3.1 Isolation and biochemical characterization
A total of 23 different species were found in the collected samples. On screening the cultures in the crude oil, four organisms were found to survive on the MSM plates. These were pure cultured and characterized using biochemical tests. The screened microorganisms belong to 4 different species of bacteria, which includes Bacillus tequilensis species (Isolate 1 - 4.32x108 CFU/ml), Bacillus subtilis species (Isolate 2 - 4.07x108 CFU/ml), Paenibacillus species (Isolate 3 - 4.28x108CFU/ml), and Microbacterium species (Isolate 4 - 4.16x108 CFU/ml) illustrated in Table 1 that were identified and distinguished employing Bergey’s protocol as a source of reference. Bacillus tequilensis is biochemically analogous to Bacillus subtilis; however, it can be discriminated by positive production of 3 rhamnose compounds, namely ornithine decarboxylase, lysine decarboxylase, and arginine dihydrolase, as well as acid production. The biochemical test results for each isolate were verified with the literature to identify the genus. The detailed list has been mentioned in table 1. The gram staining and bacterial colonies of Isolate 3 have been depicted in figure 1.
Table 1: Biochemical characteristics of the four isolates
Biochemical characterization
|
S.No
|
Parameters
|
Microorganisms
|
Isolate 1 (Bacillus subtilis)
|
Isolate 2 (Microbacterium)
|
Isolate 3 (Bacillus tequilensis)
|
Isolate 4 (Paenibacillus)
|
1
|
Colonial Characters
|
Opaque, Off-white colonies, Large, Circular, Smooth, Uneven, Slightly Raised.
|
Yellow colonies, translucent, smooth, Medium, circular, slightly raised.
|
Opaque, Uneven, Large, Circular, Smooth, yellowish colonies, Slightly Raised.
|
Thin, spreading colonies, Circular Large, , smooth, Opaque, flat.
|
2
|
Microscopic characters
|
Rod-shaped
|
Rod-shaped
|
Rod-shaped
|
Rod-shaped
|
3
|
Gram's Staining
|
Gram Positive
|
Gram Positive
|
Gram Positive
|
Gram positive
|
4
|
Pigmentation
|
Pigmentation was off - white
|
Pigmentation was Yellow
|
pigmentation was Yellowish
|
Pigmentation of off - white
|
5
|
Sucrose Fermentation
|
+ve
|
+ve
|
+ve
|
+ve
|
6
|
Casein Hydrolysis
|
+ve
|
+ve
|
+ve
|
-ve
|
7
|
Lipid Hydrolysis
|
+ve
|
+ve
|
+ve
|
+ve
|
8
|
Citrate Utilization
|
+ve
|
+ve
|
+ve
|
+ve
|
9
|
Starch Hydrolysis
|
+ve
|
+ve
|
+ve
|
+ve
|
10
|
Oxidase Activity
|
-ve
|
-ve
|
+ve
|
-ve
|
11
|
Methyl Red test
|
+ve
|
+ve
|
+ve
|
+ve
|
12
|
Lactose Fermentation
|
-ve
|
-ve
|
+ve
|
-ve
|
13
|
Glucose fermentation
|
+ve
|
+ve
|
+ve
|
+ve
|
14
|
Voges Proskeur
|
+ve
|
-ve
|
+ve
|
-ve
|
15
|
Motility
|
+ve
|
-ve
|
+ve
|
+ve
|
16
|
Indole Production
|
-ve
|
-ve
|
+ve
|
-ve
|
17
|
Gas Production from Glucose
|
+ve
|
-ve
|
+ve
|
+ve
|
18
|
Catalase Activity
|
+ve
|
+ve
|
+ve
|
+ve
|
19
|
Nitrate Reduction
|
+ve
|
-ve
|
+ve
|
+ve
|
20
|
Gelatin Hydrolysis
|
+ve
|
+ve
|
+ve
|
+ve
|
21
|
H2S Production
|
-ve
|
-ve
|
-ve
|
-ve
|
22
|
Spores
|
+ve
|
-ve
|
+ve
|
+ve
|
23
|
Urease Activity
|
-ve
|
-ve
|
-ve
|
-ve
|
3.2 Screening for biosurfactant production
The biosurfactant production was confirmed by performing various assays on the four isolates. The section discusses the respective efficiencies for each of the screened isolates.
