3.1 Strain identification and biosurfactant production
The morphological and transmitted electron microscopic (TEM) characteristics of strain revealed the small, irregular, rough and white colonies that belong to the class Firmicutes and genus Bacillus (Additional file 1: Fig. S1). This S2MT strain was identified as Bacillus nealsonii, showing 99.93% similarity by 16S rRNA ribotyping, as illustrated in the phylogenetic tree (Additional file 1: Fig. S2). Nucleotide sequence of 16S rRNA of B. nealsonii S2MT strain was submitted in the NCBI Gen-Bank database with accession number (MN128029).
For biosurfactant production, the strain S2MT exhibited positive activities in initial biosurfactant screening methods. The efficient activity of biosurfactant product in oil displacement test, 4.2 ± 0.4 cm clear zone, and drop collapse method (Additional file 1: Fig. S1). Moreover, the strain S2MT reduced the surface tension up to 34.15 ± 0.6 mN/m− 1, which is lower than the distilled water 81.3 mN/m− 1 (Fig. 1A), and expressed the emulsification activity 55 ± 0.3% in kerosene oil after 24 hours. According to Cooper and Goldenberg [23] if bacterial isolate can reduce the surface tension (40 mN/m− 1) or less it could be a promising biosurfactant producer. The above results indicate that the strain Bacillus nealsonii S2MT is a potential biosurfactant producing strain. Numerous Bacillus sp. and related genera have been reported from different types of environments i.e. extreme, hydrocarbon-contaminated terrestrial or marine environment [7] for biosurfactants production by B.licheniformis, Aeribacillus sp. Bacillus subtilis [13, 27]. However, no previous report highlighted related to biosurfactant production by Bacillus nealsonii.
3.2 Medium optimization on different carbon/nitrogen substrates and product recovery
A number of different C/N sources (as mentioned above in methods) were provided to induce the biosurfactant product yield and the glycerol/NH4NO3, the best one, contributed to produce the highest biosurfactant quantity, showing the 34.15 ± 0.6 mN/m− 1 surface tension reduction and emulsification 55 ± 0.3% (Fig. 1A). Whereas, the total dry biomass and crude biosurfactant products were obtained 4140 ± 70.71 mg/L− 1 and 1255 ± 21.21 mg/L− 1 respectively (Fig. 1D). The glycerol/Urea preceded the yeast extract as second important C/N sources (Fig. 1B, C), on which efficient surface tension reduction 36.7 ± 0.6 mN/m− 1 and 46.6 ± 2% emulsification activity was noticed together with total dry biomass 1505 ± 205.0 mg/L− 1 and crude biosurfactant 130 ± 13.43 mg/L− 1 (Fig. 1D). However, other combinations of C/N sources such as glucose with yeast extract, urea, and even NH4NO3 did not alter biosurfactant production to significant level except increased in cellular biomass, while kerosene acted as an inhibitor due to its toxicity, as depicted in (Additional file 1: Figs. S3A- I, except D, F). All this together suggested that the 2% glycerol with 0.1% NH4NO3 was the most effective C/N source for biosurfactant production by strain B. nealsonii S2MT in comparison to glucose and kerosene substrates.
Previous findings also demonstrated that the NH4NO3 with various carbon substrates have been found an efficient nitrogen source for biosurfactant production by Bacillus species. Medeot et al. (2017) achieved the maximum biosurfactant concentration (1.7 mg/mL) by Bacillus amyloliquefaciens MEP218 upon using the glucose and NH4NO3 [28]. Likewise, Fernandes et al. (2016) reported NH4NO3 combined with sucrose to get the highest concentration of biosurfactant by Bacillus subtilis RI4914 (0. 2 g/L) [29]. Also, Abdel-Mawgound et al. (2008) studied surfactin production by Bacillus subtilis BS5 using different carbon and nitrogen sources and concluded the highest biosurfactant production in NaNO3 and NH4NO3 [30]. Evidently, nitrogen source play a crucial role in biosurfactant production [15], but it depends upon the carbon/nitrogen substrates combination.
3.3 Statistical screening of critical factors for biosurfactant production
The results of surface tension (SFT) reduction and product yield, showing the effects of six factors by combining them in different proportions as design by the model are mentioned in (Table 2). Minimum SFT values 33.7, 34.2, 34.3, and 34.5 mN/m− 1 and product yield of 1300, 1110, 900, and 850 mg/L− 1 were observed in Run number. 10, 7, 13 and 19 respectively. Contrarily, the increased value of SFT was found 64, 49.5, 46.8 and 44.5 in Run no. 14, 20, 11 and 17 respectively. Whereas the product recovery was only 10, 15, 60, and 60 mg/L− 1 by the same Run numbers. It was thus found that surface tension reduction has directly proportional to the product yield.
