## 3 Results and Discussion
### Treatment Efficiency of the Contaminated Soil Under Different Bioremediation Conditions  
The bioremediation experiment revealed significant differences in the removal efficiencies of hydrocarbons between all treatments. The highest hydrocarbon removal efficiency of 75.8% was observed in the TS + NS + RPB treatment (Figure 4a). Soil respiration (0.6 mg CO\({}_{2}\) g\({}^{-1}\) h\({}^{-1}\)) (Figure 4b), DHA activity (5.2 \(\upmu\)g TPF g\({}^{-1}\) soil 24 h\({}^{-1}\)) (Figure 4c), and the highest hydrocarbon-degrading bacterial abundance (42 \(\times\) 10\({}^{6}\) CFU g\({}^{-1}\)) (Figure 4d) were obtained in the TS + NS + RPB treatment by the 90 th day of the experiment, and this activity was 44.4%, 80.6%, and 39.5% higher, respectively, than that in the CS alone treatment. At the end of the experiment on day 135, there were no significant increases in hydrocarbon-degrading bacterial population and DHA activity, and a decrease of 17.5% in soil respiration activity was observed. Figure 2: (a) Three-dimensional contour plot for maximum rhamnoid production expressed as RE (rhamnose equivalents) mg g\({}^{-1}\) cell dry weight as a function of inoculation load and crude oil (%), (b) Predicted vs. actual values for rhamnoid production based on response surface methodology (c) Growth and rhamnoid production in RSM optimized media, (d) Graph of surface tension vs. concentration of the rhamnoid. Different lower case letters after values indicate a significant difference at a \(p\)-value of 0.05 as determined by [PERSON]’s multiple range test. _Appl. Sci._**2019**, \(9\), 3773  
Figure 3: The thin-layer chromatographic slide proving mono- and di-rhammolipid presence in the extracted mixture (**a**) and Fourier transform infrared spectroscopic (FT-IR) spectra confirming the presence of a rhamnoid type biosurfactant (**b**). _Rf_–retention factor (0.31 for di-rhammolipid and 0.81 for mono-rhammolipid). Figure 4: Cont. Figure 4: _Cont._  
Plytotoxicity evaluation of treated soils based on germination studies revealed the highest germination (92%) in the TS + NS + RPB treatment, followed by the TSB + RPB treatment (Figure 4e). These treatments also supported better plant growth in these soils, which was evident by an 86.9% increase in germination percentage compared to contaminated soil (CS) (Figure S2). Soil respiration and DHA activity are the measures of total microbial activity in soil [52; 53; 54], and any change in organic matter quantity or quality can be observed as a change in soil respiration [54; 55] and/or in DHA activity [56]. The results of the present study clearly showed that the addition of rhamn improved the activity of _Shewanella_ sp. BS4 and the native soil microbial community, which in turn improved the hydrocarbon bioremediation process. Although the native soil microbes did not play a major role in the bioremediation process, the oil-polluted soil bioremediation was more efficient in the case of the simultaneous application of rhamnpid and _Shewanella_ sp. BS4. The results from this study showed that the addition of rhamnpids might enhance oil biodegradation efficiency by acting as mediators, increasing the mass transfer rate, and thus making oil compounds more bioavailable for soil-indigenous microorganisms. Also, rhamnpids may induce changes in the properties of cellular membranes of bacteria by enhancing hydrophobicity, which increases microbial adherence [57]. The single-use of rhamnpids in oil bioremediation may require the addition of nutrients, such as nitrogen or phosphorus, to the soil. For example, the augmentation of rhamnpids and nutrients enhanced crude oil reduction and increased the abundance and expression of the _alkB_ gene in soil [58]. Our results indicated that the simultaneous application of rhamnpids and rhamnpid-producing bacteria enhanced the oil bioremediation process in soil more than the individual use of these treatments.