080222). The developing solvent system used as a mobile phase comprised of Chloroform: Methanol:Water (6.5:2.5:0.4 v/v).⁵⁰ Mobile phase saturated the CAMAG twin trough glass tank (TTC 20 x 10) in 20 minutes. Different sample volumes comprising of standard Rhamnolipid (1 mg/mL) along with samples (SH-6 glycerol based rhamnolipid, SH-6 glucose-based rhamnolipid and HF-1 olive oil-based rhamnolipid-fungal isolate) in the range of 2-15 µL were applied with the help of micro syringe using a Linomat V band spotter from CAMAG Switzerland. Solvent phase was allowed to run up to 70.0 mm length of the solvent front and plates were kept at room temperature for 3 min for air-drying. An automatic developing chamber was used for the development of plates It was then derivatized using Naphthol reagent (2.5 g of Naphthol was weighed and taken in 500 mL glass beaker, to this 12.5 mL of concentrated sulphuric acid was added and 238 mL of 95% ethanol was added) and the spots were detected after heating for 2 min at 105 °C. Plates were then scanned using a scanner (4A, S/N:170422), evaluated using TLC visualizer (2, S/N:290203) under visible, UV (254 and 366 nm) light and there was a provision for image documentation.⁵⁴