Table 2
Design layout of a regular 2-level factorial model showing different influential factors and their effect on Surface tension and biosurfactants product yield
Run | Factor1 A: Temp (⁰C) | Factor2B: pH | Factor3 C: Agitation | Factor4 D: NH4NO3 (%) | Factor5 E: Yeast extract (%) | Factor6 F: NaCl (%) | Response1: SFT (mN/m) | Response2 : Yield (mg/L) |
1 | 25 | 8 | 180 | 1 | 0 | 0.5 | 37.7 | 80 |
2 | 27.5 | 7 | 140 | 0.55 | 0.1 | 0.3 | 37.4 | 400 |
3 | 25 | 6 | 180 | 0.1 | 0.2 | 0.5 | 34.9 | 380 |
4 | 27.5 | 7 | 140 | 0.55 | 0.1 | 0.3 | 37.6 | 390 |
5 | 30 | 6 | 180 | 0.1 | 0 | 0.5 | 34.9 | 380 |
6 | 25 | 8 | 100 | 1 | 0.2 | 0.1 | 36.5 | 60 |
7 | 25 | 8 | 100 | 0.1 | 0.2 | 0.5 | 34.2 | 1110 |
8 | 27.5 | 7 | 140 | 0.55 | 0.1 | 0.3 | 36.8 | 440 |
9 | 30 | 8 | 180 | 1 | 0.2 | 0.5 | 37.6 | 60 |
10 | 30 | 8 | 100 | 0.1 | 0 | 0.5 | 33.7 | 1300 |
11 | 25 | 6 | 100 | 1 | 0 | 0.5 | 46.8 | 60 |
12 | 30 | 6 | 100 | 1 | 0.2 | 0.5 | 37.6 | 30 |
13 | 30 | 8 | 180 | 0.1 | 0.2 | 0.1 | 34.3 | 900 |
14 | 25 | 6 | 100 | 0.1 | 0 | 0.1 | 64.6 | 10 |
15 | 27.5 | 7 | 140 | 0.55 | 0.1 | 0.3 | 37.1 | 380 |
16 | 30 | 6 | 100 | 0.1 | 0.2 | 0.1 | 35.4 | 50 |
17 | 30 | 8 | 100 | 1 | 0 | 0.1 | 38.2 | 60 |
18 | 25 | 6 | 180 | 1 | 0.2 | 0.1 | 38.1 | 61.6 |
19 | 25 | 8 | 180 | 0.1 | 0 | 0.1 | 34.5 | 830 |
20 | 30 | 6 | 180 | 1 | 0 | 0.1 | 49.5 | 15 |
The analysis of variance and regression of both responses (R1 = SFT (mN/m− 1) and (R2 = yield (mg/L− 1) gave details of the most significant terms of the model as shown in (Additional file 1: Table S3). The ANOVA summary of the model indicated the high signification P-value (P < 0.05) to elucidate the effect of significate model terms that affected SFT and yield. The most significant factors were pH, temperature, yeast extract and NaCl conc., with P-values (P = 0.0028), (P = 0.0036), (P = 0.0042) and (P = 0.0319) respectively. Whereas, the most significant factors with P-values on yield were NH4NO3 (P = 0.0005), temperature (P = 0.0039) and pH (P = 0.0044). The normal probability chart significantly contributed, and the Pareto chart indicates the rank wise positive and negative effects of factors on biosurfactant production based on the model (Additional file 1: Figs. S4). However, the effects of individual factors of significant model terms are depicted (Additional file 1: Figs. S5).
Response surface plot and wireframes show the effect of combine factors i.e. pH vs temperature, NH4NO3 vs. temperature, NaCl vs. temperature and NaCl vs. pH on surface tension reduction and biosurfactants yield (Figs. 2A- F). Figures 2A, D shows the surface tension value was found decreased and observed maximum biosurfactant yield, when the pH and temperature were increased. The increased pH (8) and high temperature (30 °C) may have a significant effect on biosurfactants, whereas a decreased of both factors may have an insignificant effect on biosurfactant production [31]. The NH4NO3 (0.1%) and high temperature (30 °C), and increased pH (8) and NaCl con. (0.5%) also showed a positive effect on both responses R1 and R2 (Fig. 2B, C, F). Similarly, the significant effect was found at the high NaCl conc. (0.5%) and high temperature (30 °C) on biosurfactant production (Fig. 2E), whereas, low NaCl conc. and high temperature may also have a negative effect on biosurfactants [31]. The design experiment (v. 12), based on the regular two-level factorial model suggested the equation for increase biosurfactant production as
Yield = 349.83 + 12.71 (A) + 213.37 (B) + 1.66 (C)- 283.34 (D)– 5.21 (E) + 88.34 (F) (2)
Where, the A, B, C, D, E, and F coded value of the temperature, pH, agitation, NH4NO3, Yeast extract, and NaCl respectively. After optimization conditions, the biosurfactant production was increased 3.58% more, than initial prodcution by strain S2MT.
