Microorganism and culture conditions
In the current study, Bacillus subtilis SNW3, obtained from Microbiology Research Lab, Quaid-i-Azam University, Islamabad, was previously identified and isolated from contaminated soil of Fimkessar oil field, Chakwal, Pakistan (Malik and Ahmed 2012). The bacterial sample was cultured on nutrient agar plates (Yeast extract 2.0; Beef extract 1.0; Peptone 5.0; Sodium chloride 5.0; Agar 15 g/L) incubated for 24 h at 30 °C to obtain separate pure colonies, stored for regular use at 4 °C and sub-cultured before use. The strain was preserved at -80 °C in nutrient broth (Peptone, 5; Meat extract, 1; Yeast extract, 2.0 and sodium chloride g/L) supplemented with 30% glycerol.
Cost-effective substrates for biosurfactant production.
For low cost biosurfactant production various cost-effective substrates were evaluated that includes: potato peels powder (total carbohydrate 68.7%; starch 25%; protein 18%; non-starch polysaccharide 30%; acid-soluble and acid-insoluble lignin 20% and nitrogen 1.3%) (Liang et al. 2014), molasses (total sugars 62.3%, sucrose 48.8%, starch 0.33% and ash 13.1%) (Palmonari et al. 2020), white beans powder (protein 15.62%; carbohydrates 60.47%; lipids 2.13%; crude fibre 14.15%) (Alayande et al. 2012), waste frying oil (palmitic acid 15.86%; oleic acid 29.83%; stearic acid 4.87% and linoleic acid 28.85%) (Banani et al. 2015) and nitrogen sources : sodium nitrite, urea and ammonium nitrate while, conventional media yeast extract (protein 62.5%; sugar 2.90%; fat 0.10%; ash 9.50%) was used as control. Each carbon source listed above was designed to use individually, then selected substrates were used in different combinations to achieve an optimized medium composition. Molasses used in current study was obtained from Chashma Sugar Mills Limited in Dera Ismail Khan (Pakistan). Potato peels and waste frying oil were obtained from café located at Quaid-i-Azam University Islamabad (Pakistan). Whereas white beans were obtained from National Agricultural Research Council (NARC) Islamabad Pakistan.
Inoculum
Bacillus subtilis SNW3, streaked and stored on nutrient agar plates at 4 °C was used for inoculum preparation. A loop full of culture from a single isolated colony on plate added in 100 mL nutrient broth (Peptone, 5; Meat extract, 1; Yeast extract, 2.0 and sodium chloride, 5 g/L) incubated at 30 °C for 48 h then seed culture from the nutrient broth was used as inoculum for all experiments.
Production optimization, extraction, and partial purification of biosurfactant
The strain Bacillus subtilis SNW3 was grown on conventional yeast extract media (2% w/v) and mineral salt medium (MSM) as described by Abouseoud et al. (2008) of given composition (g/L: KH2PO4, 2.0; K2HPO4, 4.0; FeSO4.7H2O, 0.025; MgSO4.7H2O, 1.0; KCl, 0.2; NaCl, 5.0; CaCl2. 2H2O, 0.02; and trace elements solution with composition of MnSO4.4H2O, 1.78; ZnSO4.7H2O, 2.32; CuSO4.5H2O, 1.0; H3BO3, 0.56; KI, 0.66 and NH4MoO4.2H2O, 0.39) for evaluation of different environmental process parameters significant for biosurfactant production at various range of temperature (15, 30, 37 and 50˚C), pH (2, 4, 6, 8, 10, 12), agitation speeds (0, 150 and 250 rpm) and inoculum size (0.5, 1, 1.5, 2 and 2.5). After that carbon and nitrogen sources used to be screened out were used separately and in different combinations with MSM while yeast extract was used as control media. All designed experiments for substrate evaluation were run with 100 mL media in 250 mL Erlenmeyer flask with pH adjusted to 7.0 ± 0.2 and kept in a shaker for 96 h of incubation at 30 ºC and 150 rpm. The cell-free supernatant obtained after centrifugation at 12,000 rpm was acidified up to pH 2.0 with 1M hydrochloric acid (HCL) and kept overnight at 4 °C. For crude surfactin, pelleted precipitates were extracted with chloroform/methanol (2:1) and concentrated by rotary evaporation (Marchut-Mikolajczyk et al. 2018).