3.2.1 BATH assay
The cell hydrophobicity was estimated by the BATH assay. Table 2 shows the affinity of the positive strains of bacterial cells towards the hydrophobic substrate. Isolate 3 (87.23±0.71) shows the highest cell adherence property, whereas Isolate 4 (62.54±1.42) was observed with the least cell adherence. Furthermore, Isolate 1 shows 84.77±0.56 adherence, whereas Isolate 2 (79.46±0.23) shows cell adherence. Positive hydrophobicity of the cells is proclaimed as a signal for the production of biosurfactant. The visualization of bacterial cells adhered to crude oil confirmed the affinity of cells in the crude oil droplet’s vicinity.Table2 discusses the BATH results for each isolate.
3.2.2 Hydrocarbon overlay agar plate
The HOA plate method identifies hydrocarbon elastic Paenibacillus species as well as shows the hydrocarbon-degrading activity of the isolated bacteria. In Table 2, Quantitative assessment of the bioemulsifiers were demonstrating the zone of clearance that Isolate 3 gave 1.7 mm, 1.6 mm, 1.5 mm, and 1.4 mm with benzene plated medium, kerosene, toluene, and hexadecane, respectively. Isolate 1 demonstrated the development across the hydrocarbon plated media with 1.4 mm diameter, 1.5 mm diameter, 1.3 mm diameter, and 0.2 mm diameter of clearance zone for toluene, benzene, hexadecane, and kerosene plated media, respectively. Isolate 3 and isolate 1 gave negative outcomes with benzene and kerosene but, however, gave great outcomes with hexadecane(Isolate 3 was 1.3mm diameter, Isolate 1 was 1.4mm diameter) and toluene(Isolate 3 was 0.6mm diameter, Isolate 1 was 1.5mm diameter). Isolate 2 was 1.3 mm diameter, 1.5 mm diameter, 1.4 mm diameter, and 0.3 mm diameter zone of clearance for toluene, benzene, hexadecane and kerosene plated media. Figure 2 shows the clearance zone observed for hexadecane in the case of Isolate 3.
Table 2: BATH assay and Hydrocarbon overlay agar plate
Microorganism
|
BATH assaya
|
Hydrocarbon overlay agar plateb
|
Kerosene
|
Hexadecane
|
Benzene
|
Toluene
|
Isolate 1
|
++
|
Nil
|
++
|
Nil
|
+
|
Isolate 2
|
++
|
Nil
|
++
|
Nil
|
++
|
Isolate 3
|
++
|
++
|
++
|
++
|
++
|
Isolate 4
|
++
|
+
|
++
|
++
|
++
|
BATH assaya: ‘+++’ - cell adhesion > 90%, ‘++’ - 60 to 89% cell adhesion, ’+’ – 40 to 59% cell adhesion.
Hydrocarbon overlay agar plateb:‘+’ - clearance zone of 0.1-1 mm, ‘++’ - clearance zone of 1.1 to 2 mm, ‘+++’ - clearance zone of 2.1 to 3.5 mm.
3.2.3 Drop collapse assay
Drop collapse assay is a hypersensitive test and can give a result with even a very small measure of biosurfactant produced. A few strains give a positive outcome with BATH assay; however, the strains test negative for drop collapse assay as their cultures with high cell hydrophobicity were act as biosurfactants themselves but were unable to produce the biosurfactant extracellularly. The analysis was performed in the duplicate set. And for conducting the analysis, 10µl of surfactant solution, and the cell-free culture broth were used. The positive outcomes with a flat droplet, as confirmed by the oil spread assay were shown by all microorganisms' strains. The drop of Isolate 1, Isolate 2, Isolate 3, and Isolate 4 collapsed in 1 min 3 sec, 56 sec, 1 min 6 sec, and 1 min 52 sec, respectively (Table 3).In order to assure the production of biosurfactants, oil spreading and surface tension experiments were performed on the microorganism’s cell-free culture broths. Figure 3 shows the drop collapse test for the four isolates.
3.2.4 Oil spreading assay
The organisms with a positive drop collapse assay were found to give positive results for the oil spreading assay. It is observed in the present analysis that the surface-active compound in the Paenibacillus isolate solution is directly proportional to the oil displacement area. Furthermore, it is a qualitative study to check the presence of surfactants. Isolate 1, Isolate 2, Isolate 3, and Isolate 4 show the clearance zone of 1.4mm, 2.4 mm, 2.1 mm, and 1.8 mm. Figure 4 depicts the oil spread assay for Isolate 3.