3.4 Stability of biosurfactant production
The effect of environmental factors on crude biosurfactants is shown in (Fig. 3A) and (Additional file 1: Table S1). As a result of temperature variation, the surface tension was found to be a decreased value of 31.5 ± 0.1 mN/m− 1 at 100ºC, however, when the temperature decreased as low as 4 °C, the surface tension was found to be increased of 44.2 ± 2.5 mN/m− 1. The biosurfactant product by S2MT strain was found highly stable at high temperature. In pH ranges, the surface tension was found to be increased at pH 3 as 40.8 ± 0.4 mN/m, whereas, the value was decreased 36.7 ± 0.4mN/m− 1 at 6 pH. The acidic condition was not in the favor of biosurfactants stability but could be caused by the precipitation of biosurfactants [12, 32]. In the case of NaCl concentration, slight differences were found in surface tension measurement; since the elevated salt concentrations gradually increased the surface tension values from 37.6 ± 0.6 mN/m− 1 to 39.65 ± 0.0.6 mN/m− 1 in 3 and 9% of NaCl concentrations respectively. The reason for the increase in surface tension might be the ionic salts form ion-dipole interactions with water, which is stronger than the gaseous phase and salt interactions, and causing the solute molecules to avoid the interface [12].
3.5 Critical micelles concentration (CMC) determination
The result of CMC is shown in (Fig. 3B). Surface-tension value of various biosurfactant solutions (0-100 mg/L− 1) were recorded. The CMC value of an obtained product was found to be at 40 mg/L, where the surface-tension was measured 81.2 mN/m− 1 to 34.5 ± 0.56 mN/m− 1, afterward, there were no much significant changes observed in surface-tension measurement. The present results of CMC were an agreement with Datta et al., (2018), who observed CMC (40 mg/L) by Bacillus subtilis MG495086 [21].
3.6 Product characterization
The crude biosurfactant was analyzed on the TLC plate by strain Bacillus nealsonii (S2MT) that indicated lipopeptide in nature. Different spots at Rf of 0.25, 0.35 and 0.75 were observed after sprayed of 0.2% ninhydrin as illustrated in (Additional file 1: Fig. S6). That might be most related to the surfactin family of the lipopeptide. Similar results were obtained by Ramyabharathi et al (2018) on Bacillus subtilis Bbv57 which produced surfactin and iturin that was confirmed on TLC with Rf value of 0.3 for surfactin and 0.7 for iturin family by comparing with standard (Sigma-Aldrich) [33]. Likewise, Yánez-Mendizábal et al. (2012) reported surfactin and iturin with Rf values 0.3 and 0.7 respectively [34]. Joy et al. (2017) also reported the pattern of TLC by Bacillus sp. (SB2) with an Rf value of 0.72 and 0.55 for lipopeptide biosurfactants [7].
Furthermore, the biosurfactant product was confirmed by LC-ESI/MS. The results of m/z peaks are summarized in (Additional file 1: Table S2). A total six different surfactin like isoforms from (C13-C15) of lipopeptide, at m/z 1008.76 and 1030.74 with retention time (Rt 12.48); 1022.78 and 1044.75 (Rt 14.26); and 1036.79 and 1058.78 with (Rt 15.70) were detected by LC/MS as shown in the chromatogram (Fig. 4). In this respect, the similar pattern was reported by Li et al. (2008) of different surfactin like homologs by B. licheniformis HSN221, when who cultivated in MSM medium with glucose, yeast extract, and ammonium nitrate [14]. Also Chen et al. (2017) reported surfactin homologs at m/z 994, 1008, 1022, 1036 with isoforms of C12, C13, C14, and C15 respectively, by B. licheniformis MB01 [13]. The above analyses revealed that the biosurfactant produced by Bacillus nealsonii S2MT strain is a lipopeptide in nature having the highest resemblance with the surfactin family. In general, surfactin like biosurfactants relating to the family of cyclic lipopeptide and mostly produced by Bacillus spp. [27].
3.7 Potential of crude biosurfactant in heavy engine oil polluted soil remediation
Petroleum hydrocarbon contaminants are major social and ecological issues. These contaminants bind to soil particles and are difficult to remove because of their strong sorption and hydrophobicity. Microbial surfactants in oil-polluted soil can emulsify these compounds and enhanced their solubility, decreased surface tension and increased oil displacement from soil particles [12, 25, 35, 36]. The potential of obtained crude biosurfactant by B. nealsonii S2MT in heavy oil-contaminated soil remediation resulted in 43.6 ± 0.08 and 46.7 ± 0.01% with the concentration 10 and 40 mg/L of crude biosurfactant respectively. However, with synthetic surfactants, sodium dodecyl sulfate (SDS) obtained 39.4 ± 0.01 and 45.3 ± 0.14% removal of contaminants with the same concentration of 10 and 40 mg/L respectively. Whereas 18.5 ± 0.07% removal of contaminants obtained with distilled water as shown in (Fig. 5).
In this respect, Souza et al. (2018) obtained 20% remediation of oil-contaminated sand by Wickerhamomyces anomalus CCMA 0358 [37], which was lower than the current study. Felix et al. (2019) used semi-purified biosurfactant by B. subtilis in diesel oil-contaminated soil remediation, and who obtained 78.5% and 81.8% removal with a concentration of 12.5 mg/L and 37.5 mg/L [12]. Another hand, 80% hydrocarbon removal from soil was achieved by using chemical surfactant SDS and Triton X-100 [38]. In contrast, the biosurfactants are natural compounds, eco-friendly, non-or-less toxic and biodegradable, and also more powerful than a synthetic one, and could be used as a crude product in bioremediation applications.