Assessment of biosurfactant production
For estimation of biosurfactant production in cell-free supernatant, various methods like oil displacement activity (ODA) was performed according to the method of Morikawa et al. (2000), emulsification index (E24%) was performed through a protocol of Cooper and Goldenberg (1987) and surface tension (SFT) was measured in mN/m by using KRUSS K20 digital Tensiometer (Kruss GmbH, Hamburg, Germany), performed at room temperature while using a platinum plate by Wilhelmy plate method according to the protocol given by manufacturer.
Structural characterization of surfactin produced.
For thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) analysis, extracted form of crude biosurfactant was used while surfactin (from sigma) was taken as standard. Crude biosurfactant components were separated on Silica coated aluminium plates, silica gel 60 F254, MERCK Germany using chloroform: methanol: acetic acid (85:10:5, v/v) visualized under the wavelength of 254 and 365 nm to find retention factor (Rf) as described by Joy et al. (2017). For determination of surfactin functional groups 10 mg crude biosurfactant was loaded and the spectrum was observed at the range of 4500–450 cm-1 using Tensor 27 (Bruker) FTIR spectrophotometer, equipped with ZnSe ATR (Marchut-Mikołajczyk et al. 2019).
Functional characterization by Antibiogram of surfactin produced.
For antibiogram analysis, the extracted surfactin (10 mg/mL) dissolved in demineralized water tested with two different antibiotics namely ciprofloxacin and clarithromycin (1 mg/mL) separately and in combination (surfactin:antibiotic 5:0.5 mg/mL) against Escherichia coli ATC 25922, poured at a concentration of 100 µL and kept at 37°C for 24 h of incubation, finally examined by diameter (mm) of the clear zone.
Determination of critical micelles concentration (CMC) and critical micelle dilution (CMD)
The CMC of the produced biosurfactant was determined by a change in surface tension reduction with biosurfactant solutions from 0.06 to 1.24 mg/mL prepared in demineralized water (Datta et al. 2018). For critical micelle dilution cell, free supernatant was diluted10-folds up to three levels (i.e. 10x, 100x, and 1000x) named as CMD-1, CMD-2, and CMD-3, respectively, and was analyzed by surface tension reduction values (Joshi et al. 2008a).
Surfactin stability studies
To elucidate the thermal stability of surfactin, the standard solutions were prepared at a concentration of 600 mg/L and incubated at different temperatures (20-121 °C) for 1 h then surface tension of test solutions was measured by the Wilhelmy plate method after cooling at room temperature. Furthermore, a stability test of produced surfactin at saline conditions was performed at different concentrations of sodium chloride NaCl (1-10%) and incubated at 30 °C for 1 hour followed by the analysis of surface tension reduction. To determine pH effect on surfactin activity different buffer solutions were added to biosurfactant standard solution, adjusted to pH 1–5 using citrate-phosphate buffer, pH 7 using phosphate buffer, and pH 9–11 using carbonate-bicarbonate buffer solutions, and check for surface tension reduction after incubation at room temperature for 30 minutes (Goswami and Deka 2019).
Exploration of surfactin for seeds germination and plant growth
The seeds of tomato (Solanum Lycopersicum), pea (Pisum sativum), chili pepper (Capsicum annuum), and lettuce (Lactuca sativa) collected from NARC Islamabad, Pakistan were surface sterilized with 10% Na–hypochlorite for 20 minutes and then washed with sterile distilled water before use. The first seed germination experiment was conducted in petri plate containing 40 seeds positioned in filter paper and cotton soaked with four different concentrations (0.1, 0.3, 0.5, and 0.7 g/100 mL) of crude surfactin solution in distilled water while 100% v/v distilled water was used as a control. These plates were kept in yellow light at 25ºC for 7 days after that relative seed germination (G, %): (No. of seeds germinated (treatment) / No. of seeds germinated (control)×100) was calculated. After the germination test seeds treated with surfactin were transferred in pots (seeds without pre-treatment with biosurfactant were used as control) and kept in a greenhouse with temperature maintained between 20 ºC to 22 ºC. Furthermore, for plant growth stimulation crude surfactin solution was added in pots at a concentration (0.1, 0.3, 0.5, and 0.7 g/100 mL) dissolved in distilled water thrice with 10 days interval while in control pots pure water was added. The emergence of plant seedlings was tested and checked for the morphological characteristic of plants like shoot length (mm), root length (mm), and dry weight (g) of plants after 40 days (Huang et al. 2017).