3.2.5 Surface tension measurement
Surface tension was measured in the cell-free culture broth, and it showed a reduction in surface tension. A direct correlation was observed between drop collapse, oil spreading, and surface tension assays. Microorganisms with slight activity in any one of the assays were active in the other two. Isolate1 to Isolate 4 gave positive results with a reduction in surface tension to 41mN/m, 38 mN/m, 36mN/m, and 42mN/m surface tension, respectively, given in Table 3.
Table 3: Oil spreading assay, Drop collapse assay, and Surface tension measurement
Microorganism
|
Drop collapse assayb
|
Oil spreading assaya
|
Surface tension measurementd
|
Isolate 3
|
++
|
+
|
++
|
Isolate 1
|
+++
|
++
|
+++
|
Isolate 4
|
++
|
++
|
+++
|
Isolate 3
|
++
|
++
|
++
|
Surface tensiond: ‘+++’- indicates surface tension which is less than 40 mN/m, ‘++’- indicates surface tension between 40 to 50mN/m,’+’- indicates surface tension between 51 to 70mN/m.
Oil spreading assaya: ‘+’ - indicates a clear zone of oil spread with a zone of 0.5-1.5 mm diameter, ‘++’ – indicates a clear zone of oil spread with a zone of 1.6 to 2.5 mm diameter, ‘+++’ – indicates a clear zone of oil spread with a zone of 2.6 to 3.5 mm diameter.
Drop collapse assayb: ‘+++’- indicates adrop collapse within 1 minute, ‘++’- indicates a drop collapse after 1minute and ‘+’ – indicates a drop collapse after 3 minutes of biosurfactant addition.
3.3. Optimization of Biosurfactant production for Paenibacillus (Isolate 3)
The pH was optimized based on the E24 index. At pH 7.0, the E24 value was 75% that decreased gradually at pH 8.0, showing a substantial effect (Figure 5(a)). Similarly, the optimum temperature was found to be35°C (E24: 73.37%) (Figure 5(b)). Isolate 3 is mesophilic and exhibits effective production at moderate temperature. Starch was settled up as the most favorable (E24: 82%) amidst the 8 different carbon sources, followed by sucrose (E24: 72%) (Figure 5(c)). Subsequently, yeast extract exhibits the maximal E24 value (82%) amidst 8 different nitrogen sources followed by Peptone (E24: 60%) (Figure 5(d)). For the production medium preparation, all optimized condition plays an essential role in increasing the yield. On varying the carbon and nitrogen sources from 1–5%, 2% of the starch (E24:84%) (Figure5 (e)) as well as 3% of the yeast extract (E24: 83%) (Figure5 (f)) gave maximum production efficiency.
3.4 Extraction of biosurfactant and dry weight
The culture inoculated in a mineral salt medium with oil was centrifuged to collect the supernatant. The collected supernatant was mixed with chloroform and methanol in the aforementioned ratio. White sediment was retained in an empty Petri plate and was weighed as mentioned in Table 4. The maximum amount of biosurfactant was produced by Paenibacillus (Isolate 3), amounting to 0.426 g per 100 mL of medium.
Table 4: Dry weight of biosurfactant produced by the organisms
Microorganism
|
Empty plate weight (g)
|
Biosurfactant containing plate weight (g)
|
Dry weight of biosurfactant (g/100ml)
|
Paenibacillus(Isolate 3)
|
23.785
|
24.211
|
0.426
|
3.5 Thin layer Chromatography
Paenibacillus dendritiformis producing biosurfactant characterization was executed using Thin Layer Chromatography. TLC plate is exposed to different reagents after the solvent completely traveled until the top of the TLC plate and affirmed unique characteristics of the spots. The color of the developed spot depends on the reaction between the developing reagent and the sample compound. The identified Rf value for the appeared spot on the TLC plate is 0.72 indicates that the compound is non-polar. Also, the resulting Rf value was nearby to the Rf values of lipopeptide previously reported by researchers for the lipopeptides produced by Paenibacillus sp. The yellow spot in the iodine vapour indicates the organic compound's presence and the lipid moiety in the structure (Figure 6). In the present analysis, we found the purple spot on treatment with Ninhydrin reagent, which indicates the peptides group's presence in the present organic lipid compound, whereas, the absence of a colored spot on treatment with Orcinol reagent assures the absence of carbohydrate compounds within the structure. Therefore, considering the entire observations ensure the presence of lipopeptide residues in the current structure of biosurfactant originated from Paenibacillus dendritiformis.
