# Speciation of the Removed Pollutants in Bioremediation of Hydrocarbon-Contaminated Soil
## 4 Discussion
The starting concentration was around 124 g\(\cdot\)kg\({}^{-1}\) of soil and the runs lasted 60 days. Their best efficiency was over 90% as an overall value with a sequential inoculation of fungal-bacterial consortia. Looking at their findings achieved with batch inoculation, it can be seen that:  
* In the first case, the removal of aliphatic compounds was comparable to ours of alkanes, whereas the removal of the aromatics was lower than 60% (our values always reach 80%);
* In the second case, where the bacterium was the same, but the fungus was changed, the removal of aromatic compounds was over 80%, showing the relevance of the biological activity of the applied fungus. These findings demonstrate that, when the strategy of inoculation is chosen, an accurate control of the adopted consortium could improve the efficiency of removal. However, the comparison with our results also demonstrates the efficacy of consortia which contain microorganisms acclimated to diesel oil or petroleum hydrocarbons for the removal of these pollutants, also evidenced by other authors [16; 17; 19; 20]. In their study on the strategies for remediation of soils contaminated with petroleum [20], [PERSON] et al. also reported the relevance of monitoring soil enzyme activities. One of these activities is the hydrolysis of fluorescein diacetate (FDA) to fluorescein, which reflects microbial activity [21; 22]. The quantity of fluorescein produced by hydrolysis in the mesocosoms at the end of the runs [8] confirmed this enzymatic activity, especially for the BS + IN mesocosm: after 228 days, the fluorescein production was 2.2 mg\(\cdot\)kg\({}^{-1}\) of soil against the value 0.9 mg/kg of soil for the BS mesocosm. At last, the microbial colonization in each mesocosm was shown by Scanning Electron Microscope images taken at different points and reported in a previous paper [8], as a further demonstration of microbial activity, even if at different extents. In the same images, the presence of biofilm can be supposed, even if no specific analyses were carried out to support this hypothesis. To this end, nowadays, the biofilm role is known and verified in many systems [23], including the removal of long-chain alkanes [24]. One of its roles is the protection of microorganisms under extreme environmental conditions, such as oligotrophic conditions, where the quantity of nutrients is scarce. In the present study, at the end of the run (228 days), the biostimulated mesocosoms had a depletion of micronutrients, as evidenced by the geophysical monitoring, and this occurred at the mesocosm bottom to a greater extent. At the same time, in both cases, the pollution removal efficiency was more than satisfactory, showing that the microbial consortia were active, and this could have occurred due to the protection of microorganisms by biofilm. Deeper investigations are required to confirm the presence of biofilms as a \"tool\" involved positively in the biodegradation process. For this purpose, the analytical task could also make use of geophysical measurements (e.g., X-ray tomography with high resolving power is suggested to scan the pore-solid interaction) as a complementary system.