Bioremediation of crude oil through various design treatments
Biodegradation efficiency of crude oil by Bacillus subtilis SNW3 was analyzed as illustrated by Rahman et al. (2002) with minor changes. An aliquot of 2 mL pre cultured Bacillus subtilis SNW3 was transferred into 250 mL of Erlenmeyer flask containing 100 mL mineral salt media with different concentrations of filter sterilized crude oil of 0.5, 1, 1.5, and 2% (v/v) as sole source of carbon and energy. For monitoring of abiotic loss of the crude oil an uninoculated media was used as control. All these flaks were incubated for 21days at 200 rpm and 35 ˚C. For monitoring the bacterial growth in crude oil, the absorbance rate was detected at (OD600 nm) by using spectrophotometer while for biosurfactant production analysis surface tension reduction was examined by tensiometer. To estimate residual crude oil in media solvent extraction method by hexane was used after that left for evaporation in a pre-weight clean beaker. For quantification of remaining crude oil degradation gravimetric analysis was performed at different time intervals by following formula proposed by Patowary et al. (2017).
Hydrocarbon degradation % = Amount of crude oil degraded /Amount of crude oil added in the media x 100
Surfactin suitability for removing hydrophobic pollutants from soil was analyzed by collecting 5-10 cm deep topsoil while following the protocol of Okop et al. (2012) and transported in a clean container to Microbiology laboratory of Quaid-i-Azam University Islamabad Pakistan. Crude oil used was collected from Pakistan petroleum limited. The soil collected was airdried and sieved with a 2 mm sieve after that 5% of crude oil was sprayed on the soil to pollute soil homogenically. The polluted soil was left undisturbed for 5 days and then divided into 200 g of equal parts and dispensed in pots. These pots were left undisturbed in the open air for a week. Then for conducting bioremediation experiments various designed treatments were established added twice throughout the remediation period: (T0) addition of distilled water as control, (T1) addition of cell-free supernatant containing surfactin, (T2) addition of cultured Bacillus subtilis SNW3 (T3) combination of cell free broth and cultured Bacillus subtilis SNW3, (T4) addition of tween 80 (T5) addition of fertilizer (NPK; 20-10-10) shown in (Table 1). The soil content of each pot was tilled twice a week for aeration with moisture maintenance at 60% and temperature of 28-30 ºC, providing all those conditions that are appropriate for crude oil-degrading microbes present in the soil. After that soil samples of 10 g were collected from different areas of the plastic pots at the 30,60, 90th day and were gravimetrically determined using formula given by Ganesh and Lin (2009).
Table 1 Design treatments for removal of crude oil from contaminated soil by surfactin and Bacillus subtilis SNW3
Treatments
|
Soil
|
Biological treatment
|
Chemical compounds
|
Crude oil concentration
|
Control (T0)
|
200 g
|
|
|
5%
|
Treatment 1 (T1)
|
200 g
|
200 mL cell free surfactin broth
|
|
5%
|
Treatment 2 (T2)
|
200 g
|
2% cultured broth 100 mL
|
|
5%
|
Treatment 3 (T3)
|
200 g
|
2% cultured broth 100 mL + 100 mL cell free surfactin
|
|
5%
|
Treatment 4 (T4)
|
200 g
|
|
10 mg/kg of tween 80
|
5%
|
Treatment 5 (T5)
|
200 g
|
|
0.8 g/kg of fertilizer
|
5%
|
Statistical Analysis
The obtained results were analyzed statistically with the use of STATISTICA software, one-way ANOVA (version 8.1). The difference between obtained results was analyzed by using the Tukeys test to find individual and control mean ± standard deviation. Significance value was set at p = 0.05 and p-values ≤ 0.05 were considered significant.