3.6 Fourier Transform Infrared Spectroscopy
The absorbance intensity will correlate proportionally to the quantity of functionality present in the activated TLC-purified biosurfactant isolate of Paenibacillus species. FTIR spectral peaks observed at 2,943, 2,911, 2,862, and 1,454 cm−1 wavenumbers confirm the –C–H stretching (−CH3, –CH2) of the long hydrophobic R chain of the lipid as the similar stretching pattern for –C–H of lipid in observed in several previously completed studies of Bacillus species producing biosurfactant (Figure 7). C=O bending of esters was indicated by the peaks at 1,739 and 1,742 cm−1 and C–O by 1,090 cm−1. Besides, 977–840 cm−1 wave number peaks indicate the presence of C=C in the fatty acid chain of the lipid fraction of the biosurfactant. Furthermore, the presence of amide and hydroxyl groups of protein in the structure was identified. Furthermore, the alimentation of the amide bond, i.e., –C=O (1630 cm−1), the carbonyl group (C=O) of amide (1,672 cm−1), and the N–H bending of primary or secondary amides (1,630 cm−1) illustrate the presence of the peptide fragments of the biosurfactant in the structure. The obtained peaks of biosurfactant indicate that lipopeptide biosurfactants were produced by P. dendritiformis; in a fatty acid chain, we found the presence of a double bond of the lipopeptide.
3.7 Stability test of biosurfactant
Optimization of biosurfactant production for Paenibacillus (Isolate 3) was carried out using RSM (response surface methodology) based on the Box–Behnken design of experiments. The R package RSM was used to carry out the analysis. The three factors considered in the analysis were pH (ph), temperature (temp), and salinity (sal). The three levels for each factor included in the design were pH 5, 7, and 10, temperature 30, 40, and 60, and salinity 4%, 6%, and 8%, encoded as -1, 0, and 1 respectively in the design. The experiment was conducted as per the Box–Behnken design and the E24 index was measured for each combination of the parameters. Table 5 shows the E24 values obtained from the design experiment.
Table 5: Results of Box-Behnken experimental design.
Experiment number
|
pH
|
Temp(°C)
|
Salinity (%)
|
E24 (%)
|
1
|
-1
|
-1
|
0
|
69.58858859
|
2
|
0
|
0
|
0
|
42.78678679
|
3
|
0
|
-1
|
1
|
32.57657658
|
4
|
1
|
0
|
1
|
52.53753754
|
5
|
1
|
0
|
-1
|
26.5015015
|
6
|
-1
|
0
|
-1
|
71.57957958
|
7
|
0
|
0
|
0
|
40.08108108
|
8
|
0
|
1
|
-1
|
49.32132132
|
9
|
1
|
-1
|
0
|
24.66366366
|
10
|
0
|
0
|
0
|
44.52252252
|
11
|
-1
|
1
|
0
|
55.3963964
|
12
|
0
|
-1
|
-1
|
73.36636637
|
13
|
-1
|
0
|
1
|
40.13213213
|
14
|
0
|
0
|
0
|
34.92492492
|
15
|
1
|
1
|
0
|
56.87687688
|
16
|
0
|
1
|
1
|
65.91291291
|
Experimental data was used to fit a linear model with a response-surface component using the R package's RSM function. While fitting the model, first-order, two-way interactions and pure quadratic terms were included. The regression equation obtained is shown below. In the equation, the first-order terms are pH, temperature (temp), and salinity (sal), while the two-way terms are ph*temp, temp*sal, and ph*sal, and the quadratic terms are ph^2 (ph square), temp^2 (temp square) and sal^2 (sal square).
E24 = 40.58 - 9.51pH + 3.41temp - 3.70sal + 11.60ph:temp + 14.37ph:sal + 14.35temp:sal + 1.72ph^2 + 9.33temp^2 + 5.39sal^2
The significance of the coefficients of the model is shown in Table 6. The *** mark in the pval column indicates that the p-value is < 0.001 while ** indicates p-value < 0.01 and * indicates p-value < 0.05, indicating statistical significance of the corresponding parameter (Table 6). The intercept, ph, ph*temp, ph*sal, temp*sal, temp^2 terms were statistically significant, and therefore they influence the E24 values. The E24 values varied from 24.6% to 73.3%, and the highest E24 was observed for pH 7, temperature 30°C, and 4% salinity (Table 7). Overall the model was found to be statistically significant (p-value 0.0008478) with F value as 19.82 (9 and 6 degrees of freedom), adjusted R-squared value as 0.91.
Table 6: Statistical significance of model parameters
Estimate or Coefficient
|
Standard error
|
Student t-test test statistics
|
pval
|
(Intercept)
|
40.57882883
|
2.243416351
|
1.84E-06***
|
ph
|
-9.51463964
|
1.586334915
|
0.00097***
|
temp
|
3.414039039
|
1.586334915
|
0.07488
|
sal
|
-3.701201201
|
1.586334915
|
0.05839
|
ph:temp
|
11.60135135
|
2.243416351
|
0.00207**
|
ph:sal
|
14.37087087
|
2.243416351
|
0.00068***
|
temp:sal
|
14.34534535
|
2.243416351
|
0.00069***
|
ph^2
|
1.722972973
|
2.243416351
|
0.47161
|
temp^2
|
9.32957958
|
2.243416351
|
0.00595**
|
sal^2
|
5.385885886
|
2.243416351
|
0.05324
|
Table 7: The results of ANOVA and lack-of-fit test of the model
Term
|
Df
|
Sum Sq
|
Mean Sq
|
F value
|
Pr(>F)
|
First order terms
|
3
|
927.0633629
|
309.021121
|
15.35000108
|
0.0032**
|
Two way interaction terms
|
3
|
2187.608863
|
729.2029545
|
36.22168641
|
0.0003***
|
Pure quadratic terms
|
3
|
476.0698311
|
158.6899437
|
7.882602973
|
0.0166*
|
Lack of fit
|
3
|
68.14782575
|
22.71594192
|
1.29454793
|
0.4185
|
Residuals
|
6
|
120.7900062
|
20.1316677
|
|
|
Pure error
|
3
|
52.64218047
|
17.54739349
|
|
|
The E24 values obtained from the design experiment are shown in two-dimensional contour plots. The x and y-axis of each contour plot indicate the parameter considered while keeping the third remaining parameter constant. The top left contour plot represents the variation in the E24 value as a pH and temperature function. In contrast, the top-right plot indicates the variation in E24 as a function of pH and salinity. The bottom contour plot indicates the variation in E24 values as a function of temp and salinity. The colour scales from green to yellow, where green indicates low E24 values while yellow indicates high E24 values. In the contour plot, the line with E24 values is also shown in figure 8.
3.8 DNA isolation, amplification and phylogenetic assessment
The phylogeny assessment of Isolate 3 was performed. DNA was isolated and amplified using PCR. After the amplification, the DNA was quantified using a nanodrop and qubit fluorometer. Qubit fluorometer estimated DNA yield as 1281.6 μgl−1and had optimal purity. Thus, the obtained amplified DNA was further used for phylogenetic assessment [30-33]. 16S rRNA sequence was 1483 base pairs long and was single-stranded bearing a molecular weight of 450176.00 Daltons. GC% and AT% was 54.96% and 45.04%, respectively. Comparatively, G was the highest, and T was the lowest. The sequence was further analyzed by BLASTn against 16S ribosomal RNA sequences (16S ribosomal Bacteria and Archaea) database using a cut-off value of >95%. The first hit shows that the query sequence matches with Paenibacillus dendritiformis strain T168 16S ribosomal RNA gene, partial sequence by 98%, whereas 4 hits were seen in the next go that showed similarity with various strains of Paenibacillus thiaminolyticus 16S ribosomal RNA gene, partial sequence. All the similar sequences were further downloaded from GenBank, and a phylogenetic tree was constructed using the neighbor-joining algorithm in Mega software. The phylogenetic tree has been depicted in figure 9.
In contemporary research, the secondary structure of 16S rRNA of Paenibacillus dendritiformis strain ANSKLAB02 has demonstrated helical regions with the interior, hairpin, bulge and multi-branched loops that may bind to23S rRNA. The free energy (ΔG) of the rRNA secondary structure for Paenibacillus dendritiformis strain ANSKLAB02was calculated to be − 131.00 kcal mol−1 been elucidated using UNAFOLD software. The structure has been depicted in figure 10. The thermodynamics result from each base of the Paenibacillus dendritiformis strain ANSKLAB02dataset shows the average energy of external closing pair helix, stack, multi-loop, bulge loop, and hairpin loop, as,-299.80 kcal/mol, −2.03 kcal/mol, 0.11 kcal/mol,0.210 kcal/mol and −1.62 kcal/mol, respectively. The closing pair and interior loop had ΔG value = − 2.11 kcal/mol. Further, the two TNA structures entropy was estimated using the RNA fold server and is depicted as a hill plot in figure 11.